Handbook of toxicology

H A N D B O O K OF Second Edition Library of Congress Cataloging-in-Publication Data Handbook of toxicology / Michael...

Author: Michael J Derelanko; Mannfred A Hollinger (eds.)

296 downloads 2914 Views 9MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form

DOWNLOAD PDF

H A N D B O O K OF Second Edition

Library of Congress Cataloging-in-Publication Data Handbook of toxicology / Michael J. Derelanko, Mannfred A. Hollinger, editors.—2nd ed. p. cm. Updated and expanded ed. of: CRC handbook of toxicology. c1995. Includes bibliographical references and index. ISBN 0-8493-0370-2 (alk. paper) 1. Toxicology—Handbooks, manuals, etc. I. Derelanko, Michael J. II. Hollinger, Mannfred A. III. Derelanko, Michael J. CRC handbook of toxicology. RA1215 .C73 2001 615.9—dc21

2001025086

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. All rights reserved. Authorization to photocopy items for internal or personal use, or the personal or internal use of specific clients, may be granted by CRC Press LLC, provided that $1.50 per page photocopied is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA. The fee code for users of the Transactional Reporting Service is ISBN 0-8493-0370-2/01/$0.00+$1.50. The fee is subject to change without notice. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. Specific permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe.

Visit the CRC Press Web site at www.crcpress.com © 2002 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-0370-2 Library of Congress Card Number 2001025086 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper

Copyright © 2002 by Taylor & Francis

Preface to the First Edition Toxicologists working in the laboratory or office rely on a large information base to design, conduct, and interpret toxicology studies and to perform risk assessments. Diverse information such as normal hematology and clinical chemistry values, reproductive indices, physiological parameters, animal housing requirements, toxicity classifications, and regulatory requirements accumulated during the toxicologist’s career are generally scattered in file cabinets and on office shelves. Although practicing toxicologists usually can locate information related to their own areas of expertise with minimal effort, obtaining reference information in less familiar areas of toxicology may require considerably more effort, possibly involving a trip to the library or a phone call to a colleague. A single basic reference source of toxicological information has not been previously available. We have attempted to fill this void with this publication. Our goal was to produce a reference book containing practical reference information useful to practicing toxicologists in the chemical and pharmaceutical industries, contract laboratories, regulatory agencies, and academia. Contributors were asked to compile reference material for their own areas of expertise which would be of value to both experts and students. The task seemed easier in concept than it proved to be in reality. It quickly became evident that limits had to be placed on the amount and detail of information included to allow for publication in a reasonable time frame. Although information for most areas of toxicology is presented, coverage of some areas is clearly missing. We encourage and welcome constructive comments on improving the information provided as well as suggestions for additional material which could be included in possible future editions of this handbook. We have designed the handbook to allow basic reference information to be located quickly. Each chapter begins with an outline of its contents. Where possible, text was purposely kept to a minimum. This book is intended only to be a basic reference source. The user requiring more detailed discussion should consult the sources cited. Much of the information provided has been previously published elsewhere. The editors and contributors cannot attest to the accuracy and completeness of such and, therefore, cannot assume any liability of any kind resulting from the use or reliance on the information presented in this handbook. Mention of vendors, trade names, or commercial products does not constitute endorsement or recommendation for use.


Preface to the Second Edition It has been approximately 10 years since we began compiling information for the first edition of the CRC Handbook of Toxicology. In reviewing the material contained in the first edition it was apparent that information such as values for physiological parameters, substance toxicity, and information related to fundamental toxicology principles and practices remain virtually timeless. On the other hand, information on such topics as regulatory requirements and guidelines, contract laboratories, and contact information such as phone numbers and addresses clearly needed updating. Moreover, although information for most areas of toxicology was included in the first edition, coverage of some toxicology specialties was clearly missing. In this respect, the CRC Handbook of Toxicology, Second Edition has been extensively updated and expanded. Nearly all of the original chapters from the first edition have been updated, with several receiving extensive revision. Additionally, coverage of inhalation toxicology, neurotoxicology, and histopathology has been expanded. Several new regulatory chapters dealing with pesticides, medical devices, consumer products, and worldwide notification of new chemicals have been added. Areas of toxicology missing from the first edition such as ecotoxicology and in vitro toxicology are now covered. Also included is a new chapter providing an extensive overview of the toxicology of metals. Since the publication of the first edition, environmental and endocrine toxicology and children’s health have become major issues that will clearly impact the field of toxicology in the future. To provide some basic information on these topics, two chapters on basic male and female endocrinology and toxicology have been included and tables have been added to the risk assessment chapter that provide information on differences in physiological and biochemical parameters between children and adults. When the first edition went to print, the Internet was in its infancy but has now become an important information-gathering tool for toxicologists. In the second edition, the authors were asked where possible to reference Web sites they consider sources of valuable information for their fields of expertise. The CRC Handbook of Toxicology contains a considerable amount of reference information. However, because of the size of the handbook and the number of tables and figures it contains, some users of the first edition reported it was not always easy to identify and locate specific information quickly. As a search aid for the second edition, headings have been added at the top of each page identifying the chapter topics. Also we included pages at the end of some of the chapters to provide additional information closely related to the subject matter of the chapter. Constructive comments on how future editions of the CRC Handbook of Toxicology can be improved are welcome. The number of chapters in the second edition has increased from the original 22 to 33 with over 200 new tables and figures added. It is said, “A picture is worth a thousand words.” Thus, as in the first edition, text has been kept to a minimum where possible and practical reference information is provided in tables and figures that are useful to practicing toxicologists in the chemical and pharmaceutical industries, contract laboratories, regulatory agencies, and academia. As before, much of the information provided has been previously published elsewhere. Although considerable effort was made to obtain the information from reliable sources, the editors and contributors cannot attest to its accuracy and completeness and, therefore, cannot assume any liability of any kind resulting from the use or reliance on the information provided in this handbook. Mention of vendors, trade names, or commercial products does not constitute an endorsement or recommendation for use. Michael J. Derelanko Mannfred A. Hollinger


Editors Michael J. Derelanko, Ph.D., D.A.B.T., F.A.T.S., is Corporate Manager of Toxicology and Risk Assessment at Honeywell International Inc., in Morristown, New Jersey. Dr. Derelanko received a B.S. degree from Saint Peter’s College in 1973. He was a National Institutes of Health predoctoral trainee in the Albert S. Gordon Laboratory of Experimental Hematology at New York University, receiving M.S. and Ph.D. degrees. He received the 1976 New York University Gladys Mateyko Award for Excellence in Biology. Following a two-year postdoctoral fellowship in gastrointestinal pharmacology at Schering-Plough Corporation, he began his career in industrial toxicology in 1980 as a research toxicologist in the laboratories of Allied Chemical Corporation. Dr. Derelanko is a Diplomate of the American Board of Toxicology and a Fellow of the Academy of Toxicological Sciences. He is a member of the Society of Toxicology, the Society for Experimental Biology and Medicine, and the honorary research society Sigma Xi. He has served on the content advisory committee of the New Jersey Liberty Science Center, has chaired or been a member of industrial and government toxicology advisory committees, is actively involved with the Chemical Industry Institute of Toxicology, and serves on the speaker’s bureau of the New Jersey Association for Biomedical Research. Dr. Derelanko has authored numerous papers in experimental hematology, gastrointestinal pharmacology, and toxicology. He has been actively involved in educating the public about toxicology, particularly at the middle school level. He has delivered invited lectures on this subject at national meetings. Dr. Derelanko’s current research interests involve understanding the toxicity of aliphatic oximes. He is coeditor along with Dr. Mannfred A. Hollinger of the first edition of the CRC Handbook of Toxicology and author of CRC’s Toxicologist’s Pocket Handbook. Mannfred A. Hollinger, Ph.D., is a Professor in the Department of Medical Pharmacology and Toxicology, School of Medicine, University of California, Davis. Dr. Hollinger is former editor of Current Topics in Pulmonary Pharmacology and Toxicology, Focus on Pulmonary Pharmacology and Toxicology, and Yearbook of Pharmacology, and assistant editor of The Journal of Pharmacology and Experimental Therapeutics. Dr. Hollinger serves on the editorial advisory board of The Journal of Pharmacology and Experimental Therapeutics, Research Communications in Chemical Pathology and Pharmacology, and The Journal of The American College of Toxicology. Dr. Hollinger is at present series editor of Pharmacology and Toxicology: Basic and Clinical Aspects for CRC Press. He is a member of the American Society of Pharmacology and Experimental Therapeutics and the Society of Toxicology. Born in Chicago, he obtained his B.S. degree from North Park College in 1961 and his M.S. (1965) and Ph.D. (1967) degrees from Loyola University, Chicago. He was employed by Baxter Laboratories from 1961 to 1963. From 1967 to 1969, Dr. Hollinger was a postdoctoral research fellow in the Department of Pharmacology, Stanford University Medical School. Since coming to Davis in 1969, Dr. Hollinger has participated in several team-taught courses to undergraduate, graduate, and medical students. While at Davis, Dr. Hollinger has published numerous research papers as well as a monograph on respiratory pharmacology and toxicology. He continues to serve as a referee for many of the principal pharmacology and toxicology journals. Dr. Hollinger was the recipient of a BurroughsWellcome Visiting Scientist Fellowship to Southampton, England in 1986 as well as a National Institutes of Health Fogarty Senior International Fellowship to Heidelberg, Germany in 1988. His current research interests deal with pulmonary fibrosis.


Contributors Mohamed B. Abou-Donia, Ph.D. Professor of Pharmacology and Neurobiology Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina

Michael J. Derelanko, Ph.D., D.A.B.T., F.A.T.S. Corporate Manager of Toxicology and Risk Assessment Honeywell International Inc. Morristown, New Jersey

Aqel W. Abu-Qare, Ph.D. Research Associate Department of Pharmacology and Cancer Biology Duke University Medical Center Durham, North Carolina

Patrick J. Devine, Ph.D. Research Associate Department of Physiology The University of Arizona Tucson, Arizona

Carol S. Auletta, D.A.B.T., R.A.C., M.B.A. Senior Director Toxicology Huntingdon Life Sciences Princeton Research Center East Millstone, New Jersey William H. Baker, D.V.M., Dipl., A.C.V.P. Associate Director of Pathology Springborn Laboratories, Inc. Spencerville, Ohio Kimberly L. Bonnette, M.S., LATG Manager of Acute Toxicology Springborn Laboratories, Inc. Spencerville, Ohio G. Allen Burton, Jr., Ph.D. Brage Golding Distinguished Professor of Research and Director Institute for Environmental Quality Wright State University Dayton, Ohio Rodger D. Curren, Ph.D. President Institute for In Vitro Sciences, Inc. Gaithersburg, Maryland


Jill Dolgin, Pharm.D. Drug Information Product Manager Product Information Department SmithKline Beecham Philadelphia, Pennsylvania Brendan J. Dunn, M.S. Manager, Toxicology and Risk Assessment Honeywell International Inc. Morristown, New Jersey Donald J. Ecobichon, Ph.D. Queen’s University Department of Pharmacology and Toxicology Kingston, Ontario, Canada Eman M. Elmasry, Ph.D. Associate Professor Faculty of Pharmacy Zagazig University Zagazig, Egypt Henry C. Fogle, B.S., M.S. Manager, International Regulatory Affairs Honeywell International Inc. Hopewell, Virginia Ramadevi Gudi, Ph.D. Study Director, Cytogenetics Studies BioReliance Corp. Rockville, Maryland

John W. Harbell, Ph.D. Vice President and Chief Scientific Officer Institute for In Vitro Sciences, Inc. Gaithersburg, Maryland

Robert V. House, Ph.D. Staff Scientist Covance Laboratories, Inc. Madison, Wisconsin

Jane E. Harris, Ph.D. Director of Toxicology BASF Corp. Princeton, New Jersey

Patricia B. Hoyer, Ph.D. Professor Department of Physiology The University of Arizona Tucson, Arizona

Steven J. Hermansky, Pharm.D., Ph.D., D.A.B.T. Associate Director Product Safety and Toxicology Schering-Plough HealthCare Products Memphis, Tennessee Kimiko Hirayama, Ph.D. Professor Kumamoto University College of Medical Science Kumamoto, Japan Richard M. Hoar, Ph.D. Consultant in Developmental Toxicology Williamstown, Massachusetts David J. Hoffman, Ph.D. Ecotoxicologist Patuxent Wildlife Research Center U.S. Geological Survey Laurel, Maryland and Adjunct Professor University of Maryland Gary M. Hoffman, B.A., D.A.B.T. Study Director and Director of Inhalation Toxicology Huntingdon Life Sciences Princeton Research Center East Millstone, New Jersey Mannfred A. Hollinger, Ph.D. Professor Department of Medical Pharmacology and Toxicology School of Medicine University of California Davis, California Copyright © 2002 by Taylor & Francis

David Jacobson-Kram, Ph.D., D.A.B.T. Vice President Toxicology and Laboratory Animal Health BioReliance Corp. Rockville, Maryland Daniel R. Lavoie, M.S. Aquatic Toxicologist Institute for Environmental Quality Wright State University Dayton, Ohio Barry S. Levine, D.Sc., D.A.B.T. Director Toxicology Research Laboratory Associate Professor of Pharmacology Department of Pharmacology University of Illinois Chicago, Illinois Karen M. MacKenzie, Ph.D., D.A.B.T. Consultant Anderson, South Carolina Rosemary C. Mandella, Ph.D., D.A.B.T. Associate Director of Toxicology Huntingdon Life Sciences Princeton Research Center East Millstone, New Jersey Dennis J. Naas, B.S. President, Owner AccuTox Consulting Services, Ltd. Midland, Michigan Rajesh K. Naz, Ph.D. Director Division of Research Department of Obstetrics and Gynecology Medical College of Ohio Toledo, Ohio

Paul E. Newton, Ph.D., D.A.B.T. Senior Study Director and Director of Inhalation Toxicology MPI Research, Inc. Mattawan, Michigan

Joseph C. Siglin, Ph.D., D.A.B.T. Director of Research Springborn Laboratories, Inc. Spencerville, Ohio

John C. Peckham, D.V.M., M.S., Ph.D. Veterinary Pathologist Experimental Pathology Laboratories, Inc. Research Triangle Park, North Carolina

Suresh C. Sikka, Ph.D., H.C.L.D. Associate Professor and Urology Research Director Tulane University Health Sciences Center New Orleans, Louisiana

William J. Powers, Jr., Ph.D., D.A.B.T. Vice President Global Preclinical Development Johnson & Johnson Raritan, New Jersey Barnett A. Rattner, Ph.D. Ecotoxicologist Patuxent Wildlife Research Center U.S. Geological Survey Laurel, Maryland and Adjunct Professor University of Maryland Dawn D. Rodabaugh, B.S. Assistant Toxicologist–Study Director Springborn Laboratories, Inc. Spencerville, Ohio

Peter T. Thomas, Ph.D. Director Toxicology Covance Laboratories, Inc. Madison, Wisconsin Valentine O. Wagner III, M.S. Study Director Bacterial Mutagenesis Studies BioReliance Corp. Rockville, Maryland Christopher W. Wilson, B.S. Assistant Toxicologist–Study Director Springborn Laboratories, Inc. Spencerville, Ohio

Richard H. C. San, Ph.D. Scientific Director Genetic Toxicology BioReliance Corp. Rockville, Maryland

Akira Yasutake, Ph.D. Chief in Biochemistry Section National Institute for Minamata Disease Kumamoto, Japan

Gene E. Schulze, Ph.D., D.A.B.T. Associate Director Department of Toxicology Bristol-Myers Squibb Pharmaceutical Research Institute Syracuse, New York

Robert R. Young, M.S. Director Toxicology Operations BioReliance Corp. Rockville, Maryland


Acknowledgments The editors and contributors thank the following individuals who helped with the preparation of the CRC Handbook of Toxicology, Second Edition by providing information, advice, constructive criticism, technical expertise, or secretarial skills: Dr. Bjorn Thorsrud, Dr. David Serota, Dr. David Dolan, Dr. George Rusch, Dr. Leigh Ann Naas, Dr. Hans Certa, Dr. George Dearlove, Dr. Gregory Kearns, Dr. Jill Merrill, Gary Roy, Donald Surprenant, Rita Levy, Renee Bolduc, Bob Nellis, Christy Calhoun, Michelle Delaurier, Ronald Brzozowski, Elise Larsen, Daniel Murray, Greg Mun, and Regina Carbon Tihan. As the second edition of the handbook contains a significant amount of information from the first edition, the editors again acknowledge the following individuals for their help in the publication of the earlier handbook: Dr. David Serota, Dr. Kurt Weingand, Dr. Walter Loeb, Dr. George Rusch, Dr. Rosemary Mandella, Dr. C. Anita Bigger, Dr. Donald Putman, Renee Brown, Rita Levy, Cynthia Nofziger, Cathy Beck, Cindy Moore, Doris Bridgeman, Isabelle Baker, Antoinette Keesey, David Keesey, Robin Larkin, Donna Blaszcak, Diane Blansett, Ellen Whiting, Sharon Harris, Farrell Merriman, Gary Roy, Joseph Townsend, Pam Errico, Elizabeth Regan, Kristin Ballard, Kim Cayz, Michael Mercieca, Jane Clark, Hans Raabe, Betsy Schadly, Skip Wagner, Robert Young, Bernette Cockrell, Christine Holzer, Trina Rode, Carol Winiarski, Dolores Yili, Georgene Rutledge, Rusty Rush, Deborah Douds, Todd Merriman, and Gregory Kowalski. The editors extend a special thanks to the authors and publishers who graciously allowed the reprinting of many of the tables and figures in the handbook.


Helpful Tips for Using This Handbook The CRC Handbook of Toxicology has been designed to allow the working toxicologist to locate basic toxicological information quickly. Where possible, text has been kept to a minimum with most of the information provided in tables and figures. The information is organized into chapters dealing with various areas of toxicology. Each chapter begins with a detailed listing of all of the major topics, tables, and figures it contains. Headings are provided on the top of each page identifying the chapter topic to allow quick location of the subject matter. Because of the large and varied amount of information in the handbook, the user seeking a specific type of information may not always find reference to it in the index. It is recommended that a user seeking, for example, information on reproductive indices used in multigeneration studies use the page headings to locate the chapter on reproductive toxicology, turn to the first page of the chapter, and scan the contents listing. The user will quickly find that Table 11.13 provides the desired information. Similarly, cage requirements for rats can be quickly found in Table 1.16 of Chapter 1 by locating this chapter on laboratory animal management and scanning its contents listing. The user is cautioned that some information contained in the handbook may change over time, particularly as relates to regulatory requirements and guidelines, addresses, and phone numbers.


Table of Contents Chapter 1 Laboratory Animal Management Joseph C. Siglin and William H. Baker Chapter 2 Acute, Subchronic, and Chronic Toxicology Carol S. Auletta Chapter 3 Dermal Irritation and Sensitization Kimberly L. Bonnette, Dawn D. Rodabaugh, and Christopher W. Wilson Chapter 4 Ocular Toxicology Brendan J. Dunn Chapter 5 Fundamental Inhalation Toxicology Paul E. Newton Chapter 6 Applied Inhalation Toxicology Gary M. Hoffman Chapter 7 Fundamental Neurotoxicology Gene E. Schulze Chapter 8 Applied Neurotoxicology Rosemary C. Mandella Chapter 9 Immunotoxicology: Fundamentals of Preclinical Assessment Robert V. House and Peter T. Thomas Chapter 10 Renal Toxicology: Renal Function Parameters for Adult Fischer-344, Sprague-Dawley, and Wistar Rats William J. Powers, Jr. Chapter 11 Reproductive Toxicology Donald J. Ecobichon Copyright © 2002 by Taylor & Francis

Chapter 12 Developmental Toxicology Karen M. MacKenzie and Richard M. Hoar Chapter 13 Endocrine Toxicology: Male Reproduction Suresh C. Sikka and Rajesh K. Naz Chapter 14 Endocrine Toxicology: The Female Reproductive System Patricia B. Hoyer and Patrick J. Devine Chapter 15 Genetic Toxicology Richard H. C. San, Ramadevi Gudi, Valentine O. Wagner III, Robert R. Young, and David Jacobson-Kram Chapter 16 Carcinogenesis Michael J. Derelanko Chapter 17 Animal Histopathology John C. Peckham Chapter 18 Animal Clinical Pathology Barry S. Levine Chapter 19 Metabolism and Toxicokinetics of Xenobiotics Mohamed B. Abou-Donia, Eman M. Elmasry, and Aqel W. Abu-Qare Chapter 20 In Vitro Methods for the Prediction of Ocular and Dermal Toxicity John W. Harbell and Rodger D. Curren Chapter 21 Ecotoxicology David J. Hoffman, Barnett A. Rattner, G. Allen Burton, Jr., and Daniel R. Lavoie Chapter 22 Metal Toxicology Akira Yasutake and Kimiko Hirayama Chapter 23 Human Clinical Toxicology Jill Dolgin


Chapter 24 Risk Assessment Michael J. Derelanko Chapter 25 Regulatory Toxicology in the United States: An Overview Michael J. Derelanko Chapter 26 Regulatory Toxicology: U.S. EPA/Chemicals (TSCA) Henry C. Fogle Chapter 27 Regulatory Toxicology: U.S. EPA/Pesticides (FIFRA) Jane E. Harris Chapter 28 Regulatory Toxicology: U.S. FDA/Pharmaceuticals William J. Powers, Jr. Chapter 29 Regulatory Toxicology: Medical Devices Steven J. Hermansky Chapter 30 Regulatory Toxicology: Consumer Products Dennis J. Naas Chapter 31 Regulatory Toxicology: Notification of New Substances in the European Union Michael J. Derelanko Chapter 32 Regulatory Toxicology: Notification of New Substances in Canada, Korea, Australia, and China Henry C. Fogle Chapter 33 Miscellaneous Information of Toxicological Significance Mannfred A. Hollinger and Michael J. Derelanko


Dedication The second edition of the Handbook of Toxicology is dedicated to toxicologists past, present, and future

Dedication of the first edition For MJD To my wife, Patricia, and my sons, Michael and Robert, for their patience and understanding; to my parents, Anne and Frank, for their encouragement and support; to my mentors, Dr. Joseph LoBue, and the late Drs. Albert Gordon and Robert Kelly, for the example they set. For MAH To Georgia Lee Hollinger, Randolph Alan Hollinger, and Christopher Hastings Hollinger, for being special contributors in their own way.


1

Laboratory Animal Management Joseph C. Siglin, Ph.D., D.A.B.T. and William H. Baker, D.V.M., D.A.C.V.P.

CONTENTS Section 1. Introduction Section 2. Animal Husbandry Section 3. Regulations and Guidelines A. Animal Welfare Act B. Public Health Service Regulations C. Guide for the Care and Use of Laboratory Animals Section 4. Institutional Programs A. AAALAC B. IACUC Section 5. Professional and Governmental Organizations A. AALAS B. ACLAM C. ASLAP D. AVMA E. ICLAS F. ILAR G. SCAW H. NIH I. DEA J. FDA K. OLAW L. APHIS M. AWI N. AWIC O. CAAT P. NABR Section 6. Organizations That Oppose the Use of Animals in Research Section 7. Animal Pain Section 8. Animal Models and Alternatives Section 9. Animal Facility Safety Section 10. Zoonotic Diseases A. Hepatitis B. Herpesvirus B C. Rabies D. Lymphocytic Choriomeningitis E. Other Zoonoses Table 1.1 Other Zoonotic Diseases Section 11. Recognition and Control of Disease


Table 1.2 Abnormal Conditions in Laboratory Animals Section 12. Animal Nutrition Table 1.3 Types and Sources of Commercial Laboratory Diets Table 1.4 Nutritional Deficiencies of Laboratory Animals A. Food and Water Requirements Table 1.5 Approximate Daily Food and Water Requirements for Various Species B. Fasting Section 13. Anesthesia and Analgesia A. General Considerations B. Controlled Substances C. Relevant Definitions D. General Principles Regarding Anesthesia, Analgesia, and Tranquilization E. Stages of Anesthesia F. Methods of Administration G. Commonly Used Anesthetic, Analgesic, and Tranquilizing Agents Table 1.6 Typical Routes and Dosages of Several Sedative, Analgesic, and Anesthetic Agents H. Species Peculiarities and Contraindications Section 14. Euthanasia A. Modes of Action B. Euthanasia Methods and Agents Table 1.7 Acceptable and “Conditionally Acceptable” Methods for Euthanasia of Several Common Laboratory Species Table 1.8 Summary of the Characteristics of Several Euthanasia Methods Section 15. Sources of Laboratory Animals Table 1.9 Names, Addresses, and Phone Numbers of Several Animal Suppliers Section 16. Species Data A. Mouse (Mus musculus) Table 1.10 Common Strains of Laboratory Mice Table 1.11 Minimum Cage Space Requirements for Mice Table 1.12 Physical and Physiological Parameters of Mice Table 1.13 Identification, Bleeding, Anesthesia, and Euthanasia Methods for Laboratory Mice Table 1.14 Various Diseases and Adverse Health Conditions of Laboratory Mice B. Rat (Ratus norvegicus) Table 1.15 Common Strains of Laboratory Rats Table 1.16 Minimum Cage Space Requirements for Rats Table 1.17 Physical and Physiological Parameters of Rats Table 1.18 Identification, Bleeding, Anesthesia, and Euthanasia Methods for Laboratory Rats Table 1.19 Various Diseases and Adverse Health Conditions of Laboratory Rats C. Guinea Pig (Cavia porcellus) Table 1.20 Minimum Cage Space Requirements for Guinea Pigs Table 1.21 Physical and Physiological Parameters of Guinea Pigs


Table 1.22 Identification, Bleeding, Anesthesia, and Euthanasia Methods for Laboratory Guinea Pigs Table 1.23 Various Diseases and Adverse Health Conditions of Guinea Pigs D. Rabbit (Oryctolagus cuniculus) Table 1.24 Minimum Cage Space Requirements for Rabbits Table 1.25 Physical and Physiological Parameters of Rabbits Table 1.26 Identification, Bleeding, Anesthesia, and Euthanasia Methods for Laboratory Rabbits Table 1.27 Various Diseases and Adverse Health Conditions of Rabbits E. Dog (Canis familaris) Table 1.28 Minimum Cage Space Requirements for Dogs Table 1.29 Physical and Physiological Parameters of Dogs Table 1.30 Identification, Bleeding, Anesthesia, and Euthanasia Methods for Laboratory Dogs Table 1.31 Various Diseases and Adverse Health Conditions of Dogs References Additional Related Information Table 1.32 Guiding Principles in the Use of Animals in Toxicology Table 1.33 General Information Sources for the Care and Use of Research Animals Table 1.34 Commonly Used Anesthetics Table 1.35 Advantages and Disadvatanges of Anesthetic Agents and Adjuncts

SECTION 1. INTRODUCTION The use of live animals continues to be an important and necessary component of research activities worldwide. To ensure the ethical and humane treatment of animals, scientists must possess a sound understanding of appropriate animal husbandry practices and must be knowledgeable of those variables which may impact and potentially confound experimental procedures and results. The purpose of this chapter is to provide the scientist with a reference source covering many of the fundamental aspects of proper laboratory animal management for species commonly utilized in toxicological research. In keeping with the desired format of this book, the information provided herein is presented in concise fashion to allow a broad coverage of animal husbandry topics and related information. For more detailed information, the reader is referred to the reference materials identified in individual sections, and at the end of this chapter.

SECTION 2. ANIMAL HUSBANDRY Animal husbandry may be simply defined as the methods used in the care and maintenance of animals. In a larger sense, however, animal husbandry encompasses all aspects of appropriate care, treatment, and management for a given species, including circadian rhythm, life span, environmental limits, breeding and reproductive patterns, nutritional and social requirements, and macro- and microenvironmental necessities. Of course, each species has its own unique peculiarities that are essential to its well-being. The scientist must be knowledgeable of these characteristics and of the various regulatory guidelines and policies that govern the use of animals in research.


SECTION 3. REGULATIONS AND GUIDELINES Over the years, the United States and other countries have developed various federal mandates and statutes designed to protect animals from illicit commerce and use. The first such federal statute in the United States was the Pet Protection Act of 1966. This act became the forerunner of what is now called the Animal Welfare Act.

A. ANIMAL WELFARE ACT The Animal Welfare Act (AWA)1 refers to the Act of August 24, 1966 (P.L. 89-544), as amended by the Acts of December 24, 1970 (P.L. 91-579), April 22, 1976 (P.L. 94-279), December 23, 1985 (P.L. 99-198), and 1990 (P.L. 101-624). The various provisions of the AWA are designed to ensure that animals used in research, for exhibition, or as pets receive humane care and treatment. The AWA also regulates the transport, purchase, sale, housing, care, treatment, and handling of such animals. The standards set forth by the AWA are considered absolute minimal standards to which people who handle animals must adhere. According to the AWA, “animal” is defined as “any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, or any other warmblooded animal, which is being used or is intended for use for research, teaching, testing, experimentation, exhibition, or as a pet.” The term dog means “all dogs including those used for hunting, security, or breeding purposes.” Regulatory authority under the AWA is implemented by the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture. Rules and regulations pertaining to implementation of the law are provided in the Code of Federal Regulations (CFR), Title 9 (Animals and Animal Products), Subchapter A (Animal Welfare), Parts 1, 2, and 3. Copies of the regulations may be obtained on line searching for “Animal Welfare Act” or at www.nal.usda.gov/awia/legislat/ usdaleg.htm. The relevant regulations and standards covered by the AWA are summarized below. Subjects Addressed by the AWA • Part 1: Definition of Terms • Part 2: Regulations Subpart A: Licensing Subpart B: Registration Subpart C: Research Facilities Subpart D: Attending Veterinarian and Adequate Veterinary Care Subpart E: Identification of Animals Subpart F: Stolen Animals Subpart G: Records Subpart H: Compliance with Standards and Holding Period Subpart I: Miscellaneous • Part 3: Standards Subparts A–F: Specifications for the Humane Handling, Care, Treatment and Transportation of Dogs and Cats, Guinea Pigs and Hamsters, Rabbits, Non-human Primates, Marine Mammals, and Other Warm-blooded Mammals According to the AWA, “each dealer, exhibitor, operator of an auction sale, and intermediate handler must comply in all respects with the regulations set forth in Part 2 and the standards set forth in Part 3 for the humane handling, care, treatment, housing, and transportation of animals.”

B. PUBLIC HEALTH SERVICE REGULATIONS In 1973, a new policy applying to all Public Health Service (PHS) awardee institutions was drafted. The policy required that institutions conducting PHS-supported research comply with the AWA Copyright © 2002 by Taylor & Francis

and the Guide for the Care and Use of Laboratory Animals.2 Each institution is also required to provide the National Institutes of Health (NIH) with an assurance which gives a detailed plan for research, training, testing, education, experimentation, or demonstration purposes. In essence, the policy requires that institutions take responsibility for the quality of their animal research programs and the conduct of investigators and animal care personnel. In 1985, the Public Health Service Policy on Humane Care and Use of Laboratory Animals by awardee institutions was updated and the final version of the policy was made effective January 1, 1986. Subsequently, Congress enacted and later revised the Health Research Extension Act November 20, 1985 (P.L. 99-158) which added several key provisions to the PHS policy. Although the policy is not law, it has the same effect because an institution must comply in order to compete for funding for animal-related research from PHS and other funding sources. Key elements of the PHS policy include: • Negotiation of Animal Welfare Assurances which include commitments by awardee institutions concerning animal care and use, training of staff, and occupational health programs for employees; • Establishment of an Institutional Animal Care and Use Committee (IACUC) with defined responsibilities; • Detailed requirements for the submission of applications for awards; • Specific record keeping requirements to ensure clear accountability for the quality of the institutional program; and • Specific reporting requirements which enable funding agencies and the NIH Office of Laboratory Animal Welfare (OLAW) to oversee the entire system. Additional information concerning the PHS policy may be obtained from the Office of Laboratory Animal Welfare, National Institutes of Health, 6705 Rockledge, Pr, RKLI, Suite 1050, MSC 7982, Bethesda, MD 20892–7982. Each institution subject to the PHS policy is expected to operate its research program in accordance with the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Research and Training. These principles are listed below. U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Research and Training • The transportation, care, and use of animals should be in accordance with the Animal Welfare Act and other applicable federal laws, guidelines, and policies. • Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge, or the good of society. • The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid results. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered. • Proper use of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals. • Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents. • Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure or, if appropriate, during the procedure. Copyright © 2002 by Taylor & Francis

• The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally, the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated. • Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their in-service training, including the proper and humane care and use of laboratory animals. • Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to the second principle (see above), by an appropriate review group such as an institutional animal care and use committee. Such exceptions should not be made solely for the purposes of teaching or demonstration.

C. GUIDE

FOR THE

CARE

AND

USE

OF

LABORATORY ANIMALS

The Guide for the Care and Use of Laboratory Animals2 was first published in 1963 under the title Guide for Laboratory Animal Facilities and Care. The Guide has been revised several times since 1963 with the latest edition prepared in 1996. The Guide provides information on common laboratory species housed under a variety of circumstances. Although the Guide is not intended to be an exhaustive review of all aspects of animal care and use, it does address a number of relevant issues, including physical construction of animal facilities, husbandry, veterinary care, sanitation, and qualifications and training of laboratory personnel. In the most recent version of the Guide (National Academy of Science ISBN-0-309-05377-3, Revised 1996), emphasis was placed on the establishment of an Animal Care and Use Committee to oversee animal care facilities and insure compliance with applicable federal, state, and local laws and regulations. Copies of the Guide may be obtained from the Institute of Laboratory Animal Resources, National Research Council, 2101 Constitution Ave., NW, Washington, D.C. 20418. www.nas.edu/cls/ilarhome.nsf. E-mail: [email protected]. FAX: 202-334-1687. Various topics covered by the Guide are listed below. Topics Covered by the Guide for the Care and Use of Laboratory Animals2 • Institutional Policies Monitoring the Care and Use of Animals Veterinary Care Personnel Qualifications and Training Personal Hygiene Occupational Health Animal Experimentation Involving Hazardous Agents Special Considerations • Laboratory Animal Husbandry Housing Animal Environment Food Bedding Water Sanitation Identification and Records Emergency, Weekend, and Holiday Care • Veterinary Care Preventative Medicine


Surveillance, Diagnosis, Treatment, and Control of Disease Anesthesia and Analgesia Surgery and Postsurgical Care Euthanasia • Physical Plant Physical Relationship of Animal Facilities to Laboratories Functional Areas Construction Guidelines Aseptic Surgery • Special Considerations Genetics and Nomenclature Facilities and Procedures for Animal Research with Hazardous Agents Farm Animals Appendices A. Selected Bibliography B. Professional and Certifying Laboratory Animal Science Organizations C. Federal Laws Relevant to Animal Care and Use D. Public Health Service Policy and Government Principles on Care and Use of Animals

SECTION 4. INSTITUTIONAL PROGRAMS A. AAALAC The American Association for Accreditation of Laboratory Animal Care (AAALAC) is a nonprofit corporation whose primary goal is to promote high-quality care and use of animals through a voluntary accreditation program. Institutions maintaining, using, importing, or breeding laboratory animals for scientific research are eligible to apply for AAALAC accreditation. The accreditation process involves inspection of the animal facilities and program by experts in laboratory animal science, who submit a comprehensive report for consideration by the Council on Accreditation. The Council reviews the report, using the Guide for the Care and Use of Laboratory Animals as a basis for determining whether full accreditation should be granted. If accreditation is granted, facilities are required to submit annual reports concerning the status of their animal facilities and animal program. Site reinspections are conducted by AAALAC representatives at intervals of 3 years or less to determine whether accreditation should be continued. The specific standards which have been established by the AAALAC Board of Trustees for accreditation are listed below. AAALAC.org. AAALAC Accreditation Standards • Care and management of laboratory animals should be directed by qualified people. • All animal care personnel should be qualified by training and experience in laboratory animal science. • Physical facilities and husbandry methods for the animals should allow their maintenance in wellbeing and comfort. • The NIH Guide is the basic guide to the establishment of specific standards for accreditation. • The accreditable unit shall comply with all statutes and regulations including, but not limited to, the prevailing standards of sanitation, health, labor, and safety of the community and state of location. • Membership in or affiliation with an organization dedicated to laboratory animal care and use is not required for accreditation.


Once a facility becomes fully accreditated, a certificate of accreditation is issued and the facility is identified on a list of accreditated facilities published in the Association’s Activities Report. Full AAALAC accreditation is accepted as partial assurance by NIH that the animal facility and program are in compliance with PHS policy. Further information on AAALAC accreditation may be obtained from AAALAC, 11300 Rockville Pike, Suite 1211, Rockville, MD 20852. Telephone: (301) 2315353. Fax: (301) 231-8282.

B. IACUC The AWA, PHS policy, and Guide for the Care and Use of Laboratory Animals all require the establishment of an Institutional Animal Care and Use Committee (IACUC) that is responsible for monitoring the facility’s animal care and use program. The AWA requires that the IACUC be appointed by the chief executive officer of the research facility and consist of a chairperson and at least two additional members as follows: • A Doctor of Veterinary Medicine, with training or experience in laboratory animal science and medicine, who has direct or delegated program responsibility for activities involving the research facility; and • An individual not affiliated in any way with the facility other than as a member of the committee, and not a member of the immediate family of a person who is affiliated with the facility. This individual should provide representation for general community interests in the proper care and treatment of animals. The Guide for the Care and Use of Laboratory Animals2 specifies an IACUC composition comparable to that required by the AWA, with the addition of “other members as required by institutional needs and by federal, state and local regulations and policies.” The PHS policy is somewhat different in that it specifically requires that the IACUC be composed of at least five members, including a veterinarian with program responsibilities, a scientist experienced in laboratory animal research, a nonscientist, and an individual who has no other association with the institution besides membership in the IACUC (the specific role and background of the fifth member is not specified). The AWA, PHS policy, and Guide for the Care and Use of Laboratory Animals all specify relatively similar functions for the IACUC, which include: • Review, at least once every 6 months, the research facility’s program for humane care and use of animals. • Inspect, at least once every 6 months, all the animal facilities, including animal study areas and satellite facilities. • Prepare and submit reports of IACUC evaluations to the institutional official. • Review and, if warranted, investigate concerns involving the care and use of animals at the facility resulting from public complaints and from reports of noncompliance received from facility personnel or employees. • Make recommendations to the institutional official regarding any aspect of the research facility’s animal program, facilities, or personnel training. • Review and approve, require modifications in (to secure approval), or withhold approval of those components of proposed activities related to the care and use of animals. • Review and approve, require modifications in (to secure approval), or withhold approval of proposed significant changes regarding the care and use of animals in ongoing activities. The IACUC may also suspend any activity involving animals which is deemed unacceptable. However, it is the intent of the guidelines above to avoid such situations through the implementation


of sensible and ethical animal programs, training, and preliminary review of proposed animal activities by the IACUC. Further information concerning IACUC authority and functions may be found in the AWA, 9 CFR, Subchapter A, Part 2, Subpart C, 2.3.1. In addition, an Institutional Animal Care and Use Committee Guidebook, prepared by the NIH Office of Laboratory Animal Welfare (OLAW), may be obtained from: Applied Research Ethics National Association, 132 Boylston Street, Boston, MA 02116, or from the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 ISBN-0-309-05377-3.

SECTION 5. PROFESSIONAL AND GOVERNMENTAL ORGANIZATIONS Information concerning various professional and governmental organizations involved in laboratory animal science, animal welfare, or related activities is provided in the following sections.

A. AALAS The American Association for Laboratory Animal Science (AALAS) is concerned with all aspects of laboratory animal care and use, and provides a means for collection and exchange of information on all phases of animal care and management. The association holds annual meetings and publishes a bimonthly journal, Laboratory Animal Science. The AALAS Animal Technician Certification Board provides for three levels of technical certification: Assistant Laboratory Animal Technician (ALAT), Laboratory Animal Technician (LAT), and Laboratory Animal Technologist (LATG). Additional information may be obtained from AALAS. Telephone: (901) 745-8620. Fax: (901) 753-0046. www.aalas.org

B. ACLAM The American College of Laboratory Animal Medicine (ACLAM) was founded in 1957 to encourage education, training, and research; to establish standards of training and experience for qualification; and to certify, by examination, qualified laboratory animal specialists as diplomates. ACLAM meets biannually in conjunction with the American Veterinary Medical Association (AVMA) and the American Association for Laboratory Animal Science (AALAS). The College emphasizes and sponsors continuing education and autotutorial programs on the use, husbandry, and diseases of animals used in research. Additional information may be obtained through the ACLAM website at www.aclam.org/foundation_contacts.

C. ASLAP The American Society of Laboratory Animal Practitioners (ASLAP) was organized to disseminate ideas, experiences, and knowledge among veterinarians involved in laboratory animal practice through education, training, and research. The Society, which was founded in 1966, is open to any graduate of a veterinary college accredited or recognized by the AVMA. ASLAP holds two educational meetings annually, one in conjunction with the AVMA annual meeting and one in conjunction with the AALAS annual meeting. Additional information may be obtained through the ASLAP Coordinator, 11300 Rockville Pike, Suite 1211, Rockville, MD 20852. Telephone: (301)231-6349. Fax: (231)231-6071. [email protected]

D. AVMA The American Veterinary Medical Association (AVMA) is the major national organization of veterinarians. The primary mission of the AVMA is the advancement of veterinary medical science, Copyright © 2002 by Taylor & Francis

including its relationship to public health and agriculture. The AVMA is the major accrediting agency for colleges of veterinary medicine. The AVMA sponsors specialization in veterinary medicine through the recognition of specialty certifying organizations such as ACLAM. The AVMA Committee on Animal Technician Activities and Training accredits 2-year programs in animal technology throughout the United States. A summary of state laws and regulations relative to veterinarians and animal technicians is available from the AVMA, 1931 North Meacham Road, Suite 100, Schaumburg, IL 60173. Telephone: (847) 925-8070. Fax: (847) 925-1329.

E. ICLAS The International Council for Laboratory Animal Science (ICLAS) is a nongovernmental organization that encourages international cooperation in laboratory animal science. ICLAS promotes the development of international standards for the care and use of laboratory animals, disseminates information concerning laboratory animals, sponsors scholarships for education, and supports programs that advance laboratory animal science in developing nations. ICLAS issues the ICLAS Bulletin every spring and autumn. Additional information may be obtained from www.iclas.org.

F. ILAR The Institute of Laboratory Animal Resources (ILAR) was founded in 1952 under the auspices of the National Research Council. ILAR’s mission is to provide expert counsel to the federal government, the biomedical research community, and the public on the scientific, technological, and ethical use of laboratory animals within the context of the interests and mission of the National Academy of Sciences. ILAR promotes the high-quality humane care of laboratory animals; the appropriate use of laboratory animals; and the exploration of alternatives in research, testing, and teaching. The most recent edition of the Guide for the Care and Use of Laboratory Animals2 was prepared by ILAR for the National Institutes of Health. For more information, contact ILAR, National Academy of Sciences, 2101 Constitution Ave., NW, Washington, D.C. 20418. Telephone: (202) 334-2590. Fax: (202) 334-1687. [email protected].

G. SCAW The Scientists’ Center for Animal Welfare (SCAW) was founded in 1979 and consists of individuals and institutions concerned with various aspects of animal welfare. SCAW promotes the principle of humane animal care and treatment in all areas of animal science. Among other activities, SCAW develops educational materials and national guidelines on humane animal experimentation; monitors animal legislation issues; and conducts workshops and surveys. For more information, contact SCAW, 7833 Walker Drive, Suite 340, Greenbelt, MD 20770. Telephone: (301) 345-3500. www.scaw.com

H. NIH The National Institutes of Health (NIH) is a federal agency that disburses funds for biomedical research and sets policy on laboratory animal welfare (PHS policy). Additional information may be obtained from: Office of Animal Care and Use, 9000 Rockville Pike, Bethesda, MD 20892. Telephone: (301) 496-5424.

I.

DEA

The Drug Enforcement Administration (DEA) of the U.S. Department of Justice is the regulatory authority responsible for the enforcement of laws pertaining to controlled substances. Licenses to


use controlled substances are obtained from this agency. Additional information may be obtained from: DEA, Registration Unit-ODRR, Washington, D.C. 20537. Telephone: (202) 307-7255.

J. FDA The U.S. Food and Drug Administration (FDA) is the federal agency responsible for enforcing the FDA Good Laboratory Practice (GLP) regulations. Additional information may be obtained from: United States FDA, Parklawn Building, 5600 Fishers Lane, Rockville, MD 20857. Telephone: (301) 443-5006. 1-888-463-6332.

K. OLAW The Office of Laboratory Animal Welfare (OLAW) of the NIH oversees compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Additional information may be obtained from: OLAW, 9000 Rockville Pike, Building 31, Room 5B63, Bethesda, MD, 20892. Telephone: (301) 496-7005.

L. APHIS The Animal, Plant and Health Inspection Service (APHIS) is the division of the U.S. Department of Agriculture that administers the federal Animal Welfare Act. Additional information may be obtained from: USDA, APHIS, 2568-A RIVA RD., ANNAPOLIS, MD 21401. Telephone: (410) 571-8692. [email protected]

M. AWI The Animal Welfare Institute (AWI) is a national organization active in laboratory animal welfare issues. The AWI encourages lay persons to serve on IACUCs and has a number of publications pertinent to laboratory animal welfare. Additional information may be obtained from: AWI, P.O. Box 3650, Washington, D.C. 20007. Telephone: (202) 337-2332. FAX: (202) 338-9478. [email protected]

N. AWIC The Animal Welfare Information Center (AWIC) is an information center of the National Agricultural Library established as result of the 1985 amendment to the Animal Welfare Act. Additional information may be obtained from: National Agricultural Library, Room 301, Beltsville, MD 20705. Telephone: (301) 504-6212.

O. CAAT The Center for Alternatives to Animal Testing (CAAT) was established in 1981 to encourage and support the development of nonanimal testing methods. The center supports grants, sponsors symposia, and publishes a variety of materials related to animal testing and alternatives. Additional information may be obtained from: Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21202. Telephone: (410) 223-1612.

P. NABR The National Association for Biomedical Research (NABR) is a nonprofit organization which was established in 1979 and merged with the National Society for Medical Research in 1985. Membership in NABR includes numerous institutions, universities, medical and veterinary schools; health agencies; academic and professional societies; and private and public research organizations.


NABR monitors legislation that could potentially impact the use of animals in research. Members of NABR may obtain copies of bills, summaries of bills, listings of current legislation, and related materials via the NABR computerized database. Additionally, a compilation of State Laws Concerning the Use of Animals in Research may be obtained through NABR. NABR supports the responsible and ethical use of laboratory animals in research, education, and product safety testing. NABR recognizes that it may not be feasible to completely replace live animals in research because whole living organisms are an indispensable element of biomedical research and testing. Still, the Association believes that animal use should be minimized whenever possible; that pain and distress should be avoided and/or minimized; and that alternatives to live animals should continue to be developed and utilized, whenever feasible. Additional information may be obtained from: Ms. Frankie Trull, President, 818 Connecticut Ave., NW, Suite 200, Washington, D.C. 20006. Telephone: (202) 857-0540.

SECTION 6. ORGANIZATIONS THAT OPPOSE THE USE OF ANIMALS IN RESEARCH A number of organizations strongly oppose the use of animals in research, or exhibit varying philosophies regarding this subject. There are, however, some deep divisions in the philosophies and strategies of these organizations. For example, animal welfare groups such as the humane societies tend to be most concerned with the proper care and treatment of animals, pet adoption, and humane euthanasia. On the other hand, “animal rights” groups are primarily concerned with establishing the “legal rights” of animals. These latter groups outwardly oppose the use of animals in research and the “exploitation” of animals for sport or food. The activities of these groups have challenged the research community to better inform and educate the public about the critical need for animals in research. Additional standing goals for scientists include: • Reduction of the number of animals used through thoughtful selection of techniques and models. • Relief of any unavoidable discomfort to animals. • Improvement of animal facilities and assurance that personnel are fully informed and properly trained. • Elimination or reduction of experimental procedures that cause pain or distress. • Utilization of nonanimal alternatives whenever and wherever possible.

SECTION 7. ANIMAL PAIN In accordance with the AWA, Guide for the Care and Use of Laboratory Animals, and Public Health Service Policy for the Humane Care and Use of Laboratory Animals, veterinarians and investigators must identify and eliminate sources of pain and distress, with the exception of those procedures that are essential to the research in question and approved by the IACUC. Although it is widely agreed that laboratory animals need not experience substantial pain or distress, there is a general lack of agreement on the specific meaning of such terms as comfort, wellbeing, discomfort, stress, fear, anxiety, pain, and distress. Nonetheless, provisional definitions for these terms have been developed3 and are presented below. • Comfort: A state of physiological, psychological, and behavioral equilibrium in which an animal is accustomed to its environment and engages in normal activities, such as feeding, drinking, grooming, social interaction, sleeping-waking cycles, and reproduction. • Wellbeing: A positive mental state that reflects the level of welfare and comfort of an animal. Copyright © 2002 by Taylor & Francis

• Discomfort: A minimal change in an animal’s adaptive level or baseline state as a result of changes in its environment or biological, physical, social, or psychotic alterations. Physiological or behavioral changes that indicate a state of stress might be observed, but are not marked enough to indicate distress. • Stress: The effect produced by external (physical or environmental) events or internal (physiological or psychological) factors, referred to as stressors, which induce an alteration in an animal’s biological equilibrium. • Anxiety and fear: Emotional states that are traditionally associated with stress. They can be adaptive in that they inhibit an organism’s actions that could lead to harm or cause it to act in ways allowing it to escape from potentially harmful situations. • Pain: Results from potential or actual tissue damage. Pain can be considered a potent source of stress, that is, a stressor. It can also be considered a state of stress itself, however, and can lead to distress and maladaptive behaviors. • Distress: An adverse state in which an animal is unable to adapt completely to stressors and the resulting stress and shows maladaptive behaviors. It can be evident in the presence of various experimental or environmental phenomena, such as abnormal feeding, absence or diminution of postprandial grooming, inappropriate social interaction with conspecifics or handlers, and inefficient reproduction. With regard to the issues of animal discomfort and pain, researchers should consider the following questions before undertaking any live animal experiment: • Will the procedure yield results that are beneficial to animal or human health and wellbeing? • Has a literature search been performed to ensure that the proposed procedures do not unnecessarily duplicate previous experiments? • Is the species and number of animals appropriate for the purpose of the experiment? • Is the discomfort to the animals limited to that which is unavoidable in the conduct of the experiment? • Have appropriate analgesic, anesthetic, and tranquilizing drugs been considered to minimize pain and discomfort? • Has the method of euthanasia been considered? • Are the individuals performing the experimental procedures and caring for the animals properly trained? In general, procedures that cause minimal pain or discomfort to humans and place the animal in minimal distress are considered acceptable.

SECTION 8. ANIMAL MODELS AND ALTERNATIVES Animal models may be broadly classified as experimental, negative, or spontaneous. An experimental model is one in which an experimentally induced condition mimics a human disease. A negative model, on the other hand, is one in which a particular condition cannot be produced, and is therefore studied to better understand the reason for the protective or resistant effect(s). A spontaneous model is one in which the animal naturally develops a disease or some other condition of interest. In considering a particular animal model for an experiment, the investigator must first ensure that there are no acceptable nonanimal alternatives for the planned research. Once this has been clearly established and documented (e.g., by detailed literature search and review), special consideration must be given toward species availability, husbandry and technical expertise, space and caging requirements, special environmental requirements, genetic characteristics, nutritional Copyright © 2002 by Taylor & Francis

requirements, microbial etiology and life span of the animal, and reproductive, anatomic, physiological, and behavioral characteristics of the species. An alternative model is defined as any technique that reduces or eliminates the need for live animals and thereby prevents potential pain and distress in animals. Such alternative models include computer and mathematical simulations, microbiological systems, tissue/organ culture, epidemiological surveys, and plant analysis. A major drawback common to many of these alternative models is the lack of complex physiological interactions that occur in the whole animal. Nonetheless, the potential for reduction and replacement of live animals provides strong incentive for the continued development and validation of alternative models.

SECTION 9. ANIMAL FACILITY SAFETY The Occupational Safety and Health Act (OSHA), which is administered by the U.S. Department of Labor, is not specifically directed at laboratories and research operations. However, the regulations apply to all work places and cover fire, electrical, and mechanical safety, and exposure to chemicals, radiation, and noise. In general, research laboratories have not been subjected to the frequent and rigorous OSHA inspections which are common to industries with intrinsically high accident rates. However, there now exist specific OSHA regulations concerning occupational exposures to toxic substances in research laboratories. These standards require that the laboratory develop a “Chemical Hygiene Program” designed to provide employee protection in the specific circumstances of the individual laboratory. In addition, there are requirements for training of employees, worker availability to reference materials concerning chemical hazards, and a provision for medical consultation and examination.

SECTION 10. ZOONOTIC DISEASES4,10 Zoonotic diseases are those which are transmissible from animals to humans under natural conditions. A few of the better known zoonotic diseases are described in the following sections.

A. HEPATITIS Hepatitis A virus can infect chimpanzees, gorillas, patas monkeys, celebres, apes, woolly monkeys, and some tamarins. However, chimps recently introduced into captivity are the most common source of infection for humans. The incubation period for the virus may be 15–50 days, followed by abrupt onset of fever, anorexia, nausea, and jaundice. The severity of the disease is related to age, with fatality quite low among hospitalized patients. Lifelong immunity is conferred by development of an IgG immune response. Disease control measures involve quarantine, adequate protective clothing, sanitation, and personal hygiene. As an additional measure, the PHS recommends immunoprophylaxis (i.e., administration of immune serum globulin every 4 months) for personnel in close contact with newly imported chimps.

B. HERPESVIRUS B Herpesvirus B, which is caused by Herpesvirus simiae, represents the most serious health hazard to humans from nonhuman primates. In the natural host of the virus, the Macaca spp, the disease is mild and similar to that of herpes simplex in humans, with the development of tongue and lip ulcers which heal in 7–14 days. In contrast, in infected humans, disease symptoms may be similar to polio, with rapid flaccid paralysis leading to death, or permanent paralysis in survivors. Transmission usually occurs through a bite from an infected animal, or by exposure of the broken skin or mucus membranes to infected saliva or infected tissues. Because of the potential danger to humans, all macaques should be viewed as potential carriers, and protective clothing should be


worn at all times which protects the handler from bites and scratches. Although antiserum is available, its effectiveness is questionable. Note: Herpes simplex in man can be transmitted to lower primates with generalized disease in owl monkeys, tree shrews, lemurs, marmosets, and tamaris.

C. RABIES In the United States, the skunk and bat are the largest natural reservoirs of the rabies virus. The virus is transmitted through the saliva of infected animals via bites, scratches, abrasions, or across mucus membranes. In dogs, the virus is present in the saliva for 1–14 days before clinical symptoms manifest. In humans, the disease is almost always fatal, even when proper treatment is begun shortly after exposure. The most important disease control measure for domestic animals is vaccination. For humans, preexposure vaccination should be made available for all persons working with potentially infected animals.

D. LYMPHOCYTIC CHORIOMENINGITIS Mice, hamsters, and humans serve as natural hosts for the virus causing lymphocytic choriomeningitis, and wild mice are a natural reservoir for the virus, which is the only latent virus in mice that naturally infects humans. The incidence of the disease may be 100% in wild populations, and may become 100% in breeding colonies if preventive measures are not instituted. However, only persistently infected mice and acutely infected hamsters are known to transmit the virus, which may be passed in the urine, feces, saliva, and nasal secretions of carrier animals. Lifelong infection with high concentrations in all organs is often observed in fetal and newborn infected mice. Hamsters, on the other hand, may remain infected for long periods, but eventually eliminate the virus. There are four different recognized forms of the disease in mice. In the cerebral form, death may occur with no previous symptoms on the fifth or sixth day after inoculation. In the visceral form, death may also occur after several days, but is often preceded by conjunctivitis and ruffled fur. In the late-onset form which occurs in neonatally infected mice, animals may seem healthy until 9–12 months of age when signs of chronic illness manifest, including ruffled fur, weight loss, and hunched posture. A form resulting in early death of neonatally exposed mice may also occur under poorly understood conditions. Infection of humans often results in mild influenza-like symptoms, and may or may not involve the central nervous system. Other than direct virus isolation, a rise in antibody titer serves as the most conclusive diagnosis of infection.

E. OTHER ZOONOSES Other zoonoses are described briefly in Table 1.1.

TABLE 1.1 Other Zoonotic Diseases Disease Monkey pox Benign epidermal monkey pox (BEMP) Yaba virus

Description Related to smallpox; clinical signs in humans include fever, headache, sore throat, and rash Primarily affects macaques and Leaf monkeys; circumscribed elevated lesions on eyelids, face, and elsewhere; in humans, disease regresses in 2–3 weeks Caused by a poxvirus transmitted via a mosquito vector; virus has been inoculated into humans, but natural transmission has not been recorded; infected animals develop benign histiocytomas that eventually regress


TABLE 1.1 (Continued) Other Zoonotic Diseases Disease Contagious ecthyma (ORF)

Yellow fever Hentaviral Diseases (Korean hemorrhagic fever, epidemic hemorrhagic fever, Hentavirus pulmonary syndrome, nephropathia epidemica) Measles

Rickettsialpox

Murine typhus Rocky Mountain Spotted Fever

Q fever

Psittacosis

Brucellosis

Leptospirosis

Tuberculosis

Description Caused by poxvirus of sheep and goats; characterized by epithelial proliferation and necrosis in the skin and mucus membranes of urogenital and gastrointestinal tracts; in humans, seen as painful nodules on hands which resolve in 1–2 mo Caused by an RNA flavivirus transmitted by mosquitos; classic lesion is massive hepatic midzonal necrosis; disease severity varies among species of nonhuman primates Caused by Hantaan virus carried by wild rodents worldwide, disease involves fever and renal and/or pulmonary involvement with headaches, diarrhea, nausea, vomiting, and possible hemorrhagic symptoms

Caused by a morbillivirus of the family Paramyxoviridae. Highly contagious disease with incubation time of 9–11 days; rash begins in oral cavity and spreads over face, neck, chest, and body; natural immunity develops after capture, but vaccination may be necessary for naive animals. Measles is not a natural disease of macaques, but is acquired through contact with humans. Caused by Rickettsia akarii; domestic mice are natural host, and vector is the mite, Allodermanyssus sanguineus; self-limiting disease in man characterized by fever, headache, myalgia, lymphadenopathy, leukopenia, eschar-like lesions, and generalized rash Caused by Rickettsia typhi: transmission to humans via rat fleas; clinical signs similar to those of rickettsialpox Caused by Rickettsia rickettssii; transmitted by ticks (Dermacentor spp.) as vectors and reservoir hosts; mammalian hosts include wild rodents, lagamorphs, and dogs; disease in humans includes fever, headache, myalgia, and generalized hemorrhagic rash Caused by Coxiella burnetti; disease is widespread in sheep; dogs, cats, and chickens can become infected; organism is shad in urine, feces, milk, and placenta of asymptomatic ungulates; incubation is 2–3 wk and results in febrile systemic disease; most cases resolve in 2 wk Caused by Chlamydia psittaci; hosts include mice, guinea pigs, rabbits, cats, lambs, calves, birds, and frogs; disease includes conjunctivitis, pneumonitis, pericarditis, hepatitis, enteritis, urethritis, and arthritis; in humans, may be asymptomatic or present after 1–2 weeks of incubation, frequently with respiratory symptoms Caused in the laboratory by Brucella canis, due to use of random-source dogs; oral and transcutaneous routes of infection occur in the laboratory; brucellosis should be suspected when dog has history of abortion or infertility; source of infection is not known in most human cases. Other Brucella species may be contracted through the use of other species in the laboratory environment, i.e., goats, sheep, pigs, cattle Leptospira spp. bacteria are found worldwide and divided into serovars based on DNArelatedness; reservoirs are wild and domestic animals including rats, swine, cattle, and dogs; transmission primarily via contact with skin, especially if abraded, or mucous membranes with infected urine-contaminated materials; may be clinically inapparent or present with fever of sudden onset, headache, chills, severe myalgia, conjunctival suffusion or may present with a diphasic fever, meningitis, rash, hemolytic anemia, hemorrhage into skin and mucous membranes, hepatorenal failure, jaundice, mental confusion/depression, myocarditis, and pulmonary involvement. Caused by Mycobacterium acid-fast bacilli; natural reservoirs are cattle, birds, and humans, with many other species susceptible: outbreaks occur in nonhuman primates, with Old World species more susceptible than New World monkeys and great apes; tuberculosis can occur in every organ system, although respiratory system is most familiar form


TABLE 1.1 (Continued) Other Zoonotic Diseases Disease Campylobacteriosis

Salmonella

Shigellosis

Streptobacillus moniliformis Yersinia

Dermatophilosis

Erysipeloid

Listeriosis

Pseudomonas Dermatomycoses (ringworm)

Toxoplasmosis

Amebiasis

Balantidiasis Giardiasis

Description Caused by Campylobacter spp. which has been isolated from dogs, cats, hamsters, ferrets, nonhuman primates, rabbits, swine, cattle, sheep, chickens, turkeys, and wild birds; disease in humans is self-limiting and usually brief; clinical symptoms include abdominal pain, fever, and diarrhea Caused by over 1600 serotypes worldwide; two most common in laboratory colonies are Salmonella typhimunum and Salmonella enteritidis, due primarily to contaminated laboratory feed; acute gastroenteritis is most common presenting symptom; some cases proceed to septicemia after bacterial invasion of gut wall Caused by Shigella spp., including S. flexneri, S. sonnie, and S. dysenteriae, all found in nonhuman primates; humans are main reservoir; nonhuman primates acquire disease after contact with infected primates or through contaminated facilities, food, or water; children may exhibit more severe disease with symptoms of dysentery with blood and mucus in feces Common in wild rodents, rare in laboratory rats, causes rat-bite fever (Haverhill Fever) in man, organism inhabits oropharynx of rat and is transmitted by bite Species which are zoonotic in laboratory animals include Y. pseudotuberculosis, Y. enterocolitica, and Y. pestis; Y. pseudotuberculosis and Y. enterocolitica produce mesenteric lymphadenitis, septicemia, and appendicitis in humans; infection can occur through feces-contaminated food, or through direct contact with infected animals Caused by Dermatophilus congolensis; experimentally transmitted to mice, guinea pigs, and rabbits; produces circumscribed patches of alopecia in infected animals with exudative dermatitis; organism may persist in the fur and infect humans Caused by Erysipelothrix rhusiopathiae in swine, lambs, calves, poultry, fish, and wild and laboratory mice: produces inflammatory lesions of the skin with occasional concurrent septicemia; pigs are the most common source in the laboratory Caused by Listeria monocytogenes; laboratory species most commonly affected are ruminants, guinea pigs, rabbits, and chinchillas; in normal hosts, disease may be expressed as pustular or papular cutaneous lesions or an acute, mild, febrile illness, sometimes with influenza-like symptoms; pregnant woman and fetuses are at risk with the potential for in utero infections and abortion Opportunistic organism, especially for immunosuppressed animals; transmission from the caretakers or animals has been documented, but not the reverse Caused by three genera of fungi: Microsporum, Trichophyton, and Epidermophyton; frequently the animals are asymptomatic and not identified until caretaker develops the disease; transmission occurs by direct or indirect contact with infected animal; dermatomycosis is usually self-limiting in humans and presents as scaling, erythema, and occasional vesicles in the skin Caused by Toxoplasma gondii; felines develop intestinal infection followed by shedding of oocysts resulting in transmission to humans; human infection is common, but clinical symptoms rare; congential infection can lead to systemic disease with neuropathological lesions Caused by Entamoeba histolytica; parasite is commonly found in feces of normal monkeys and apes, but may also cause severe clinical disease; most cases of human disease exhibit no clinical symptoms; mild diarrhea to acute bloody or mucoid dysentery with fever or chills may occur after invasion of colon wall Caused by Balantidium coli: common in domestic swine and also found in humans, great apes, and several monkey species; most infections are asymptomatic Caused by Giardia spp.; found worldwide among all classes of vertebrates with no apparent host specificity; dogs and nonhuman primates may serve as reservoirs for human infection; in humans, infection often causes chronic or intermittent diarrhea, with light-colored, soft, and mucoid stools


TABLE 1.1 (Continued) Other Zoonotic Diseases Disease Pneumocystis pneumonia

Cyptosporidiosis

Description Caused by Pneumocystis carinii; latent infections occur in rodents, lagomorphs, nonhuman primates, and domestic and zoo animals; zoonotic transmission has not been proven but may be possible; disease occurs in immunodeficient individuals or those with other debilities; frequently fatal and characterized by alveolitis as lungs fill with white foamy fluid containing parasites Caused by Cryptospridium parvum; affects epithelial cells of GI, biliary, and respiratory tract of humans, birds, fish, reptiles, rodents, cats, dogs, cattle, and sheep; diarrhea is major symptom in man, remitting in < 30 days in most immunologically competent people; may be fatal in immunodeficient persons

SECTION 11. RECOGNITION AND CONTROL OF DISEASE Adequate veterinary care and daily observation of animals are essential for the recognition and control of disease. Diseases are transmitted by the following routes: • Vector: A living carrier that transfers an infective agent from one host to another. • Fomite: An inanimate object that is not intrinsically harmful, but is able to harbor pathogenic microorganisms. • Genes: Inheritable abnormalities and mutations may result in disease states. There are several procedures that can be instituted to control disease. Some routine procedures are listed below. • Closely observe each animal at the time of receipt, and reject any animal(s) exhibiting abnormal physical, behavioral, or physiological conditions. • Isolate and quarantine each new shipment of animals until their health status can be verified. • Establish procedures that maintain barriers between animals and personnel (e.g., gloves, masks, and protective clothing); between animals and animals (e.g., changing gloves and disinfecting equipment between animals); and between animals and equipment (e.g., disinfect cleaning utensils and sanitize caging). • Establish animal health and monitoring programs matched to the quality and types of animals and needs of the research laboratory. Daily observation of animals allows early detection of signs of disease. While checking the general physical condition of each animal, the caretaker should also look for any signs of injury and/or abnormal physiological findings. Observations of any of the conditions listed in Table 1.2 should be

TABLE 1.2 Abnormal Conditions in Laboratory Animals Abnormal physical conditions

Nonspecific signs of injury Abnormal physiological findings

Dehydrated, emaciated, listless, prostrate, dyspnea, alopecia, circling/head tilt, coughing, sneezing, discharges, scratching, unkempt, abscess/tumor(s), diarrhea, few or no feces, blood in feces, worms in feces, vomitus, bloody vomitus, worms in vomitus Limping, paralysis, ataxia, dilated pupils, convulsions, fractures, hemorrhage, wounds, contusions Lack of urine, excess urination, few or no feces, anorexia, decreased water intake, excessive water intake


followed by diagnosis, prognosis, and treatment, after consultation with the attending veterinarian. If necessary, animals should be euthanized to control disease and alleviate pain and distress.

SECTION 12. ANIMAL NUTRITION All animals require regular amounts of clean pure water and food. Fortunately, there are a variety of “complete balanced diets” available commercially for various laboratory species. These diets have been designed to provide the necessary fats, carbohydrates, proteins, fiber, vitamins, and minerals needed by the particular species. Researchers often select “certified” diets for use in their laboratories because these have been assayed for levels of various potential contaminants (e.g., aflatoxins and heavy metals; chlorinated hydrocarbons and polychlorinated biphenyls; and organophosphate pesticides). Similarly, in many laboratories, the water supplied to the animals is analyzed at regular intervals to ensure potability and absence of contaminants which may negatively impact animal health and research objectives. It is advisable that researchers closely review and retain all reports of food and water analyses. Some of the various types and sources of commercial laboratory diets are listed in Table 1.3. Various nutritional deficiencies which may affect laboratory animals are presented in Table 1.4.

TABLE 1.3 Types and Sources of Commercial Laboratory Diets Source Purina Mills, Inc. 505 N. 4th St. Richmond, IN 47374 (765) 962-9561 www.labdiet.com

Species

Diet Types

Rat/mouse/hamster

5001 Laboratory Rodent Diet; 5002 Certified Rodent Diet; 5008 Formulab Diet; 5010 Laboratory Autoclavable Rodent Diet; 5014 Certified Autoclavable Rodent Diet; 5L36 Certified Rodent OptiDiet; 5P07 Prolab RMH 1000; 5P06 Prolab RMH 2000; 5P14 Prolab RMH 2500; 5R24 Autoclavable Prolab RMH 2500; 5P00 Prolab RMH 3000; 5P04 Autoclavable Prolab RMH 3500; 5053 Pico*Lab Rodent Diet 20; 5061 Pico-Vac Lab Rodent Diet; 5P75 and 5P76 Prolab Isopro RMH 3000 5012 Rat Diet 5015 Mouse Diet; 5020 Mouse Diet 9F; 5021 Autoclavable Mouse Breeder Diet; 5058 PicoLab Rodent Diet 20; 5062 Pico-Vac Mouse Diet 20 5304 Autoclavable Rabbit Diet; 5321 Laboratory Rabbit Diet; 5322 Certified Rabbit Diet; 5325 Certified High Fiber Rabbit Diet; 5326 Laboratory Rabbit Diet HF; 5P25 Prolab Hi-Fiber Rabbit; 5p26 Prolab Rabbit Diet 5025 Guinea Pig Diet; 5026 Certified Guinea Pig Diet; 5L08 Guinea Pig Diet, Autoclavable 20; 5P18 Prolab Guinea Pig 5080 Laboratory Mini-Pig Starter Diet; 5L80 Laboratory Mini-Pig HF Grower Diet; 5081 Laboratory Mini-Pig Grower Diet; 5082 Laboratory Mini-Pig Breeder Diet; 5084 Laboratory Porcine Grower Diet; 5P94 Prolab Mini-Pig Diet 5006 Laboratory Canine Diet; 5007 Certified Laboratory Canine Diet; 5L18 Laboratory High Density Canine Diet; 5P40 Prolab Canine 1600; 5P41 Prolab Canine 2000 5003 Laboratory Feline Diet 5280 Ferret Diet; 5L14 High Density Ferret Diet 5065 Laboratory Chick Diet S-G; 5070 Laboratory Cage Layer Diet 5508 Rumilab Diet

Rat Mouse

Rabbit

Guinea Pig Mini-Pig

Dog

Cat Ferret Avian Ruminant


TABLE 1.3 (Continued) Types and Sources of Commercial Laboratory Diets Source

Species

Primate

Harlan Teklad P.O. Box 44220 Madison, WI 53744-4220 Toll Free: (800) 483-5523 Voice: (608) 277-2070 FAX: (608) 277-2066 www. Harlan.com/teklad/ global/index

Rodents

Rabbits Guinea Pig Primates

Cats

Diet Types 5037 & 5038 Monkey Diet Jumbo and Monkey Diet; 5040 New World Primate Diet; 5045 & 5047 High Protein Monkey Diet and Jumbo; 5048 Certified Primate Diet; 5049 & 5050 Laboratory Fiber-Plus Monkey Diet and Jumbo; 5052 Fiber-Balance Monkey Diet; 5K91 Certified Hi-Fiber Primate; 5P46 Prolab Primate 18 *Pico diets are irradiated 2014 Protein Rodent Maintenance Diet (14%) 2014S Protein Rodent Maintenance Diet (14%) (Sterilizable) 2016 Protein Rodent Diet (16%) 2016S Protein Rodent Diet (16%) (Sterilizable) 2018 Protein Rodent Diet (18%) 2018S Protein Rodent Diet (18%) (Sterilizable) 2030 Rabbit Diet 2031 High Fiber Rabbit Diet 2040 Guinea Pig Diet 2041 High Fiber Guinea Pig Diet 2050 Protein Primate Diet (20%) 2055 Protein Primate Diet (25%) 2021 Protein Dog Diet (21%) 2025 Protein Dog Diet (25%) 2027 Protein Dog Diet (27%) 2060 Cat Diet

The above diets are standard diets. Harlan Teklad also provides services to custom design diets such as the examples listed below: purified mineral deficient vitamin deficient adjusted calories adjusted protein amino acid diet adjusted carbohydrate adjusted fat essential fatty acid atherogenic deficient basal mixes rabbit diets with cholesterol isoflavone reduced basal mixes

TABLE 1.4 Nutritional Deficiencies of Laboratory Animals Nutritional Deficiency

Species Affected

Vitamin A

All species

Vitamin C Vitamin D

Primates and guinea pigs All species

Vitamin E Vitamin K

All species All species (the guinea pig may be an exception)


Symptom(s) Night blindness, dryness and thickness of cornea and conjunctiva, skin lesions Scurvy conditions, breakdown of connective tissues Lameness, enlargement of long bones with softening and deformation of all bones Weak muscles, poor growth, low reproduction Slow blood clotting time

TABLE 1.4 (Continued) Nutritional Deficiencies of Laboratory Animals Nutritional Deficiency

Species Affected

Vitamin B1 Vitamin B2

All species All species

Nicotinic acid

All species

Vitamin B6 Biotin

All species Mice (raw egg whites or sulfur drugs can result in a deficiency for any mammal) All species All species All species All species All species All species All species All species All species All species All species All species Guinea pig

Folic acid Choline Vitamin B12 Calcium Phosphorus Magnesium Sodium Chlorine Potassium Iron Copper Iodine Cobalt

A. FOOD

AND

Symptom(s) Gastrointestinal, nervous, cardiovascular symptoms Skin lesions or mucous membrane lesions, cardiac problems in dogs, collapse, comma Skin, gastrointestinal, nervous symptoms, inflammation of the mouth in dogs Convulsions, nausea, dermatitis, anemia Skin lesions

Anemia, diarrhea in primates Weight loss, reduced reproduction and lactation Anemia Lameness Lameness Low blood pressure, nervous symptoms Reduced growth, eye disturbances, low protein digestion Abnormal fluid and pH balances Reduced appetite and growth Anemia Anemia, hair loss, dermatosis Weak newborns, decreased basal metabolism rate Anemia

WATER REQUIREMENTS

Approximate daily food and water requirements for various species are presented in Table 1.5. Most toxicology studies employ ad libitum feeding conditions in which animals are allowed to regulate their own dietary intake to meet energy requirements. However, the use of ad libitum

TABLE 1.5 Approximate Daily Food and Water Requirements for Various Species Species

Daily Food Requirement

Daily Water Requirement

Mouse Rat Hamster Guinea pig Rabbit Cat Dog Primate

3–6 g 10–20 g 7–15 g 20–30 ga 75–100 g 100–225 g 250–1200 g 40 g/kga

3–7 ml 20–30 ml 7–15 ml 12–15 ml/100 g 80–100 ml/kg 100–200 ml 100–400 ml/day 350–1000 ml

a Like humans, guinea pigs and nonhuman primates require a continuous supply of vitamin C (ascorbic acid) in the diet.


feeding for long-term rodent bioassays has recently received increased attention since it appears that this practice impacts longevity, carcinogenesis, and overall animal health.

B. FASTING Like humans, animals are often fasted in preparation for blood collection. Generally, fasting periods of 18–24 hours may be safely utilized for most species. However, for mice, fasting periods of 18–24 hours may lead to severe debility, dehydration, and even death. Therefore, it is recommended that fasting periods of no longer than 4–6 hours be used for this species.

SECTION 13. ANESTHESIA AND ANALGESIA* A. GENERAL CONSIDERATIONS As stated earlier, investigators using live animals must employ appropriate anesthetic, analgesic, and sedative agents when necessary to control pain and distress, unless use of such agents would interfere with the specific objectives of the research. If these agents are not used, both the Animal Welfare Act and Guide for the Care and Use of Laboratory Animals require that the procedures be directly supervised by the responsible investigator in accordance with all regulations and guidelines governing these situations. If pain-relieving procedures are not employed, the investigator must provide well-documented evidence demonstrating that the use of such agents would interfere with the results of the study.

B. CONTROLLED SUBSTANCES To comply with these regulations, it is imperative that appropriate pain-relieving agents be available and that appropriate methods of administration and dosages be established. Because many painrelieving agents are controlled substances, the use and handling of these agents are regulated by the Controlled Substances Act (84 Stat. 1242; 21 U.S.C. 801). This statute is specifically administered by the U.S. Drug Enforcement Agency (DEA). Among other requirements, users of controlled substances must ensure that drug supplies are adequately protected (stored in a secure cabinet or safe) and inventoried in accordance with the requirements of the statute.

C. RELEVANT DEFINITIONS • Analgesia: The relief of pain without loss of consciousness. • Tranquilization: A state of behavioral change in which the animal is relaxed, unconcerned by its surroundings, and often indifferent to minor pain. • Sedation: Mild state of central nervous system (CNS) depression in which the animal is awake, but calm. • Local anesthesia: Loss of sensation in a limited area. • Regional anesthesia: Insensibility in a larger but still limited area. • Preanesthesia: A state produced by the concomitant use of several drugs to decrease anxiety without producing excessive drowsiness, to facilitate smooth, rapid induction of general anesthesia without prolonging emergence, provide amnesia for the perioperative period while maintaining cooperation prior to loss of consciousness, relieve preoperative and postoperative pain, and minimize some of the undesirable effects of anesthesia, i.e., salivation, bradycardia, and postanesthetic vomiting. • General anesthesia: A state of controlled and reversible unconsciousness characterized by lack of pain (analgesia), lack of memory (amnesia), and relatively depressed reflex responses without affecting the animal’s vital systems, i.e., respiration and circulation. * See Additional Related Information at the end of this chapter.


• Surgical anesthesia: Generally referred to as a surgical plane of anesthesia representing Stage III, plane 2 of the classical stages and planes of anesthesia; a medium depth of anesthesia suitable for most surgical procedures.

D. GENERAL PRINCIPLES REGARDING ANESTHESIA, ANALGESIA, AND TRANQUILIZATION The health of the animal should be carefully evaluated before instituting any anesthetic, analgesic, or tranquilizing procedure, and the specific drug(s) selected should provide the minimal level of CNS depression necessary. In addition, before undertaking any procedure, the investigator should closely consider the effect of the technique on experimental objectives, including potential drug interactions and interferences with test substance(s) (e.g., competing metabolic pathways, etc.).

E. STAGES

OF

ANESTHESIA

Indicators of anesthesia are commonly divided into four classic stages based on the depth of consciousness, presence or absence of reflex reactions, and degree of CNS and physiological depression. Stage I is characterized by disorientation, normal or panting respiration (20–30 breaths/min., unchanged heart rate, centrally positioned eyeball, normal pupil size, pupillary response to light, good muscle tone, and the presence of all reflexes. Stage II is marked by “excitement” with possible struggling, vocalization, paddling, chewing, or yawning; irregular respiration with possible holding of breath or hyperventilation; increased heart rate; centrally positioned eyeball or possible nystagmus (rapid involuntary oscillation of eyeball), possible dilation of pupils; pupillary response to light; good muscle tone; and presence of all reflexes with some possibly exaggerated. These first two stages represent presurgical anesthetic depths. Stage III anesthesia is subdivided into four different “planes” of progressively deeper unconsciousness. In plane 1 (light anesthesia), respiration is regular with a rate of 12–20 breaths/min.; pulse is strong (>90 beats per min., [bpm]); the animal may respond with movement, eyeballs may be centrally positioned or there may be nystagmus; pupil size is normal and responds to light; muscle tone is good and swallowing reflex is poor or absent and others present but diminished. In plane 2 (medium or surgical anesthesia), respiration may be shallow at 12–16 breaths/min.; heart rate >90 bpm; heart and respiration rates may increase in response to surgical activity; eyeballs may be ventrally rotated; pupil size moderately dilated; pupillary light response sluggish; muscle tone relaxed and patellar, ear flick, palpebral and corneal reflexes may be present, but others absent. In plane 3 (deep anesthesia), respiration is shallow at 3, the entry in the final column of Table 19-3 may be used (except for five tabular entries where an additional increment in the third decimal place is indicated). The estimate for Example 19-3 is P10 = 0.602 + (.831 × .301) = 852. For N greater than 6, P10 may be estimated by computing for the last N trials the mean of the test levels corrected by a factor which is dependent on the constants A and C of Table 19-4. The estimate is ΣXi d + ( A + C) N N where the Xi’s are the test levels and A and C are obtained from Table 19-4. In Table 19-4 no refers to the number of ’s and nx to the number of X’s in the final N trials. The standard error of this estimated mean is approximately σ 2 / N . The additional adjustment C, which improves this estimate, particularly for the smaller sample sizes, is based on the initial trials. This adjustment has little effect except in a few cases with small probability of occurrence, that is, where there are great differences of the number of X’s and ’s in the final N trials and where the series starts with a compensating run of ’s or X’s, respectively. If the use of this adjustment has any appreciable effect on the estimate it is advisable to investigate the possible disagreement of the experimental situation with the assumptions of this model; for example, the assumption that the interval may be very much smaller than σ or that the sampling is not all from the same population.


0370_frame_C02 Page 117 Thursday, July 12, 2001 9:47 AM

Log dose 1.204 .903 .602 .301 0

Results of tests ×

×

×

FIGURE 19-4 Results of six tests in an up-down experiment.

TABLE 19-3 Values of k for estimating P50 from up-and-down sequence of trials of nominal length N. The estimate of P50 is Xf + kd, where Xf is the final test level and d is the interval between dose levels. If the table is entered from the foot, the sign of k is to be reversed.

2 × 3 × ×× 4 × × × ×× ××× 5 × ×× ×× ×× × ×× × ×× ××× ×××× 6 × ×× ×× ××× ×× ××× ××× ×××× ×× ××× ××× ×××× ××× ×××× ×××× ×× ×××

k For Test Series Whose First Part Is –.377 .894 .028 .315 –.432 1.140 .506 –.154 –.860 .741 .182 .381 –.142 1.549 1.000+1 –.547 –1.246 .381 –.142 .040 –.453 1.248 .758 –.263 –.752 .954 .504+1 .681 .252+1 2.014+1 1.496+1

× ×× × × × × × ×× ×× × × × × × × × × × × × × × × × ×× ×× × ×× ×× × ××× × × ×× × ×× × ×

× ×× ××× ×××× − k For Series Whose First Part Is

Second part of series

–.500 .842 –.178 .299 –.500 1.000 .194 –.157 –.878 .701 .084 .305 –.305 1.288 .555 –.547 –1.250 .372 –.169 .022 –.500 1.169 .611 –.296 –.831 .831 .296 .500 –.043 1.603 .893


–.388 .890 .000 .314 –.439 1.122 .449 –.154 –.861 .737 .169 .372 –.169 1.500 .897 –.547 –1.247 .380 –.144 .039 –.458 1.237 .732 –.266 –.763 .935 .463 .648 .187 1.917 1.329

–.378 .894 .026 .315 –.432 1.139 .500 –.154 –.860 .741 .181 .380 –.144 1.544 .985 –.547 –1.246 .381 –.142 .040 –.453 1.247 .756 –.263 –.753 .952 .500 .678 .244 2.000 1.465

.55σ .76σ .67σ

.61σ

.36σ

Standard Error of P50

Sample Size N

Second part of series


The estimates are given in Table 19-3 for each possible configuration of responses, with the assumption that the proportion of successes is given by a normal cumulative distribution. For estimates as given in Table 19-3 it is assumed that d = σ. Fortunately, estimates for this design with N ≥ 3 prove to have standard errors which depend very little on the actual value of σ and, in addition, are almost independent of the starting level and of µ. This is approximately true even when the spacing d differs from σ.

TABLE 19-4 Values of A and C for approximate estimate of P50 for N > 6. The estimate is ΣNx ⋅ d(A + C)/N, where the Xx’s are the test levels of the final N trials with n nonresponses and nx responses and d is the interval between dose levels. C = 0 for a series whose first part is a single or ×. n − nx 5 4 3 2 1 0 −1 −2 −3 −4 −5

C For Test Series Whose First Part Is

A 10.8 7.72 5.22 3.20 1.53 0 −1.55 −3.30 −5.22 −7.55 −10.3

n× − n

−A

0 0

0 0

0 0

.03 .10 .16 .44 .55 1.14 1.77 2.48 3.5

.03 .10 .17 .48 .65 1.36 2.16 3.36 4.8

.03 .10 .17 .48 .65 1.38 2.22 3.52 5.2

××

0 0 .03 .10 .17 .48 .65 1.38 2.22 3.56 5.3

××× ×××× ××××× −C For Test Series Whose First Part Is

C. HISTORICAL CONTROL DATA — RODENT BODY WEIGHTS AND FOOD CONSUMPTION The following are representative mean values from several chronic studies conducted in our laboratory. (Age at week – 1 is approximately 5 weeks.)

TABLE 2.33 Body Weight and Food Consumption — CD-1 Mice Males

Females

Week

Body Weight (g)

Food Consumption (g/kg/day)

Body Weight (g)


−1 0 1 2 3 4 5 6

23.64 27.46 28.82 30.00 31.02 31.92 32.46 33.36

— 235.55 212.60 198.36 196.14 196.60 182.76 181.46

19.24 21.62 22.66 24.24 24.80 25.92 26.32 27.06

— 293.20 267.00 262.42 273.80 252.74 269.70 245.48


TABLE 2.33 (Continued) Body Weight and Food Consumption — CD-1 Mice Males

Females

Week

Body Weight (g)


Body Weight (g)


7 8 9 10 11 12 13–14 17–18 21–22 25–26 27–30 31–34 35–38 39–42 43–46 47–50 51–54 55–58 59–62 63–66 67–70 71–74 75–79

34.44 34.40 34.88 35.08 35.36 36.14 36.30 37.62 38.00 38.24 38.40 39.40 39.66 40.26 40.03 40.40 39.90 40.16 39.80 40.46 40.24 39.88 40.56

170.20 180.72 169.70 166.00 166.90 153.94 166.34 146.16 152.86 153.40 149.40 134.50 135.48 136.42 131.80 126.46 128.72 135.80 137.88 133.70 133.18 142.22 142.66

27.80 27.96 28.50 28.90 29.26 29.34 29.80 30.98 31.54 32.28 32.30 33.12 33.48 33.28 34.10 34.34 35.10 34.46 35.20 34.76 34.78 35.14 35.48

247.56 240.86 222.38 216.24 211.50 209.74 218.14 209.00 204.54 190.88 190.95 177.30 166.90 180.42 165.70 156.52 149.16 173.96 158.84 172.72 161.12 167.62 171.02

TABLE 2.34 Body Weight and Food Consumption — Sprague-Dawley Rats Males

Females

Week

Body Weight (g)


Body Weight (g)


−1 0 1 2 3 4 5 6 7 8 9 10 11 12 13

132.46 188.64 236.12 286.94 327.78 362.78 393.70 417.26 432.70 446.16 457.80 475.74 481.42 497.12 508.12

— 146.00 115.98 101.12 90.28 82.02 75.12 68.98 63.32 62.08 58.44 60.96 57.14 59.78 56.14

103.82 139.14 161.56 182.80 203.46 222.10 232.70 243.04 250.68 255.40 261.36 268.62 274.52 278.24 282.46

— 122.30 122.58 111.00 104.30 94.04 87.44 84.82 77.12 76.44 73.40 75.84 73.34 73.26 70.68


TABLE 2.34 (Continued) Body Weight and Food Consumption — Sprague-Dawley Rats Males

Females

Week

Body Weight (g)


Body Weight (g)


17–18 21–22 25–26 29–30 33–34 37–38 41–42 45–46 49–50 53–54 57–58 61–62 65–66 69–70 72–74 77–78 81–82 85–86 89–90 93–94 97–98 101–102

549.84 577.62 603.22 616.76 635.46 651.96 660.34 678.16 695.72 703.72 720.88 728.74 735.76 735.04 737.54 736.58 738.04 733.70 725.80 723.62 721.50 703.84

50.30 47.62 45.04 44.36 43.94 42.60 41.12 40.12 39.24 39.20 37.70 37.56 38.00 38.46 37.80 39.34 38.48 39.22 38.52 37.92 37.48 35.60

299.70 311.82 327.22 334.22 348.96 363.38 374.96 389.65 409.40 412.76 430.90 441.18 455.06 460.24 465.70 467.70 471.72 473.98 479.42 490.06 494.10 498.56

67.42 63.30 62.18 61.84 61.74 58.44 56.40 52.63 52.34 51.28 49.16 48.82 48.26 48.24 47.78 46.78 47.74 47.38 47.82 45.68 45.04 44.10

TABLE 2.35 Body Weight and Food Consumption — Fischer 344 Rats Males

Females

Week

Body Weight (g)


Body Weight (g)


−1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 16

86.03 118.27 151.50 183.60 211.07 228.17 244.10 256.27 263.53 275.23 281.90 290.47 296.60 300.23 301.10 319.07

— 137.73 112.73 99.33 85.73 77.73 75.07 67.13 63.07 60.40 56.17 54.20 55.63 54.40 54.25 51.10

68.43 89.90 108.07 123.70 135.70 142.80 150.60 155.77 162.07 164.40 166.47 170.20 172.23 173.07 174.75 181.53

— 146.93 124.63 107.00 96.43 90.93 89.03 80.47 72.73 69.67 68.20 66.27 67.40 69.10 64.10 65.63


TABLE 2.35 (Continued) Body Weight and Food Consumption — Fischer 344 Rats Males

D. EFFECT

Females

Week

Body Weight (g)


Body Weight (g)


20 24 28–30 32–34 36–38 40–42 44–46 48–50 52–54 56–59 60–62 64–66 68–70 72–74 76–78 80–82 84–86 88–90 92–94 96–98 100–102

331.67 343.40 355.70 366.23 374.67 382.90 384.13 385.90 396.23 400.87 401.90 404.87 406.57 410.20 404.93 394.77 393.60 397.07 391.43 388.77 388.03

48.17 47.97 46.67 45.33 44.23 44.07 42.63 38.17 44.17 43.57 40.97 43.77 44.37 43.67 46.00 43.60 43.37 43.73 42.93 42.47 43.33

186.87 193.67 201.90 206.70 211.37 215.40 218.90 223.53 228.47 236.17 241.47 246.37 253.80 259.53 265.07 262.80 264.47 270.70 275.13 277.17 277.70

62.63 62.07 59.87 60.00 61.10 58.27 56.00 57.07 57.37 55.23 50.63 53.43 54.60 54.57 54.77 51.50 50.37 51.50 50.83 49.63 51.57

OF

DECREASED BODY WEIGHTS

ON

ORGAN WEIGHTS

OF

TABLE 2.36 Effect of Decreased Body Weights on Relative Organ Weightsa of Ratsb Decrease

No Change

Liver (?)

Heart Kidneys Prostate Spleen Ovaries

Increase Adrenal glands (?) Brain Epididymides Pituitary Testes Thyroid (?) Uterus

Relative weights = organ/body weight ratios (?) ? = Differences slight or inconsistent. b For absolute weights, all except thyroids decrease. Summary of results reported in: Schwartz, E. R. et al.,10 Scharer, K.11 a

RATS


E. RODENT SURVIVAL RATES TABLE 2.37 Monthly Survival Rates of Untreated CD-1 Mice in Chronic Toxicity Studies Conducted Between 1985 and 1992 Males

Females

Month of Study

Mean % Survival

±SD

No. of Studies

Mean % Survival

±SD

No. of Studies

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

99.7 99.6 99.2 98.7 98.1 97.8 97.4 96.6 95.7 94.8 93.4 92.6 89.5 86.7 83.1 76.8 69.4 59.7

0.7 0.8 1.3 1.5 1.6 1.5 1.6 2.2 2.6 3.2 3.2 3.7 7.0 7.5 7.5 9.0 10.6 12.9

21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21 21

99.9 99.7 99.5 99.4 99.4 99.0 98.4 98.1 97.5 96.9 96.0 95.1 92.9 90.0 87.3 81.9 76.0 67.0

0.4 0.7 0.9 0.9 0.9 1.5 1.9 1.9 2.0 2.0 2.3 3.4 4.3 4.3 4.3 6.3 7.8 10.4

20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20

TABLE 2.38 Eighteen-Month Survival Rates of Untreated CD-1 Mice in Chronic Toxicity Studies Between 1985 and 1992 Survivorshipb Male

Female

Study Code

Route of Administration

Termination Date

Supplier

Incidence

%

Incidence

%

A B C D E F G H I J K M N O

Diet Diet Diet Diet Diet Gavage Diet Diet Diet Diet Gavage Diet Diet IM

3/87 6/87 1/88 3/88 4/88 11/88 1/89 8/89 1/90 3/90 4/90 7/90 12/90 1/91

CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K

26/48 30/50 38/65 27/50 46/69 38/66 24/47 33/49 32/50 30/48 25/59 35/51 25/50 19/48

54 60 58 54 67 58 51 67 64 63 43 68 50 40

27/49 32/50 46/65 33/50 56/69 33/67 26/47 27/46 28/45 — 42/59 39/50 31/49 25/49

55 64 71 66 81 49 55 59 62 — 71 78 63 51

a


TABLE 2.38 (Continued) Eighteen-Month Survival Rates of Untreated CD-1 Mice in Chronic Toxicity Studies Between 1985 and 1992 Survivorshipb Study Code


P Q R S T U V a b

Male

Female

Termination Date

Suppliera

Incidence

%

Incidence

%

8/91 9/91 4/92 5/92 6/92 9/92 11/92

CR-P CR-P CR-K CR-K CR-K CR-K CR-K

37/50 46/50 24/50 32/50 26/60 28/50 39/48

74 92 48 64 43 56 81

36/50 42/50 34/49 30/50 49/60 38/48 33/49

72 84 69 60 82 79 67

Diet Diet Diet Diet Diet Diet Diet

Supplier: CR-K, Charles Rive-Kingston, NY, CR-P, Charles River-Portage, MI. Animals killed accidentally were excluded from calculations of survivorship.

100

90

MEAN MONTHLY SURVIVAL (%)

80

70

60

50

40

30 Males

+

20

Females

10

0 0

1

2

3

4

5

6

7

8

9

10

11

12

13

MONTH OF STUDY FIGURE 2.3 Mean monthly survival — mice.

14 15

16

17

18


TABLE 2.39 Monthly Survival Rates of Untreated Sprague-Dawley Rats in Chronic Toxicity Studies Conducted Between 1984 and 1992 Males Month of Study

Mean % Survival

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

99.8 99.4 99.1 98.9 98.6 98.1 97.7 97.2 96.9 96.5 95.3 94.5 93.2 91.9 89.9 86.4 82.9 78.5 72.0 66.4 57.8 50.3 43.0 34.7

Females

±SD

No. of Studies

Mean % Survival

±SD

No. of Studies

0.6 1.0 1.3 1.4 1.9 2.1 2.1 2.1 2.1 2.0 2.6 3.2 4.3 4.0 5.1 6.5 7.6 8.7 9.9 9.7 9.0 9.9 10.6 8.9

19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 18 18 18 18

99.8 99.8 99.7 99.5 99.4 99.1 99.1 98.8 98.2 97.8 97.1 96.2 94.9 93.5 91.2 88.8 84.5 80.6 74.4 68.9 61.7 56.5 49.7 42.3

0.6 0.6 1.0 1.1 1.1 1.4 1.4 1.4 1.9 2.1 2.7 3.0 3.2 3.7 4.4 5.0 6.3 7.7 7.9 9.7 11.2 12.2 12.7 12.2

19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19 19

TABLE 2.40 Twenty-Four-Month Survival Rates of Untreated Sprague-Dawley Rats in Chronic Toxicity Studies Conducted Between 1984 and 1992 Survivorshipb Study Code


DD EE FF GG HH II JJ KK LL MM

Diet Diet Diet Diet Diet Diet Diet Gavage Diet Gavage

Male

Female

Termination Date

Supplier

Incidence

%

Incidence

%

7/86 7/86 10/86 12/87 1/88 3/88 5/88 11/88 4/89 10/89

CR-P CR-P CR-K CR-P CR-K CR-K CR-P CR-K CR-K CR-K

27/55 20/70 20/55 20/63 21/50 15/50 27/60 20/60 21/55 17/60

49 29 36 32 42 30 45 33 38 28

22/55 31/70 25/55 31/63 21/50 24/50 28/50 19/60 24/54 14/60

40 44 45 49 42 48 56 32 44 23

a


TABLE 2.40 (Continued) Twenty-Four-Month Survival Rates of Untreated Sprague-Dawley Rats in Chronic Toxicity Studies Conducted Between 1984 and 1992 Survivorshipb Study Code


NN OO PP QQ RR SS TT UU VV

Diet Diet Diet IntraMuscular Diet Diet Diet Diet Diet

a b

Male

Female

Termination Date

Suppliera

Incidence

%

Incidence

%

11/89 7/90 8/90 12/91 5/92 4/92 10/92 11/92 12/92

CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K CR-K

22/52 20/52 17/53 — 21/50 7/46 26/58 13/60 13/49

42 38 32 — 42 15 45 22 27

23/50 19/54 21/52 41/50 21/50 16/50 22/60 21/60 17/50

46 35 40 82 42 32 37 35 34

Supplier: CR-K, Charles River-Kingston, NY; CR-P, Charles River-Portage, MI. Animals killed accidentally were excluded from calculations of survivorship.

100

90

MEAN MONTHLY SURVIVAL (%)

80

70

60

50

40

30 Males 20

Females

10

0

0

4

8

12

16

MONTH OF STUDY FIGURE 2.4 Mean monthly survival — rats.

20

24


REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Bruce, R.D., An up-and-down procedure for acute toxicity testing, Fund Appl. Toxicol., 5, 151, 1985. SYNAPSE, American Society of Laboratory Animal Practitioners, Vol. 24, 1991. Laboratory Manual For Basic Biomethodology of Laboratory Animals, MTM Associates, Inc. Freireich, E.J. et al., Quantitative comparison of toxicity of anti-cancer agents in mouse, rat, dog, monkey and man, Cancer Chemother. Rep., 50, 219, 1966. Chan, P.K. and Hayes, A.W., Principles and methods for acute toxicity and eye irritancy, in Principles and Methods of Toxicology, 2nd ed., Hayes, A.W., Ed., Raven Press, New York, 1989. Finney, D.J., Probit Analysis, 3rd ed., Cambridge University Press, London, 1971. Litchfield, J.T. and Wilcoxon, F., A simplified method for evaluating dose-effect experiments, J. Pharmacol. Erp. Ther., 96, 99, 1949. Miller, L.C. and Tainter, M.L., Estimation of the ED50 and its error by means of logarithmic-probit graph paper, Proc. Soc. Exp. Biol. Med., 57, 261, 1944. Dixon, W.J. and Massay, M.J., Eds., Introduction to Statistical Analysis, 3rd ed., McGraw-Hill, New York, 1969. Schwartz, E., Tomaben, J.A., and Boxill, G.C., The effects of food restriction on hematology, clinical chemistry and pathology in the albino rat, Toxicol. Appl. Parmacol., 25, 515, 1973. Scharer, K., The effect of underfeeding on organ weights of rats. How to interpret organ weight changes in cases of marked growth retardation in toxicity tests, Toxicol., 7, 45, 1977.

3

Dermal Irritation and Sensitization Kimberly L. Bonnette, M.S., L.A.T.G., Dawn D. Rodabaugh, B.S., and Christopher W. Wilson, B.S.

CONTENTS Section 1. Comparison of Table 3.1 Table 3.2 Section 2. Comparison of Table 3.3 Table 3.4 Table 3.5 Table 3.6

Section 3.

Section 4.

Section 5.

Section 6.

Major Study Designs Comparison of Dermal Irritation Study Designs Comparison of Sensitization Study Designs Regulatory Guidelines Quick Reference Chart for Common U.S. Test Guidelines Quick Reference Chart for Common Foreign Test Guidelines Quick Reference Chart for Miscellaneous Test Guidelines Comparison of Excerpts from Selected Dermal Irritation Test Guidelines Table 3.7 Comparison of Excerpts from Selected Sensitization Test Guidelines Materials and Procedures for Performing Dermal Irritation Studies A. The Occluded Dermal Irritation Test in Rabbits B. The Semioccluded Dermal Irritation Test in Rabbits C. The Nonoccluded Dermal Irritation Test in Rabbits D. The Corrosivity Test in Rabbits E. The Photoirritation Test in Rabbits F. The Photoirritation Test in Guinea Pigs Materials and Procedures for Performing Dermal Sensitization Studies A. The Modified Buehler Sensitization Test in Guinea Pigs B. The Standard Buehler Sensitization Test in Guinea Pigs C. The Guinea Pig Maximization Test D. The Murine Local Lymph Node Assay E. The Photosensitization Test in Mice F. The Photosensitization Test in Guinea Pigs Comparison of Scoring Systems Table 3.8 Draize Dermal Irritation Scoring System Table 3.9 Human Patch Test Dermal Irritation Scoring System Table 3.10 Chamber Scarification Dermal Irritation Scoring System Table 3.11 Magnusson Sensitization Scoring System Table 3.12 Split Adjuvant Sensitization Scoring System Table 3.13 Buehler Sensitization Scoring System Table 3.14 Contact Photosensitization Scoring System Table 3.15 Human Patch Test Sensitization Scoring System Comparison of Classification Systems Table 3.16 Environmental Protection Agency (EPA) Method of


Calculating the Primary Irritation Index (PII) for Dermal Irritation Studies Table 3.17 Federal Hazardous Substances Act (CPSC-FHSA) Method of Calculating the Primary Irritation Index (PII) for Dermal Irritation Studies Table 3.18 European Economic Community’s (EEC) Method of Calculating the Primary Irritation Index (PII) for Dermal Irritation Studies Table 3.19 Environmental Protection Agency (EPA) Dermal Classification System Table 3.20 Environmental Protection Agency (EPA) Standard Evaluation Procedure Dermal Classification System Table 3.21 Federal Fungicide, Insecticide, and Rodenticide Act (EPA-FIFRA) Dermal Classification System Table 3.22 European Economic Community (EEC) Dermal Classification System Table 3.23 Federal Hazardous Substances Act (CPSC-FHSA) Dermal Classification System Table 3.24 Draize Dermal Classification System Table 3.25 Department of Transportation (DOT) and International Maritime Organization (IMO) Packing Group Classification System Table 3.26 Maximization Sensitization Classification System Table 3.27 Optimization Sensitization Classification System Section 7. Materials that Produce Dermal Irritation and/or Sensitization Table 3.28 Common Materials Utilized as Positive Controls Table 3.29 Materials Categorized by their Ability to Produce Dermal Irritation or Sensitization Table 3.30 Dermal Irritants and Sensitizers Listed by Occupation Section 8. Glossary of Common Terminology References Additional Related Information Table 3.31 Relative Ranking of the Skin Permeability in Different Animal Species Table 3.32 In Vivo Human Percutaneous Absorption Rates of Some Neat Chemical Liquids Table 3.33 In Vitro Human Percutaneous Permeability Coefficients of Aqueous Solutions of Some Industrial Chemicals


SECTION 1. COMPARISON OF MAJOR STUDY DESIGNS TABLE 3.1 Comparison of Dermal Irritation Study Designs1–5 Category Number of animals Species Preliminary screens

Control group Preparation of skin Application site Application area Patch types Occlusion Preparation of test substance

Dose level Exposure interval No. of applications Test substance residue removal Observation intervals Minimum observation period Maximum observation period

EPA-OPPTS (870.2500) At least three healthy adults Albino rabbit recommended May not test if: pH ≤ 2, ≥ 11.5, dermal LD50 < 200 mg/kg, dermal limit test at 2000 mg/kg did not produce irritation, a validated and accepted in vitro test demonstrates corrosive properties, or corrosive potential is predicted from structure–activity relationships. None required Clip or shave ≈ 24 hr before test Use only healthy, intact skin Dorsal area of trunk ≈ 6 cm2 Gauze patch held loosely in contact with skin using nonirritating tape Semiocclusive dressing Solids: pulverized if necessary, moistened sufficiently with water or suitable vehicle to ensure good skin contact Liquids: generally used undiluted Solids: 0.5 g Liquids: 0.5 ml 4 h (3 min, 1 h if corrosion is anticipated) One Water or another appropriate solvent 30–60 min, 24, 48, and 72 h after patch removal 72 h 14 days

Category

Japanese-MAFF (Current)

Number of animals Species Preliminary screens Control group Preparation of skin

At least six young adults Albino rabbit May not test if: pH ≤ 2, ≥ 11.5 None required Clip or shave ≈ 24 hr before test

Application site Application area Patch types

Dorsal area of trunk ≈ 6 cm2 Gauze patch held loosely in contact with skin using nonirritating tape Semiocclusive dressing preferred; occlusive may be appropriate Solids: Pulverized if necessary, moistened sufficiently with water or suitable vehicle Liquids: Generally used undiluted

Occlusion Preparation of test substance

Dose level

Solids: 0.5 g Liquids: 0.5 ml


Japanese-MAFF (Draft) At least three young adults White rabbit May not test if: pH ≤ 2, ≥ 11.5 None required Clip ≈ 24 hr before test Use only healthy, intact skin Dorsal area of trunk ≈ 6 cm2 Gauze patch held in contact with skin using nonirritating tape Semiocclusive dressing preferred; occlusive may be appropriate Solids: crushed if necessary, moistened thoroughly with water or suitable vehicle to ensure good skin contact Liquids: Applied undiluted Solids: 0.5 g Liquids: 0.5 ml

TABLE 3.1 (Continued) Comparison of Dermal Irritation Study Designs1–5 Category

Japanese-MAFF (Current)

Exposure interval

4h

No. of applications Test substance residue removal Observation intervals

One Water or another appropriate solvent 30–60 min, 24, 48, and 72 h after patch removal 72 h 14 days

Minimum observation period Maximum observation period Category Number of animals Species Preliminary screens

Control group Preparation of skin Application site Application area Patch types Occlusion Preparation of test substance

Dose level Exposure interval No. of applications Test substance residue removal Observation intervals Minimum observation period Maximum observation period

Japanese-MAFF (Draft) 4 h (3 min, 1 h, 4 h to the first animal if severe potential irritation/corrosion is anticipated) One Water or another appropriate solvent 30 or 60 min, 24, 48, and 72 h after patch removal 72 h 14 days

European-OECD

European-EEC

Generally three healthy adults, one animal may sometimes be used Albino rabbit recommended Do not test if: pH ≤ 2, ≥ 11.5, material is highly toxic by dermal route, dermal limit test at 2000 mg/kg did not produce irritation, in vitro test indicate corrosive properties None required Closely clip ≈ 24 h before test; use only healthy, intact skin Dorsal area of trunk ≈ 6 cm2 Gauze patch held loosely in contact with skin using nonirritating tape Semiocclusive Solids: Pulverized if necessary, moistened with smallest amount of water or suitable vehicle to ensure good skin contact Liquids: Applied undiluted Solids: 0.5 g Liquids: 0.5 ml 4 h, may be reduced to 1 h or 3 min One Water or another appropriate solvent 60 min, 24, 48, and 72 h after patch removal 72 h Not indicated

At least three healthy adults, one animal may sometimes be used Albino rabbit recommended Do not test if: pH ≤ 2, ≥ 11.5, material is highly toxic by dermal route, dermal limit test at 2000 mg/kg did not produce irritation, in vitro test indicate corrosive properties None required Clip or shave ≈ 24 h before test; use only healthy, intact skin Dorsal area of trunk ≈ 6 cm2 Gauze patch held loosely in contact with skin using nonirritating tape Semi-occlusive or occlusive Solids: Pulverized if necessary, moistened sufficiently with water or suitable vehicle to ensure good skin contact Liquids: Applied undiluted Solids: 0.5 g Liquids: 0.5 ml 4 h, may be reduced to 1 h or 3 min One Water or another appropriate solvent 60 min, 24, 48, and 72 h after patch removal 72 h 14 days


TABLE 3.2 Comparison of Sensitization Study Designs.6–11 Category Acceptable test methods

Species Number and sex Control animals

Dose level Preparation of skin Observation of animals

Body weights

EPA OPPTS 870.2600 a

Buehler Test, Guinea Pig Maximization Test (GPMT),a Open Epicutaneous Test, Mauer Optimization Test, Split Adjuvant Technique, Freund’s Complete Adjuvant Test, Draize Sensitization Test Guinea pig Dependent on method used Periodic (every 6 months) use of a positive control substance with an acceptable level of reliability for the test system selected is recommended; irritation controls may or may not be used Dependent on method used Clipping, shaving, or depilation depending on method used Skin reactions are to be graded and recorded after the challenge exposure at the time specified by the methodology selected (usually 24, 48, and 72 h) Initial and terminal body weights required

Japanese-MHW Adjuvant and Patch Test; Buehler Test;a Draize Test; Freund’s Complete Adjuvant Test; Maximization Test;a Open Optimization Test; Split Adjuvant Test Guinea pig Dependent on method used Positive controls are required; preferred substances include p-phenylenediamine, 1-chloro-2,4-dinitrobenzene, neomycin sulfate, and nickel sulfate Dependent on method used Dependent on method used Skin reactions are to be noted at 24, 48, or 72 h after challenge exposure

Initial and terminal body weights required

Category

Japanese-MAFF (current)

Japanese-MAFF (draft)

Acceptable test methods

Buehler Test,a Guinea Pig Maximization Test,a other methods may be used provided that they are well validated and scientific justification is given Guinea pig Dependent on method used Positive control are required to evaluate the responsivity of the test system


Draize Test, Freund’s Complete Adjuvant Test, Mauer Optimization Test, Buehler Test,a Open Epicutaneous Test, Guinea Pig Maximization Test,a Split Adjuvant Technique Guinea pig Dependent on method used Use of a positive control substance for the reliability of the test system selected is recommended Dependent on method used Dependent on method used Dependent on method used

Body weights


Species Number and sex Control animals

Category Acceptable test methods

Species Number and sex

Dependent on method used Dependent on method used Skin reactions are to be graded and recorded after the challenge exposure at the time specified by the methodology selected (usually 24, 48, and 72 h) Initial and terminal body weights required

European-OECD a

Buehler Test, Guinea Pig Maximization Test,a other methods may be used provided that they are well validated and scientific justification is given Guinea pig Dependent on method used


European-EEC a

Buehler Test, Guinea Pig Maximization Test,a other methods may be used provided that they are well validated and scientific justification is given Guinea pig Dependent on method used

TABLE 3.2 (Continued) Comparison of Sensitization Study Designs.6–11 Category

European-OECD

European-EEC

Control animals

Mild-to-moderate positive controls are required every 6 months (response of at least 30% in an adjuvant test and 15% in a nonadjuvant test should be expected); preferred substances are hexylcinnamicaldhyde, mercaptobenzothiazol, and benzocaine; others are accepted with justification


Dependent on method used Clipping, shaving, or depilation depending on method used 24 and 48 h after patch removal at challenge

Body weights


Mild-to-moderate positive controls are required every 6 months (response of at least 30% in an adjuvant test and 15% in a nonadjuvant test should be expected); preferred substances are phenylenediamine, 2,4-dinitrochlorobenzene, potassium dichromate, neomycin sulfate, and nickel sulfate, or others that are known sensitizing substances from the literature Dependent on method used Clipping, shaving, or depilation dependent on method used All skin reactions from induction and challenge procedures should be recorded and reported Initial and terminal body weights required

a

These test methods are the most widely used.

SECTION 2. COMPARISON OF REGULATORY GUIDELINES TABLE 3.3 Quick Reference Chart for Common U.S. Test Guidelines1–15 Specific Section for Regulatory Group Dermal

Reference

Dermal Irritation

Dermal Sensitization

U.S. Environmental Protection Agency (EPA)

Health Effects Test Guidelines, August 1998

OPPTS 870.2500

OPPTS 870.2600

U.S. Consumer Product Safety Commission (CPSC)

Subchapter C—Federal Hazardous Substances Act Regulations, 16 CFR Part 1500, January 1993 (FHSA) 49 CFR, Part 173, October 1998 (DOT) Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, Redbook II, 1993 (FDA) The U.S. Pharmacopeia, USP24 and The National Formulary, NF 19, January 1, 2000 (USP)

Section 1500.41

Section 1500.3

Sections 173.136, 173.137

Not specified

Not specified

Not specified

Chapter 88

Not Specified

U.S. Department of Transportation (DOT) U.S. Food and Drug Administration (FDA)

U.S. Pharmacopeia (USP)


TABLE 3.4 Quick Reference Chart for Common Foreign Test Guidelines2,4,5,7,8,10,11,16 Specific Section for Regulatory Group Government of Canada, Environment Canada, and Health and Welfare Canada (CEPA) European Organization for Economic Cooperation and Development (OECD) European Economic Community (EEC) Japanese Ministry of Forestry and Fisheries (MAFF)

Japanese Ministry of Forestry and Fisheries (MAFF) Japanese Ministry of Health and Welfare (MHW)

Dermal Irritation

Reference Canadian Environmental Protection Act, Guidelines for the Notification and Testing of New Substances: Chemicals and Polymers, March 1993 Guidelines for Testing of Chemicals, Section 4, Health Effects, July 1992 Part B: Methods for the Determination of Toxicity, December 1992 Agricultural Chemicals Laws and Regulations, Testing Guidelines for Toxicology Studies, January 1985 Guidelines on the Compiling of the Results on Toxicity (draft) December 1987 1990 Guidelines for Toxicity Studies of Drugs Manual, September 1989

Dermal Sensitization

Section 5.1

Section 5.1

Subsection 404

Subsection 406

No. L 383 A/124, B.4

No. L 383 A/131, B.6

pp. 25–26

pp. 27–29

pp. 9–11

pp. 16–19

Not specified

Chap. 7, pp. 75–80

TABLE 3.5 Quick Reference Chart for Miscellaneous Test Guidelines17–23 Regulatory Group

Reference

Study Type

International Maritime Organization (IMO)

International Maritime Dangerous Goods Code

Dermal corrosion

Occupational Safety and Health Administration (OSHA)

OSHA’s Hazard Communication Standard, 29 CFR 1900.1200, Appendix A, August 1987

Dermal irritation and sensitization

American Society for Testing and Materials (ASTM)

Annual Book of ASTM Standards, F719 (13.01), E993 (11.04), F720 (13.01)


The Cosmetic, Toiletry and Fragrance Association, Inc. (CTFA)

CTFA Safety Testing Guidelines, Sections II and IV



TABLE 3.6 Comparison of Excerpts from Selected Dermal Irritation Test Guidelines EPA OPPTS 870.2500 (a) Scope — (1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 USC 136, et seq.) and the Toxic Substances Control Act (TSCA) (15 USC 2601). (2) Background. The source of materials used in developing this harmonized OPPTS test guideline are 40 CFR 798.4470 Primary Dermal Irritation; OPP 81-5 Primary Dermal irritation (Pesticide Assessment Guidelines, Subdivision F — Hazard Evaluation; Human and Domestic Animals); EPA report 540/09-82-025, 1982; and OECD 404 Acute Dermal Irritation/Corrosion. (b) Purpose. Determination of the irritant and/or corrosive effects on skin of mammals is useful in the assessment and evaluation of the toxic characteristics of a substance where exposure by the dermal route is likely. Information derived from this test serves to indicate the existence of possible hazards likely to arise from exposure of the skin to the substance. (c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792 — Good Laboratory Practice Standards (GLP) apply to this test guideline. The following definitions also apply to this test guideline. “Dermal corrosion” is the production of irreversible tissue damage in the skin following the application of a test substance. “Dermal irritation” is the production of reversible inflammatory changes in the skin following the application of a test substance. “Pharmacological effect” means any chemically induced physiological changes in the test animal. “Target Organ” means any organ of a test animal showing evidence of an effect of chemical treatment. (d) Principle of the test methods. (1) The substance to be tested is applied in a single dose to the skin of several experimental animals, each animal serving as its own control [except when severe irritation/corrosion is suspected and the stepwise procedure is used (see paragraph (f)(1)(iii))]. The degree of irritation is read and scored at specified intervals and is further described to provide a complete evaluation of the effects. The duration of the study should be sufficient to permit a full evaluation of the reversibility or irreversibility of the effects observed but need not exceed 14 days. (2) When testing solids (which may be pulverized if considered necessary), the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle, to ensure good contact with the skin. When vehicles are used, the influence of the vehicle on irritation of skin by the test substance should be taken into account. Liquid test substances are generally used undiluted. (e) Initial considerations. (1) Strongly acidic or alkaline substances, for example, with a demonstrated pH of 2 or less, or 11.5 or greater, need not be tested for primary dermal irritation, owing to their predictable corrosive properties. (2) It is unnecessary to test materials which have been shown to be highly toxic (LD50 less than 200 mg/kg) by the dermal route or have been shown not to produce irritation of the skin at the limit test dose level of 2000 mg/kg body weight. (3) It may not be necessary to test in vivo materials for which corrosive properties are predicted on the basis of results from well validated and accepted in vitro tests. If an in vitro test is performed before the in vivo test, a description or reference to the test, including details of the procedure, must be given together with results obtained with the test and reference substances. (4) It may not be necessary to test materials for which corrosive potential is predicted from structure-activity relationships. (f) Test procedures — (1) Animal selection — (i) Species and strain. The albino rabbit is recommended as the preferred species. If another mammalian species is used, the tester should provide justification/reasoning for its selection. (ii) Number of animals. At least three healthy adult animals (either sex) should be used unless justification/reasoning for using fewer animals is provided. It is recommended that a stepwise procedure be used to expose one animal, followed by additional animals to clarify equivocal responses. (iii) Stepwise exposure of animals. A single rabbit may be used if it is suspected that the test material might produce severe irritation/corrosion. Three test patches are applied concurrently or sequentially to the animal. The first patch is removed after 3 min. If no serious skin reaction is observed, the second patch is removed after 1 h. If observations indicate that exposure can be continued humanely, the third patch is removed after 4 h and the responses graded. If a corrosive effect is observed after an exposure of up to 4 h, then further animal testing is not required. If no corrosive effect is observed in one animal after a 4-h exposure, the test is completed using two additional animals, each with one patch only, for an exposure period of 4 h. If it is expected that the test substance will not produce severe irritancy or corrosion, the test may be started using three animals, each receiving one patch for an exposure period of 4 h.


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines (2) Control animals. Separate animals are not recommended for an untreated control group. Adjacent areas of untreated skin of each animal may serve as a control for the test. (3) Dose level. A dose of 0.5 ml of liquid or 500 mg of solid or semisolid is applied to the test site. (4) Preparation of test area. Approximately 24 h before the test, fur should be removed from the test area by clipping or shaving from the dorsal area of the trunk of the animals. Care should be taken to avoid abrading the skin. Only animals with healthy intact skin should be used. (5) Application of the test substance. (i) The recommended exposure duration is normally 4 h unless corrosion is observed [see paragraph (f)(1)(ii)]. Longer exposure may be indicated under certain conditions (e.g., expected pattern of human use and exposure). At the end of the exposure period, residual test substance should generally be removed, where practicable, using water or an appropriate solvent, without altering the existing response or the integrity of the epidermis. (ii) When vehicles are used, the influence of the vehicle on irritation of skin by the test substance should be taken into account. If a vehicle is used, it should not alter the absorption, distribution, metabolism, retention or the chemical properties of the test substance nor should it enhance, reduce, or alter its toxic characteristics. Although water or saline is the preferred agent to be used for moistening dry test materials, other agents may be used providing the use is justified. Acceptable alternatives include: gum arabic, ethanol and water, carboxymethyl cellulose, polyethylene glycol, glycerol, vegetable oil, and mineral oil. (iii) The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with nonirritating tape. In the case of liquids or some pastes, it may be necessary to apply the test substance to the gauze patch and apply that to the skin. The patch should be loosely held in contact with the skin by means of a suitable semiocclusive dressing for the duration of the exposure period. Access by the animal to the patch and resultant ingestion/inhalation of the test substance should be prevented. 6. Observation period. The duration of the observation period need not be rigidly fixed. It should be sufficient to fully evaluate the reversibility or irreversibility of the effects observed. It need not exceed 14 days after application. 7. Clinical examination and scoring. (i) After removal of the patch, animals should be examined for signs of erythema and edema and the responses scored within 30–60 min, and at 24, 48, and 72 h after patch removal. (ii) Dermal irritation should be scored and recorded according to the grades provided in the guidelines. Further observations may be needed, as necessary, to establish reversibility. In addition to the observation of irritation, any lesions and other toxic effects should be fully described. (g) Data and reporting — (1) Data summary. Data should be summarized in tabular form, showing for each individual animal the irritation scores for erythema and edema at 30 to 60 min, and 24, 48, 72 h after patch removal, any other dermal lesions, a description of the degree and nature of the irritation, corrosion and reversibility, and any other toxic effects observed. (2) Evaluation of results. The dermal irritation scores should be evaluated in conjunction with the nature and reversibility or otherwise of the responses observed. The individual scores do not represent an absolute standard for the irritant properties of a material. They should be viewed as reference values which are only meaningful when supported by a full description and evaluation of the observations. (3) Test report. In addition to the reporting recommendations as specified under 40 CFR part 792, subpart J, the following specific information should be reported: (i) Species, strain, sex, age, and source of test animal. (ii) Rationale for selection of species (if species is other than the species preferred or required by the OPP toxicology data requirements for pesticide registration). (iii) Tabulation of erythema and edema data and any other dermal lesions/responses for each individual animal at each observation time point (e.g., 30–60 min and 24, 48, 72 h until end of test/reversibility). (iv) Description of any lesions observed. (v) Narrative description of the degree and nature of irritation or corrosion observed. (vi) Description of any systemic effects observed. (vii) Description of any pretest conditioning, including diet, quarantine, and treatment of disease. (viii) Description of caging conditions including number (and any change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animal. (ix) Manufacturer, source, purity, and lot number of test substance. (x) Physical nature and, where appropriate, concentration and pH value for the test substance.


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines (xi) Identification and composition of any vehicles (e.g., diluents, suspending agents, and emulsifiers) or other materials used in administering the test substance. (xii) A list of references cited in the body of the report, i.e., references to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results. Japanese-MAFF (Current) 1. Purpose. The purpose of this study is to obtain the data which will make the basis to establish safe handling procedures in use. 2. Test Substance. The end-use product should be used. However, strongly acidic or alkaline substances (approximately pH 2 or less or pH 11.5 or greater) might not be tested. 3. Test Animals. At least six of young adult albino rabbits should be used. 4. Exposure Conditions. (1) Approximately 24 hours before the test, fur should be removed from the dorsal area of the trunk of the test animals by clipping or shaving. (2) When testing solids (which may be pulverized if considered necessary), the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle, to ensure good contact with the skin. When vehicles are used, the influence of the vehicle should be taken into account. Liquid test substances are generally used undiluted. (3) A dose of 0.5 ml of liquid or 0.5 g of solid or paste is applied to the test site. (4) The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In the case of liquids or some pastes, it may be necessary to apply the test substance to the gauze patch and then apply that to the skin. The patch should be loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. (However, the use of occlusive dressing maybe considered appropriate in some cases.) (5) Exposure duration is for four hours. At the end of the exposure period, residual test substance should generally be removed by using water or an appropriate solvent. 5. Clinical Examination and Scoring. After removal of the patch, animals should be examined for signs of erythema and edema and the responses scored within 30 minutes or 60 min, and then at 24, 48 and 72 h after patch removal. Dermal irritation is scored and recorded according to the grades in Table 3.8. Further observations may be needed, as necessary, to establish reversibility. It need not normally exceed 14 days after application. In addition to the observation of irritation, any lesions and other toxic effects should be fully described. Japanese-MAFF (Draft) 1. Objective This test seeks to provide information on potential skin irritation or corrosion forming a basis for establishing the safe method of handling the agrochemical during use. 2. Test substance The preparation. However this test should not be undertaken with strongly acidic or alkaline materials (generally those up to pH 2 and from pH 11.5) as these may be expected to be corrosive. 3. Test animal species and age Young adult white rabbits are used. Three or more are used. 4. Method of administration (1) About 24 h before the test, the hair in the dorsal region of the trunk of the test animals is clipped short. Care is taken not to damage the skin and only animals with healthy, undamaged skin are used. (2) If the test substance is a solid, it is moistened thoroughly with water or a suitable vehicle to ensure good contact with the skin. If necessary it may also be crushed. Care must also be taken that the vehicle used has no effects on the test. Test substances in liquid form are applied undiluted. (3) 0.5 ml of liquid test substance and 0.5 g of solid or paste test substances are applied to the test site.


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines (4) The test substance is applied to a small area of skin (about 6 cm2), covered with a gauze patch and secured with nonirritant tape. For liquid or paste materials, a method may also be adopted in which the test substance is applied to the gauze patch and the gauze patch applied to the skin. Contact between the patch and skin is maintained with a suitable semiocclusive dressing during the exposure period (an occlusive dressing may be used in some cases). Untreated skin of the animal is taken as the control. (5) The exposure period is normally 4 hours. Test substance remaining at the end of the period of application is removed with water or a suitable vehicle. 5. Points to note regarding administration (1) If severe potential irritation/corrosion is suspected (1) If the test substance is suspected of being severely irritating or corrosive, a test is conducted in one animal. 1) If the test substance is suspected of being corrosive, three test patches are applied simultaneously to one animal. The first patch is removed after three min. If no strong skin reaction is observed, the second patch is removed after one hour. If it is judged in terms of the humane treatment of the animal at this stage that the exposure can be extended to 4 h, the third patch is removed after 4 h and the reactions are graded. If a strong irritant reaction is observed after 3-min or 1-h exposure, the remaining patches are removed and the test stopped immediately. The three patches may also be applied successively to different sites on the same animal for examination. 2) If severe irritation is suspected with the test substance, one patch is applied to one animal for 4 h. 3) If no severe irritation or corrosion is observed after a 4-h exposure, two more animals are tested with one patch each for 4 h. (2) If it is anticipated that severe irritation/corrosion will not occur with the test substance, the test is started using three animals and one patch is applied to each for 4 h. 6. General condition and scoring The animals are examined and scored for signs of erythema and edema 30 or 60 minutes, and 24, 48, and 72 h after removal of the patch. Skin irritation is scored and recorded in accordance with the evaluation scores given in the appendix. If necessary, subsequent examinations are given to demonstrate reversibility. There is generally no need to go beyond 14 days. In addition to examining for irritation, any serious injury or other toxic actions are recorded thoroughly. European-OECD Introduction 1. OECD Guidelines for Testing of Chemicals are periodically reviewed in light of scientific progress. In the review, special attention is given to possible improvements in relation to animal welfare. This updated version of the original guideline 404 (adopted in 1981) is the outcome of a meeting of OECD experts held in Paris in May 1991. 2. The main differences between this and the original version of the guidelines are (a) the inclusion of data from in vitro tests in the information on which a decision not to proceed to an in vivo test can be based; and (b) the possibility to use one animal in a first step of the in vivo procedure allowing certain chemicals to be exempted from further testing. 3. Definitions used are set out in the Annex. Initial Considerations 4. In the interests of animal welfare, it is important that the unnecessary use of animals is avoided, and that any testing which is likely to produce severe responses in animals is minimized. Consequently, test substances meeting any of the following criteria should not be tested in animals for dermal irritation/corrosion: (i) Materials that have predictable corrosive potential based on structure-activity relationships and/or physicochemical properties such as strong acidity or alkalinity, e.g., when the material to be applied has a pH of 2 or less or 11.5 or greater (alkaline or acidic reserve (1) should also be taken into account); (ii) materials which have been shown to be highly toxic by the dermal route; (iii) materials which, in an acute dermal toxicity test (2), have been shown not to produce irritation of the skin at the limit test dose level of 2000 mg/kg body weight. In addition, it may not be necessary to test in vivo materials for which corrosive properties are predicted on the basis of results from in vitro tests (3).


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines Principle of the In Vivo Test 5. The substance to be tested is applied in a single dose to the skin of one or more experimental animals, untreated skin areas of the test animal(s) serving as control. The degree of irritation is read and scored at specified intervals and is further described in order to provide a complete evaluation of the effects. The duration of the study should be sufficient to evaluate fully the reversibility of the effects observed. Animals showing severe distress and/or pain at any stage of the test must be humanely killed. Description of the In Vivo Method Selection of Animal Species 6. Several mammalian species may be used. The albino rabbit is the preferred species. Number and Sex of Animals 7. Three healthy adult animals are required for the complete test. Male and/or female animals can be used. Additional animals may be used to clarify equivocal responses. Sometimes the test can be performed with one animal only. Housing and Feeding Conditions 8. Animals should be individually housed. The temperature of the experimental animal room should be 20ºC (± 3ºC) for rabbits, 22ºC (± 3ºC) for rodents and the relative humidity 30–70%. Where the lighting is artificial, the sequence should be 12 h light, 12 h dark. Conventional laboratory diets are suitable for feeding and an unrestricted supply of drinking water should be available. Preparation of the Animals 9. Approximately 24 h before the test, fur should be removed by close-clipping the dorsal area of the trunk of the animals. Care should be taken to avoid abrading the skin and only animals with healthy intact skin should be used. 10. Some strains of rabbit have dense patches of hair which are more prominent at certain times of the year. Such areas of dense hair growth should not be used as patch sites. Procedure Application of the Test Substance 11. The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In the case of liquids or some pastes, it may be necessary to apply the test substance to the gauze patch and then apply that to the skin. The patch should be loosely held in contact with the skin by means of a suitable semi-occlusive dressing for the duration of the exposure period. Access by the animal to the patch and resultant ingestion/inhalation of the test substance should be prevented. 12. Liquid test substances are generally used undiluted. When testing solids (which may be pulverized if considered necessary), the test substance should be moistened with the smallest amount of water, or where necessary a suitable vehicle, needed to ensure good contact with the skin. When vehicles are used, the influence of the vehicle on irritation of the skin by the test substance should be taken into account. 13. At the end of the exposure period, normally 4 h, residual test substance should be removed, where practicable, using water or an appropriate solvent without altering the existing response or the integrity of the epidermis. Dose Level 14. A dose of 0.5 ml of liquid or 0.5 g of solid or semi-solid is applied to the test site. Exposure of One Animal 15. If it is suspected that the test substance might produce severe irritancy/corrosion, a single animal test should be employed. When it is suspected that the substance may cause corrosion, three test patches are applied simultaneously to the animal. The first patch is removed after three minutes. If no serious skin reaction is observed, the second patch is removed after one hour. If the observations at this stage indicate that the exposure can humanely be allowed to extend to four hours, the third patch is removed after four hours and the responses are graded. If a corrosive effect is observed after either three minutes or one hour exposure, the test is immediately terminated by removal of the remaining patches. Alternatively, three patches may be applied sequentially. When it is suspected that the substance may cause severe irritancy, a single patch should be applied to the animals for four hours.


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines Exposure of a Further Two Animals 16. If neither a corrosive effect nor a severe irritant effect is observed after a four hour exposure, the test should be completed using two additional animals, each with one patch only, for an exposure period of four hours. Exposure of Three Animals 17. If it is expected that the test substance will not produce severe irritancy or corrosion, the test may be started using three animals, each receiving one patch for an exposure period of four hours. Observation Period 18. The duration of the observation period should not be fixed rigidly but should be sufficient to evaluate fully the reversibility of the effects observed. Clinical Observations and Grading of Skin Reactions 19. Animals should be examined for signs of erythema and oedema and the responses scored at 60 min, and then at 24, 48, and 72 h after patch removal. Dermal irritation is scored and recorded according to the grades in the table below. Further observations may be needed to establish reversibility. In addition to the observation of irritation, all lesions and other toxic effects should be recorded and fully described. Data and Reporting Data 20. Data should be summarized in tabular form, showing for each individual animal the irritation scores for erythema and oedema at 60 minutes, 24, 48, and 72 hours after patch removal, all lesions, a description of the degree and nature of irritation, corrosion or reversibility, and any other toxic effects observed. Test Report 21. The test report must include the following information: Test substance: • physical nature and, where relevant, physicochemical properties; • identification data. Vehicle: • justification for choice of vehicle. Test animals: • species/strain used; • number, age and sex of animals; • source, housing conditions, diet, etc.; • individual weights of animals at the start and at the conclusion of the test. Test conditions: • technique of patch site preparation; • details of patch materials used and patching technique; • details of test substance preparation, application and removal. Results: • tabulation of irritation response data for each individual animal for each observation time period (e.g., 60 min, 24, 48, and 72 h after patch removal); • description of all lesions observed; • narrative description of the degree and nature of irritation observed, and any histopathological findings; • description of any other toxic effects in addition to dermal irritation/corrosion. Discussion of the results: If an in vitro test is performed before the in vivo test, the description or reference of the test, including details of the procedure, must be given together with results obtained with the test and reference substances.


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines European-EEC 1.4 Principle of the Test Method Initial Considerations Careful consideration needs to be given to all the available information on a substance to minimize the testing of substances under conditions that are likely to produce severe reactions. The following information may be useful when considering whether a complete test, a single-animal study, or no further testing is appropriate. i) Physiochemical properties and chemical reactivity. Strongly acidic or alkaline substances (demonstrated pH of 2 or less or 11.5 or greater, for example) may not require testing for primary dermal irritation if corrosive properties can be expected. Alkaline or acidic reserve should also be taken into account. ii) If convincing evidence of severe effects in well validated in vitro tests is available, a complete test may not be required. iii) Results from acute toxicity studies. If an acute toxicity test by the dermal route has been conducted with the substance at the limit test dose level (2,000 mg/kg body weight), and no skin irritation was observed, further testing for skin irritation may be unnecessary. In addition, testing of materials which have been shown to be highly toxic by the dermal route is unnecessary. The substance to be tested is applied in a single dose to the skin of several experimental animals, each animal serving as its own control. The degree of irritation is read and graded after a specific interval, and is further described to provide a complete evaluation of the effects. The duration of the observations should be sufficient to evaluate fully the reversibility of the effects observed. Animals showing severe and enduring signs of distress and pain may need to be humanely killed. 1.6 Description of the Test Method 1.6.1. Preparations Approximately 24 hours before testing, fur should be removed, by clipping or shaving, from the dorsal area of the trunk of the animal. When clipping or shaving the fur, care should be taken to avoid abrading the skin. Only animals with healthy intact skin should be used. Some strains of rabbit have dense islets of hair which are more prominent at certain times of the year. Test substances should not be applied to these zones of dense hair growth. When testing solids (which may be pulverized if considered necessary) the test substance should be moistened sufficiently with water or, where necessary, a suitable vehicle, to ensure good contact with the skin. When vehicles are used, the influence of the vehicle on irritation of skin by the test substance should be taken into account. Liquid test substances are generally used undiluted. 1.6.2. Test Conditions 1.6.2.1. Experimental Animals Although several mammalian species may be used, the albino rabbit is the preferred species. 1.6.2.2. Number of Animals If it is suspected from in vitro screening results or other considerations that the substance might produce necrosis (i.e., be corrosive) a single-animal test should be considered. If the results of this test do not indicate corrosivity, the test should be completed using at least two additional animals. For the complete test, at least three healthy adult animals are used. Separate animals are not required for an untreated control group. Additional animals may be required to clarify equivocal responses. 1.6.2.3. Dose Level Unless there are contra-indications 0.5 ml of liquid or 0.5 g of solid or semi-solid is applied to the test site. Adjacent areas of untreated skin of each animal serve as controls for the test. 1.6.2.4. Observation Period The duration of the observation period should not be fixed rigidly. It should be sufficient to evaluate fully the reversibility or irreversibility of the effects observed, but need not normally exceed 14 days after application.


TABLE 3.6 (Continued) Comparison of Excerpts from Selected Dermal Irritation Test Guidelines 1.6.3. Procedure Animals should be caged individually. The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In the case of liquids or some pastes it may be necessary to apply the test substance to the gauze patch and then apply that to the skin. The patch should be loosely held in contact with the skin by means of a suitable occlusive or semi-occlusive dressing for the duration of the exposure period. Access by the animal to the patch and resultant ingestion/inhalation of the test substance should be prevented. At the end of the exposure period, residual test substance should be removed, where practicable, using water or an appropriate solvent, without altering the existing response or the integrity of the epidermis. Exposure duration normally is four hours. If it is suspected that the substance might produce necrosis (i.e., be corrosive), the duration of exposure should be reduced (e.g., to one hour or three minutes). Such testing may also employ a single animal in the first instance and, if not precluded by the acute dermal toxicity of the test compound, three patches may be applied simultaneously to this animal. The first patch is removed after three minutes. If no serious skin reaction is observed, the second patch is removed after one hour. If the observations at this stage indicate that a four-hour exposure is necessary and can be humanely conducted, the third patch is removed after four hours and the responses are graded. In this case (i.e. when a four-hour exposure has been possible), the test should then be completed using at least two additional animals, unless it is not considered humane to do so (e.g., if necrosis is observed following the four hour exposure). If a serious skin reaction (e.g., necrosis) is observed at either three minutes or one hour, the test is immediately terminated. Longer exposures may be indicated under certain conditions, e.g., expected pattern of human use and exposure. 1.6.3.1. Observation and Grading Animals should be observed for signs of erythema and oedema and the response graded at 60 minutes, and then at 24, 48, and 72 hours after patch removal. Dermal irritation is graded and recorded according to the system in Table 3.8. Further observations may be needed if reversibility has not been fully established within 72 hours. In addition to the observation of irritation, any serious lesions such as corrosion (irreversible destruction of skin tissue) and other toxic effects should be fully described. Techniques such as histopathological examination or measurement of skin-fold thickness may be used to clarify doubtful reactions or responses masked by staining of the skin by test substance. 2. Data Data should be summarized in tabular form, showing for each individual animal the irritation gradings for erythema and oedema throughout the observation period. Any serious lesions, a description of the degree and nature of irritation, reversibility or corrosion and any other toxic effect observed should be recorded. 3. Reporting 3.1 Test Report The test report shall, if possible, include the following information: • species, strain, source, environmental conditions, diet, etc.; • test conditions (including the relevant physicochemical properties of the chemical, the technique of skin preparation and cleansing, and the type of dressing: occlusive or semi-occlusive); • tabulation of irritation response data for each individual animal for each observation time period (e.g., 1, 24, 48, and 72 hours, etc., after patch removal); • description of any serious lesions observed, including corrosivity; • description of the degree and nature of irritation observed and any histopathological findings; • description of any toxic effects other than dermal irritation, • discussion of the results; • interpretation of the results.


TABLE 3.7 Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 EPA OPPTS 870.2600 This guideline is one of a series of test guidelines that have been developed by the Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency for use in the testing of pesticides and toxic substances, and the development of test data that must be submitted to the agency for review under Federal regulations. The Office of Prevention, Pesticides and Toxic Substances (OPPTS) has developed this guideline through a process of harmonization that blended the testing guidance and requirements that existed in the Office of Pollution Prevention and Toxics (OPPT) and appeared in Title 40, Chapter I, Subchapter R of the code of Federal Regulations (CFR), the Office of Pesticide Programs (OPP) which appeared in publications of the National Technical Information Service (NTIS), and the guidelines published by the Organization for Economic Cooperation and Development (OECD). The purpose of harmonizing these guidelines into a single set of OPPTS guidelines is to minimize variations among the testing procedures that must be performed to meet the data requirements of the U.S. Environmental Protection Agency under the Toxic Substance Control Act (15 U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 136, et seq.). (a) Scope. (1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 USC 136, et seq.) and the Toxic Substances Control Act (TSCA) (15 USC 2601). (2) Background. The source materials used in developing this harmonized OPPTS test guideline are the OPPT 40 CFR 798.4100 Dermal Sensitization; OPP 81-6 Dermal Sensitization (Pesticide Assessment Guidelines, Subdivision F-Hazard Evaluation; Human and Domestic Animals); EPA report 540/09-82-025, 1982; and OECD 406 Skin Sensitization. (b) Purpose. In the assessment and evaluation of the toxic characteristics of the substance, determination of its potential to provoke skin sensitization reactions is important. Information derived from test for skin sensitization serves to identify the possible hazard to a population repeatedly exposed to the test substance. While the desirability of skin sensitization testing is recognized, there are some real differences of opinion about the best method to use. The test selected should be a reliable screening procedure which should not fail to identify substances with significant allergenic potential, while at the same time avoiding false negative results. (c) Definitions. (1) “Challenge exposure” is an experimental exposure of a previously treated subject to a test substance following an induction period, to determine if the subject will react in a hypersensitive manner. (2) “Induction exposure” is an experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state. (3) “Induction period” is a period of at least 1 week following an induction exposure during which a hypersensitive state is developed. (4) “Skin sensitization” (“allergic contact dermatitis”) is an immunologically mediated cutaneous reaction to a substance. In the human, the responses may be characterized by pruritis, erythema, edema, papules, vesicles, bullae, or a combination of these. In other species, the reactions may differ and only erythema and edema may be seen. (d) Principle of the test method. Following initial exposure(s) to a test substance, the animals are subsequently subjected, after a period of not less that 1 week, to a challenge exposure with the test substance to establish whether a hypersensitive state has been induced. Sensitization is determined by examining the reaction to the challenge exposure and comparing this reaction to that of the initial induction exposure. The test animals are initially exposed to the test substance by intradermal and/or epidermal application (induction exposure). Following a rest period of 10 to 14 days (the induction periods), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure is compared with that demonstrated by control animals that undergo sham treatment during induction and then receive the challenge exposure. (e) Test procedures. (1) Any of the following seven test methods is considered to be acceptable. It is realized, however, that the methods differ in their probability and degree of reaction to sensitizing substances. (i) Buehler test; (ii) Guinea pig maximization test; (iii) Open epicutaneous; (iv) Mauer optimization test; (v) Split adjuvant technique; (vi) Freund’s complete adjuvant test; (vii) Draize sensitization test;


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 (2) The GPMT of Magnusson and Kligman, which uses adjuvant, and nonadjuvant Buehler test are given preference over other methods. Although strong preference is given to either the Buehler test or the GPMT, it is recognized that other tests may give useful results. If other tests are used, the tester should provide justification/reasoning for their use, methods and protocols must be provided, and each test should include a positive and a negative control group. (f) Screening tests. The mouse ear swelling test (MEST) or the local (auricular) lymph node assay (LLNA) in the mouse may be used as screening tests to detect moderate to strong sensitizers. If a positive result is seen in either assay, the test substance may be designated a potential sensitizer, and it may not be necessary to conduct a further test in guinea pigs. If the LLNA or MEST does not indicate sensitization, the test substance should not be designated a nonsensitizer without confirmation in an accepted test with guinea pigs. (g) Animal selection. (1) Species and strain. The young adult guinea pig is the preferred species. Commonly used laboratory strains should be employed. If other species are used, the tester should provide justification/reasoning for their selection. (2) Housing and feeding. The temperature of the experimental animal room should be 20±3°C with the relative humidity 30–70%. Where the lighting is artificial, the sequence should be 12 h light/12 h dark. Conventional laboratory diets may be used with an unlimited supply of drinking water. It is essential that guinea pigs receive an adequate amount of ascorbic acid. (3) Number and sex. The number and sex will depend on the method used. If females are used, they should be nulliparous and not pregnant. (4) Control animals. (i) The sensitivity and reliability of the experimental technique used should be assessed every 6 months in naive animals by the use of positive control substance known to have mild-to-moderate skin-sensitizing properties. In a properly conducted test, a response of at least 30% in an adjuvant test and at least 15% in a nonadjuvant test should be expected for mild-to-moderate sensitizes. Preferred substances are hexylcinnamicaldhyde (CAS No. 101-86-0), mercaptobenzothiazole (CAS No. 149-30-4), benzocaine (CAS No. 94-09-7), dinitro-chloro-benzene (CAS No. 97-00-7), or DER 331 epoxy resin. There may be circumstances where, given adequate justification, other control substances meeting the above criteria may be used. (ii) Depending upon the test selected, animals may be used as their own controls, but usually there will be a separate group of sham-treated animals that are exposed to the test substance only after the induction period, whose reactions are compared to those of the animals that have received both induction and challenge exposures. Control groups which provide the best design should be used. Some cases may best be served by both naive and vehicle control groups. (5) (i) The dose level will depend upon the method selected. (6) (i) Skin reactions should be graded and recorded after the challenge exposures at the time specified by the methodology selected. This is usually at 24 and 48 h. Additional notations should be made as necessary to fully describe unusual responses. (ii) Regardless of method selected, initial and terminal body weights are to be recorded. (7) Procedures. (i) The procedures to be used are those described by the methodology chosen. (h) Data and reporting. Data should be summarized in tabular form, showing for each individual animal the skin reaction, results of the induction exposure, and the challenge exposure at times indicated by the method chosen. As a minimum, the erythema and edema should be graded and any unusual findings should be recorded. (1) Evaluation of the results. The evaluation of results will provide information on the proportion of each group that became sensitized and the extent (slight, moderate, severe) of the sensitization reaction in each individual animal. (2) Test report. In addition to the information required by 40 CFR part 158 (for pesticides) and 40 CFR part 792 subpart J (for toxic substances), the test report shall include the following information: (i) A description of the methods used and the commonly accepted name; (ii) Information on positive control study, including: (A) Positive control used; (B) Method used; and (C) Time conducted. (iii) The number, species, strain, age, source, and sex of the test animals; (iv) Individual weights of the animals at the start of the test and at the conclusion of the test; (v) A brief description of the grading system; and (vi) Each reading made on each individual animal. (vii) The chemical identification and relevant physicochemical properties of the test substance.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 (viii) The vehicles used for induction and challenge, and justification for their use, if other than water or physiological saline. Any material that might reasonably be expected to react with or enhance or retard absorption of the test substance should be reported. (ix) The total amount of test substance applied for induction and challenge, and the technique of application in each case. (x) Description of any pretest conditioning, including diet, quarantine, and treatment of disease. (xi) Description of caging conditions including number (and any change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of test animal. (xii) Histopathological findings, if any. (xiii) Discussion of results. (xiv) Manufacturer, source, purity, and lot number of test substance. (xv) Physical nature and, where appropriate, concentration and pH value for the test substance. (xvi) A list of references cited in the body of the report, i.e., references to any published literature used in developing the test protocol, performing the testing, making and interpreting observations, and compiling and evaluating the results. Japanese-MHW 2. Selection of Test Methods These test methods are given as examples because they have been adopted in most laboratories, and because that they all represent well established assay techniques with a high degree of reproductibility. Generally, original reports pertaining to the individual test methods are cited herein, although some of these tests are in use with modifications. Testing procedure need not be limited to those cited herein, and in cases where any other test method is employed, justification of its application should be stated along with citation of the appropriate literature. (a) Adjuvant and patch test The test comprises intradermal injections of FCA and abrasion of the skin, topical application of the test substance onto the scratched region, and covering of the test site with an occlusive patch for sensitization. The topical challenge is made without a covering. The test is used for such test substances which are not injectable intradermally. (b) Buehler test This test also employs topical application of the test substance. The test site is covered with an occlusive patch and a wrapping and the topical challenge is carried out with a wrap, as during induction, to enhance penetration and prevent evaporation of the test substance. (c) Draize test The test method was the first predictive sensitization assay accepted by regulatory agencies. It is characterized by the intradermal introduction of a dilution of the test substance for sensitization, and a challenge by subsequent intradermal injection. (d) Freund’s complete adjuvant test The test comprises intradermal injections of the test substance incorporated in a 1:1 mixture of FCA and distilled water. (e) Maximization test The maximization test, as described in the Guidelines, combines FCA, sodium lauryl sulfate, intradermal injection and occlusive topical application of the test substance during the sensitization period. (f) Open epicutaneous test This test closely simulates the conditions of drug use in humans by utilizing repeated topical application of the test substance. (g) Optimization test The optimization test is analogous to Draize test but involves the use of FCA for sensitization and an intradermal challenge with covering of the test site. (h) Split adjuvant test The test utilizes skin damage caused by the application of dry ice onto a shaved area of skin, and FCA as an adjuvant. The test substance is applied topically with a dressing.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 Whichever test method is selected, it is impracticable to accomplish a perfect prediction of the sensitizing potential of a substance in humans based solely on results of the test, but the test results may provide important information valid for the extrapolation of data to the conditions of human use. In the current Guidelines, the spirit of concern for research animals is incorporated. Test methods, for example, are roughly divided into two groups: those involving the use of an adjuvant and those which do not, so as to systematically minimize the types of tests to be adopted. Thus, scientific considerations have been made for the reduction of the number of animals used in tests. They are, in brief, 1) reduction of the number of animals used in tests, 2) limitation of types of testing methods and delineation of test methods that may be selected, 3) classification of the test methods according to assay sensitivity, and so forth. 3. Selection of Test Animals Primarily, animals highly susceptible to the sensitizing action of the test substance are to be selected as a test system. In all the test methods mentioned above, regardless, the animal species used is the guinea pig. Young, healthy adult albino guinea pigs (usually between 1 and 3 months of age) weighing not more than 500 g at the start of the test are used as a rule. They may be male or female, or of both sexes, and, in the case of females, the animals should be nonpregnant and nulliparous. This animal species is selected primarily for the reasons that guinea pigs are known to elicit, if at all, reactions similar to those that occur in man and that a substantial amount of background laboratory data has been accumulated for this species. 4. Number of Animals An extreme reduction in the number of animals used in tests may render statistical data analysis meaningless. Only the minimum number of animals required for test groups (groups subject to sensitization with the test substance) and control groups (positive control and control groups) are stated in these Guidelines. If any influence of the minimized number of animals on test results is anticipated, the number should be increased appropriately. The above stated minimum number of animals (5/group) may suffice only for such circumstances where the response is either obviously negative or strongly positive. It follows that, otherwise, each test group need consist of at least 10 animals and each control group of at least 5 animals. 5. Positive Controls Positive controls are required as references for comparative assessments of the responsiveness of animals used and of the sensitizing potency of the drug substance being tested. Compounds currently in use for this purpose include: p-phenylenediamine (CAS No. 106-50-3), 1-chloro-2,4-dinitrobenzene (CAS No. 97-00-7), neomycin sulfate (CAS No. 1405-10-3) and nickel sulfate (CAS No. 7786-81-4), but any other suitable sensitizers documented in the biomedical literature may also be used. 6. Test Methods Detailed accounts are given of the maximization test (Magnusson and Kligman) and the adjuvant and patch test, under Description of the Test Procedure in the Guidelines, while the other six test methods are only cited. All the test methods mentioned in the Guidelines may be regarded as essentially equivalent and none given a preference to others. That is, any of the test methods mentioned above may be adopted. The maximization test and adjuvant and patch test are selected as examples to be detailed on the rationale that those involving the use of Freund’s complete adjuvant are likely to be superior in assay sensitivity to those not using it. It is most desirable to conduct the testing stepwise in evaluating a substance for skin sensitizing potential. In the first step, one of the five tests incorporating the use of the adjuvant is to be performed to ascertain if the property of the test substance is to be further assessed by comparison with a known sensitizing substance or by conducting a test not involving the use of an adjuvant so as to permit evaluation of the intensity of sensitization by the test substance. All these tests are designed to determine the potential of test substances to induce hypersensitivity by, in general, exposure (sensitization) of experimental animals to the test substance and a challenge exposure (elicitation) after a subsequent rest period of about 2 weeks. Test results are for sensitizing potential interpreted by comparing cutaneous responses of experimental animals with those of controls. Each of the test methods described has advantages and drawbacks and, therefore, it is most desirable that the tests by performed properly by personnel well versed in these aspects.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 In preparing animals for tests, they should be randomly allocated to experimental groups. Test sites on the skin must be clipped free of hair or shaven prior to administration of the test substance. Study conditions must be carefully set up since test results may vary with the conditions. 7. Dose Levels In case where graded dose levels appropriate to the assay are employed, the physicochemical properties of the test substance, such as the solubility at test concentrations and the tolerated local or systemic dose need be taken into account. The concentrations of the test substance for sensitizing and challenge chosen for the assay may be justified whenever deemed necessary. 8. Observation Parameters Body weights of animals must be recorded at least at the initiation and at the completion of testing. All animals should be observed for any signs of skin irritation during the sensitization period. Skin reactions are to be noted at 24, 48, or 72 hours after challenge exposure, and interpreted for sensitizing activity of the test substance. The reactions must be rated as specified for each test. All observed cutaneous reactions and any adverse findings noted must be recorded. 9. Reporting of Test Results It is advisable that test results be summarized by tabulation or other means in such a way that the skin reactions of individual animals at respective periods of observation can be clearly recognized. In reporting the test results, data concerning the following parameters must be included: 1) Strain of guinea pigs used. 2) Number, age in weeks, and sex(es) of animals used. 3) Individual animal body weights at the start and at the completion of test. 4) All reactions observed in animals, along with details of reactions if any scoring system or classification scheme is employed. 5) Evaluation of test results, and comments. 10. Evaluation of Test Results The skin sensitizing potential of the test substance should be evaluated according to the reactions observed in animals in the test group and in each control group. Interpretation of the test results must be based on evaluation of the potential of the test substance to sensitize the skin. Basically, it is to be made according to the evaluation criteria specified in the literature reporting the test method. In cases where the incidence of a positive skin reaction is to be assessed, it is advisable that increased numbers of animals be used in test and control groups, and that data obtained be processed by an appropriate statistical procedure. What should be noted here is that the tests mentioned herein, unfortunately, are not necessarily adequate as assays for predicting the sensitizing potential of the test substance in humans. To evaluate the sensitizing activity of the material, therefore, the material is first to be subjected to any one of the test methods involving the use of an adjuvant and determined thereby as to whether it has sensitizing activity or not. If the material has proven to be positive, then it should be further assessed, preferably by a test method not involving the use of an adjuvant in order to make practical risk assessment and classification of the test substance. Japanese-MAFF (Current) 1. Purpose The purpose of this study is to obtain the data which will make the basis to establish safe handling procedures in use. 2. Test Substance The end-use product should be used. However, strongly acidic or alkaline substance (approximately pH 2 or pH 11.5 or greater) might not be tested. 3. Test Animals At least one mammalian species should be used. The young adult guinea pig is the preferred species.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 4. Test Methods Any of the following seven test methods is considered to be acceptable. Use of a positive control substance for the reliability for the test system selected is recommended. Draize Test Freund’s Complete Adjuvant Test Mauer Optimisation Test Buehler Test Open Epicutaneous Test Guinea Pig Maximization Test Split Adjuvant Technique Japanese-MAFF (draft) 1. Objective This test seeks to provide information on potential skin sensitization forming a basis for establishing the safe method of handling the agrochemical during use. 2. Test substances Basic compound and preparation 3. Test animal species, age, and sex Young adult guinea pigs are used. Females are nulliparous, nonpregnant animals. 4. Test methods The test methods which are undertaken with a relatively high frequency are the guinea pig maximization test (GPM method) and the Buehler test (Buehler method). However, if information about sensitization is available, the test method therein may be substituted. A positive control group is also provided in order to evaluate the responsivity of the test system. 5. Test procedures Both the GPM method and the Buehler method are described in detail in the guidelines. (1) Number of animals. This is dependent on the method used. (2) Dose settings. The maximum concentration of the test substance used in exposure for sensitization is one to which there is satisfactory resistance systemically but producing mild to moderate skin irritation. The maximum concentration of test substance used for challenge exposure is the highest at which no irritation is shown. Two or three animals are used to determine the appropriate concentration of the test substance. (3) Sensitization. Procedures are dependent on the method used. (6) Examination. About 21 h after removing the patches, the challenged area is shaved if necessary. Three hours later (about 48 h after the start of application of the challenging patches), the skin is examined for any reaction which is then recorded in accordance with the grades shown in the table. Skin reactions are observed and recorded again 24 h after the first examination. Table: Evaluation criteria for challenge patch test reaction No visible change Diffuse or patchy erythema Moderate and chronic erythema Marked erythema and oedema

0 1 2 3

(8) Examination of general condition. All skin reactions and all abnormal findings which occur as a result of sensitization and challenge are recorded. European-OECD Introduction 2. Currently, quantitative structure–activity relationships and in vitro models are not yet sufficiently developed to play a significant role in the assessment of the skin-sensitization potential of substances which therefore must continue to be based on in vitro models.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 3. The guinea pig has been the animal of choice for predictive sensitization tests for several decades. Two types of tests have been developed: adjuvant tests in which sensitization is potentiated by injection of Freund’s complete adjuvant (FCA), and nonadjuvant tests. In the original guideline 406, four adjuvant test and three nonadjuvant tests were considered to be acceptable. In this updated version, the guinea pig maximization test (GPMT) of Magnusson and Kligman which uses adjuvant and the nonadjuvant Buehler test are given preference over other methods and the procedures are presented in detail. It is recognized, however, that there may be circumstances where other methods may be used to provide the necessary information on sensitization potential. 4. The immune system of the mouse has been investigated more extensively than that of the guinea pig. Recently, mouse models for assessing sensitization potential have been developed that offer the advantages of having an end point which is measured objectively, being of short duration, and treating a minimal number of animals. The mouse ear swelling test (MEST) and the local lymph node assay (LLNA) appear to be promising. Both assays have undergone validation in several laboratories and it has been shown that they are able to detect reliably moderate to strong sensitizes. The LLNA or the MEST can be used as a first stage in the assessment of skin sensitization potential. If a positive result is seen in either assay, a test substance may be designated as a potential sensitizer, and it may not be necessary to conduct a further guinea pig test. However, if a negative result is seen in the LLNA or MEST, a guinea pig test (preferably a GPMT or Buehler test) must be conducted using the procedure described in this guideline. 5. Definitions: “Skin sensitization” (allergic contact dermatitis): An immunologically mediated cutaneous reaction to a substance. In the human, pruritis, erythema, edema, papules, vesicles, bullae, or a combination of these may characterize the responses. In other species the reactions may differ and only erythema and edema may be seen. “Induction exposure” An experimental exposure of a subject to a test substance with the intention of inducing a hypersensitive state. “Induction period” A period of at least 1 week following an induction exposure during which a hypersensitive state may develop. “Challenge exposure” An experimental exposure of a previously treated subject to a test substance following an induction period, to determine if the subject reacts in a hypersensitive manner. General Principle of Sensitization Tests in Guinea Pigs 6. The test animals are initially exposed to the test substance by intradermal injection and/or epidermal application (induction exposure). Following a rest period of 10 to 14 days (induction period), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure in the test animals is compared with that demonstrated by control animals which undergo sham treatment during induction and receive the challenge exposure. Elements Common to Sensitization Tests in Guinea Pigs Sex of Animals 7. Male and/or female healthy young adult animals can be used. If females are used they should be nulliparous and nonpregnant. Housing and Feeding Conditions 8. The temperature of the experimental animal room should be 20ºC (± 3ºC) and the relative humidity 30–70%. Where the lighting is artificial, the sequence should be 12 h light, 12 h dark. For feeding, conventional laboratory diets may be used with an unlimited supply of drinking water. It is essential that guinea pigs receive an adequate amount of ascorbic acid. Preparation of the Animals 9. Animals are acclimatized to the laboratory conditions for at least 5 days prior to the test. Before the test, animals are randomized and assigned to the treatment groups. Removal of hair is by clipping, shaving or possibly by chemical depilation, depending on the test method used. Care should be taken to avoid abrading the skin. The animals are weighed before the test commences and at the end of the test. Reliability Check 10. The sensitivity and reliability of the experimental technique used should be assessed every six months by use of substances which are known to have mild-to-moderate skin sensitization properties.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 11. In a properly conducted test, a response of at least 30% in an adjuvant test and at least 15% in a non-adjuvant test should be expected for mild/moderate sensitisers. Preferred substances are hexyl cinnamic aldehyde (CAS No. 101-86-0), mercaptobenzothiazole (CAS No. 149-30-4) and benzocaine (CAS No. 94-09-7). There may be circumstances where, given adequate justification, other control substances meeting the above criteria may be used. Removal of the Test Substance 12. If removal of the test substance is considered necessary, this should be achieved using water or an appropriate solvent without altering the existing response or the integrity of the epidermis. Description of the Guinea Pig Methods 13.-36. Both the guinea pig maximization test and the Buehler test methods are described in the guideline. Data and Reporting Data 37. Data should be summarized in tabular form, showing for each animal the skin reactions at each observation. Test Report 38. The test report must include the following information: Test substance: — physical nature and, where relevant, physicochemical properties — identification data Vehicle: — justification of choice of vehicle Test animals: — strain of guinea pig used — number, age, and sex of animals — source, housing conditions, diet, etc. — individual weights of animals at the start and at the conclusion of the test Test conditions — technique of patch site preparation — details of patch materials used and patching technique — result of pilot study with conclusion on induction and challenge concentrations to be used in the test — details of test substance preparation, application, and removal — vehicle and test substance concentrations used for induction and challenge exposures and the total amount of substance applied for induction and challenge. Reliability check: — a summary of the results of the latest reliability check including information on substance, concentration, and vehicle used. Results: — on each animal including grading system — narrative description of the nature and degree of effects observed — any histopathological findings Discussion of results — If a screening assay is performed before the guinea pig test, the description or reference of the test, including details of the procedure, must be given together with results obtained with the test and reference substances. European-EEC Method 1.1 Introduction Remarks: The sensitivity and ability of tests to detect potential human skin sensitizers are considered important in a classification system for toxicity relevant to public health. There is no single test method which will adequately identify all substances with a potential for sensitizing human skin and which is relevant for all substances.


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 Factors such as the physical characteristics of a substance, including its ability to penetrate the skin, must be considered in the selection of a test. Tests using guinea pigs can be subdivided into the adjuvant-type tests, in which an allergic state is potentiated by dissolving or suspending the test substance in Freunds Complete Adjuvant (FCA), and the non-adjuvant tests. Adjuvant-type tests are likely to be more accurate in predicting a probable skin sensitizing effect of a substance in humans than those methods not employing Freunds Complete Adjuvant and are thus the preferred methods. The Guinea Pig Maximization Test (GPMT) is a widely used adjuvant-type test. Although several other methods can be used to detect the potential of a substance to provoke skin sensitization reaction, the GPMT is considered to be the preferred adjuvant technique. With many chemical classes, non-adjuvant tests (the preferred one being the Buehler test) are considered to be less sensitive. In certain cases there may be good reasons for choosing the Buehler test involving topical application rather than the intradermal injection used in the Guinea Pig Maximization Test. Scientific justification should be given when the Buehler test is used. The Guinea Pig Maximization Test (GPMT) and the Buehler test are described in this method. Other methods may be used provided that they are well-validated and scientific justification is given. Regardless of the methods used, the sensitivity of the strain of guinea pig being used for skin sensitization testing must be checked at regular intervals (six months) using a known mild to moderate sensitizer and a satisfactory number of positive responses obtained. 1.3. Reference Substances The following substances, diluted as necessary, are recommended, as well as any other sensitizing substance known either from the literature or which belongs to the group of the substance being tested. • p-phenylenediamine CAS No. 106-50-3 • 2,4-dinitrochlorobenzene CAS No. 97-00-7 • potassium dichromate CAS No. 7778-50-9 • neomycin sulphate CAS No. 1405-10-3 • nickel sulphate CAS No. 7786-81-4 1.4. Principle of the Test Methods Following initial exposure to a test substance (the ‘induction’ period) the animals are subjected approximately two weeks after the last induction exposure to a ‘challenge’ exposure to the test substance in order to establish if a hypersensitive state has been induced. Sensitization is determined by examining the skin reaction to the challenge exposure. 1.5 Quality Criteria None. 1.6 Description of the Test Method The guinea pig maximization test (GPMT) and the Buehler test are described in the guideline. 2. Data (GPMT and Buehler test) Data should be summarized in tabular form, showing for each animal the skin reactions at each observation. 3. Reporting (GPMT and Buehler test) 3.1 Test Report (GPMT and Buehler test) The test report shall, if possible, include the following information: — strain of guinea pig used — test conditions, vehicle and test substance concentrations used for induction and challenges — number, age, and sex of animals


TABLE 3.7 (Continued) Comparison of Excerpts from Selected Sensitization Test Guidelines6–11 — individual weights of animals at the start and at the conclusion of the test — discussion of the results — interpretation of the results 3.2 Evaluation and Interpretation (GPMT and Buehler test) There are limitations in the extent to which the results of animal and in vitro tests can be extrapolated directly to humans and this must be borne in mind when tests are evaluated and interpreted. When available, evidence of adverse effects in humans may be of relevance in determining the potential effects of chemical substances on the human population.

SECTION 3. MATERIALS AND PROCEDURES FOR PERFORMING DERMAL IRRITATION STUDIES24–33 A. THE OCCLUDED DERMAL IRRITATION TEST 1.

IN

RABBITS

Materials

a. Gauze Dressing Type: Ace-Tex Corporation nonsterilized Thickness: 4-ply gauze dressing Size: 1 × 1 inch b. Tape — Nonirritating Type: Blenderm® . . . (Medical-Surgical Division/3M, St. Paul, MN) Size: 1 inch wide c. Occlusive Materials Type: Impervious material (e.g., plastic wrap) d. Binding Materials Elastic wrap Type: Rubber elastic bandage: Ace® Bandage or Coban® (Medical-Surgical Division/3M, St. Paul, MN), or Expandover® (Sherwood Medical, St. Louis, MO) Size: Adequate wrapping of the entire test site e. Securing Materials Type: Zonas® (Johnson & Johnson Medical Inc., Arlington, TX) porous athletic tape Size: 2 inches wide f.

Elizabethan or similar Collars: Optional.

g. Animal Species New Zealand white rabbits. 2.

Procedures

The hair is removed from a sufficient area on the rabbit’s back on the day before dosing. Care should be taken to avoid abrading the skin during the clipping procedure. On the day of dosing, the test site (approximately 1 × 1 inch square of intact skin) should be designated and the gauze patch (1 × 1 inch) should be secured to the animal on at least two cut edges of the gauze patch, using the nonirritating Blenderm tape. The test substance, either 0.5 ml or 0.5 g, should be administered under the gauze dressing and the remaining cut edges secured to the animal’s back with nonirritating tape. Copyright © 2002 by Taylor & Francis

Liquids are administered as received, powders should be moistened with a suitable vehicle before application (e.g., distilled water). If the test article is a solid or powder that does not work well as a paste (e.g., does not spread well), 0.5 g of the test article will be applied to an approximate 1 × 1 in. 4-ply gauze patch and will be moistened with the appropriate amount of distilled water or suitable vehicle (generally 0.5 ml) and the gauze patch applied to the test site. An impervious sheet of material (e.g., plastic wrap) is then wrapped around the trunk of the animal. The elastic wrap is then wrapped around the animal’s torso and is secured in place using the Zonas athletic tape. The Zonas is wrapped around the outermost portion of the elastic wrap at the cranial and caudal ends. An Elizabethan or similar collar may then be placed around the animal’s neck. After the designated time of exposure (i.e., 4 or 24 hours), the tape, elastic wrap, impervious wrap, and gauze patch are removed and the test site is delineated using an indelible marker. The test site should then be rinsed with a suitable vehicle (e.g., distilled water). At the appropriate grading intervals (e.g., 1, 24, 48, and 72 hours after patch removal), the animals should be examined and scored for signs of erythema and edema according to the Draize dermal grading system. Grading of the test sites may be continued after the 72-hour scoring interval if irritation persists (e.g., day 7, day 10, day 14).

B. THE SEMIOCCLUDED DERMAL IRRITATION TEST 1.

IN

RABBITS

Materials

a. Gauze Dressing Type: Ace-Tex Corporation nonsterilized Thickness: 4-ply gauze dressing Size: 1 × 1 inch b. Tape — Nonirritating Type: Blenderm® Size: 1 inch wide c. Binding Materials Elastic wrap Type: Rubber elastic bandage : Ace® Bandage, Coban®, or Expandovert® Size: Adequate wrapping of the entire test site d. Securing-Materials Type: Zonas® porous athletic tape Size: 2 inches wide e.

Elizabethan or similar collars: Optional.

f. Animal Species New Zealand White Rabbit 2.

Procedures

The hair is removed from a sufficient area on the rabbit’s back on the day before dosing. Care should be taken to avoid abrading the skin during the clipping procedure. On the day of dosing, the test site (approximately 1 × 1 inch square of intact skin) should be designated and the gauze patch (1 × 1 inch) should be secured to the animal on at least two cut edges of the gauze patch using the nonirritating Blenderm tape. The test substance, either 0.5 ml or 0.5 g, should be administered under the gauze dressing and the remaining cut edges secured to the animal’s back with nonirritating tape. Liquids are administered as received. Powders should be moistened with a suitable vehicle prior to application (e.g., distilled water). If the test article is a solid or powder


that does not work well as a paste (e.g., does not spread well), 0.5 g of the test article will be applied to an approximate 1 × 1 inch 4-ply gauze patch and will be moistened with the appropriate amount of distilled water or suitable vehicle (generally 0.5 ml) and the gauze patch applied to the test site. The elastic wrap is then wrapped around the animal’s torso and secured in place using the Zonas athletic tape. The Zonas tape is wrapped around the outermost portion of the elastic wrap at the cranial and caudal ends. An Elizabethan or similar collar may then be placed around the animal’s neck. After the designated exposure time (i.e., 4 or 24 h), the tape, elastic wrap, and gauze patch are removed and the test site is delineated using an indelible marker. The test site should then be rinsed with a suitable vehicle (e.g., distilled water). At the appropriate grading intervals (e.g., 1, 24, 48, and 72 h after patch removal), the animals should be examined and scored for signs of erythema and edema according to the Draize dermal grading system. Grading of the test sites may be continued after the 72-h scoring interval if irritation persists (e.g., day 7, day 10, day 14).

C. THE NONOCCLUDED DERMAL IRRITATION TEST 1.

Materials

a.

Elizabethan or similar collars or restrainer

IN

RABBITS

b. Animal Species New Zealand White Rabbit 2.

Procedures

The hair is removed from a sufficient area on the rabbit’s back on the day before dosing. Care should be taken to avoid abrading the skin during the clipping procedure. On the day of dosing, the test site (approximately 1 × 1 inch square of intact skin) should be designated and the test substance (0.5 ml or 0.5 g) should be administered to that area. Liquids are administered as received, powders should be moistened with a suitable vehicle before application (e.g., distilled water). The animal should then be placed in a restraining device or an Elizabethan or similar collar should be applied. After the designated exposure time (i.e., 4 or 24 hours), the test site should be delineated with an indelible marker and rinsed with a suitable vehicle (e.g., distilled water). At the appropriate grading intervals (e.g., 1, 24, 48, and 72 hours following patch removal), the animals should be examined and scored for signs of erythema and edema according to the Draize dermal grading system. Grading of the test sites may be continued after the 72-hour scoring interval if irritation persists.

D. THE CORROSIVITY TEST 1.

IN

RABBITS

Materials

a. Gauze Dressing Type: Ace-Tex Corporation nonsterilized Thickness: 4-ply gauze dressing. Size: 1 × 1 inch b. Tape-Nonirritating Type: Blenderm® Size: 1 inch wide c. Binding Materials Elastic wrap Type: Rubber elastic bandage: Ace® Bandage or Coban®


d. Securing Materials Type: Zonas® porous athletic tape Size: 2 inches wide e.

Elizabethan or similar collars: Optional.


Procedures

The hair is removed from a sufficient area on the rabbit’s back on the day before dosing. Care should be taken to avoid abrading the skin during the clipping procedure. On the day of dosing, each test site (approximately 1 × 1 inch square of intact skin) should be selected based on the number of exposure periods required (i.e., 3 minutes, 1 hour, and/or 4 hours). The 1 × 1 inch gauze dressing should be secured to the animal’s back at the designated exposure site on at least two cut edges of the gauze patch using the nonirritating Blenderm tape. The test substance, either 0.5 ml or 0.5 g, should be administered under the gauze dressing at each exposure site and the remaining cut edges secured to the animal’s back. Liquids and powders should be administered as received. If the test article is a solid or powder that does not work well as a paste (e.g., does not spread well), 0.5 g of the test article will be applied to an approximate 1 × 1 inch 4-ply gauze patch and will be moistened with the appropriate amount of distilled water or suitable vehicle (generally 0.5 ml) and the gauze patch applied to the test site. The elastic wrap is then wrapped around the animal’s torso and secured in place using the Zonas athletic tape. The Zonas is wrapped around the outer most portion of the elastic wrap at the cranial and caudal ends. If more than one exposure interval is utilized, the elastic wrap overlying the gauze dressings may be delineated using an indelible marker. This should aid in the unwrapping process. An Elizabethan or similar collar may then be applied to the animal. After the designated exposure interval (i.e., 3 min, 1 h, and/or 4 h), a window can be cut into the elastic wrap overlying the gauze patch for the appropriate test site and the gauze patch removed. The test site should be delineated with an indelible marker and then rinsed with a suitable vehicle (e.g., distilled water). If more than one exposure interval is utilized, the cut out window of the elastic wrap should be secured to the animal with additional Blenderm tape. This will help prevent possible disruption of the remaining site(s). These steps are repeated until the last exposure interval is completed, at which time the entire elastic wrap is removed. At the appropriate grading intervals (e.g., after patch removal and at 24, 48, and 72 h after patch application), the animals should be examined and scored for signs of erythema and edema according to the Draize dermal grading system. Grading of the test sites may be continued after the 72-h scoring interval if irritation persists.

E. THE PHOTOIRRITATION TEST 1.

IN

RABBITS

Materials

a. UVA Bulbs Eight (nonoccluded procedure) or four (occluded procedure) Sylvania (Osram Sylvania, Danvers, MD) F-40/350 BL blacklight flourescent or equivalent. b. UVA/UVB Photometer IL 1350 radiometer/photometer. c. Irradiation Deflector Aluminum foil.


d. Materials for Nonocclusive Procedure Rabbit stocks or other restraining device e.

Materials for Occlusive Procedure 1. Occlusive Materials Type: Impervious material: plastic wrap or other suitable plastic wrap 2. Binding Materials Type: Rubber elastic bandage: Ace® bandage, or Coban®, or Expandover® Size: Adequate wrapping of the entire test site 3. Securing Materials Type: Zonas® porous athletic tape Size: 2 inches wide 4. Tape — Nonirritating Type: Blenderm® Size: 1 inch wide 5. Gauze Dressing Type: Ace-Tex Corporation nonsterilized Thickness: 4-ply gauze dressing Size 1 × 1 in. square


Procedures

a. Preliminary Procedures The photoirritation study is conducted using three or six adult New Zealand White rabbits per group. Animals of either sex should be utilized for the test and positive control (if utilized) groups. On the day before dose administration, the animals selected for study should have the fur clipped for the dorsal area of the trunk of each animal using a small animal clipper. Care should be taken to avoid abrading the skin during the clipping procedure. b. Nonoccluded Procedure On the following day (day 0), the test article (or positive control material, e.g., Oxsoralen Lotion, 1% 8-MOP from CN Pharmaceuticals, Inc., Cosa Mesa, CA, if used) will be applied initially to one small area of intact skin on the right side of each animal as follows: a 0.025 ml dose of the test article (or positive control material, if used) will be applied to one approximate 2.5 cm × 2.5 cm test site on the animals. The test sites will be delineated with a marker. The test sites will remain unoccluded. Immediately after application, the animals will be placed in stocks. Animals will remain in stocks until the completion of the UVA light exposure period and completion of the left site dose period. Approximately 2 h after test article application, excess test article may be removed using dry gauze to have adequate UVA light exposure. The test site may be re-delineated, if needed. Each animal will be wrapped with aluminum foil. An approximate 2.5 × 2.5 cm square section will be cut in the aluminum foil to expose the test site on the right side. Treatment sites on the right side will then be exposed to a target dose of 5 or 10 J/cm2. UVA light (320 to 400 nm) will be emitted from a bank of eight Sylvania F-40/350 BL blacklight fluorescent tubes. The peak emission of the light source will be 360 nm. After the completion of the UVA light exposure period, the foil will be removed. Any residual test article (from right test sites) will be removed with gauze moistened in deionized water (or appropriate solvent) followed by dry gauze. Animals will be removed from the stocks and the test article (or positive control material) will be applied to an area


of intact skin approximate 2.5 × 2.5 cm on the left side of each animal. The test sites will be delineated with a marker. The test site will remain unoccluded. The animals will be returned to the stocks until the completion of the exposure period. Approximately 2 h after test article application on the second test site (on the left side), any residual test article (from the left test site) will be removed with gauze moistened in deionized water (or appropriate solvent) followed by dry gauze. The test sites may be re-delineated, if needed. The animals will be returned to their cages. c. Occluded Procedure On the day of dose administration (day 0), the test article (or positive control material, if utilized) will be applied to two small areas of intact skin of each animal as follows: 0.5 ml or 0.5 g aliquots of the test substance should be applied to two separate areas of intact skin, one on the left side and one on the right side of each test animal. The test substance is held in contact with the skin using an approximately 1 × 1 in. square gauze patch secured to the animal with a nonirritating tape. An impervious plastic wrap is placed over the trunk of the animal and further wrapped with an elastic wrap. The elastic wrap is then secured to the animal using the athletic tape. Animals will be placed in stocks following dosing and will remain in stocks until completion of UVA light exposure period. Approximately 2 h after chamber application, the elastic wrap, plastic wrap, and gauze patch located on the right side of each animal will be removed. Excess test article may be removed using dry gauze to have adequate UVA light exposure. The test site may be re-delineated (if needed). The patch on the left side of the animal will remain undisturbed. Each animal will be wrapped with aluminum foil. An approximate 2.5 × 2.5 cm square section will be cut in the aluminum foil to expose each test site on the right side. Treatment sites on the right side will then be exposed to a target dose of 5 or 10 J/cm2. UVA light (320 to 400 nm) will be emitted from a bank of four or eight Sylvania F-40/350 BL blacklight fluorescent tubes. The peak emission of the light source will be 360 nm. After the completion of the UVA light exposure period, the foil and chamber (from the left side) will be removed. Any residual test article (from all sites) will be removed with gauze moistened in deionized water (or appropriate solvent) followed by dry gauze. Animals will be returned to their cages.

F. THE PHOTOIRRITATION TEST 1.

IN

GUINEA PIGS

Materials

a. UVA Bulbs Eight Sylvania (Osram Sylvania, Danvers, MD) F-40/350 BL blacklight flourescent or equivalent. b. UVA/UVB Photometer IL 1350 radiometer/photometer. c. Irradiation Deflector Aluminum foil. d. Materials for Nonocclusive Procedure Type: Buehler restainer or similar device Type: Dental dam e.

Materials for Occlusive Procedure 1. Occlusive Materials Type: 25 mm Hilltop® Chamber 2. Binding Materials Type: Buehler restainer or similar device Type: Dental dam


f. Animal Species Hartley-derived albino guinea pig 2.

Procedures

a. Preliminary Procedures The photoirritation study is conducted using six adult Hartley-derived albino guinea pigs per group. Animals of either sex should be utilized for the test and positive control (if utilized) groups. On the day before dose administration, the animals selected for study should have the fur clipped for the dorsal area of the trunk of each animal using a small animal clipper. Care should be taken to avoid abrading the skin during the clipping procedure. b. Nonoccluded Procedure On the following day (day 0), immediately prior to application, the animals will be placed in individual restrainers. The dental dam will be pulled taut over the back and secured to the bottom of the restrainer. An approximate 2.5 × 2.5 cm window will be cut into the right side of the dental dam to ensure that the test sites remain unoccluded. The restrainers will be adjusted as necessary to minimize any discomfort of the animals. Animals will remain in restrainers until the completion of the UVA light exposure period and completion of the left site dose period. The test article (or positive control material, e.g., Oxsoralen Lotion 1% 8-MOP from CN Pharmaceuticals, Cosa, CA, if used) will be applied initially to one small area of intact skin on the right side of each appropriate animal as follows: a 0.025 ml dose of the test article (or positive control material, if used) will be applied to one approximate 2.5 × 2.5 cm test site on the animals. The test sites will then be delineated with a marker. The test sites will remain unoccluded. Approximately 2 h after test article application, excess test article may be removed using dry gauze in order to have adequate UVA light exposure. The test sites may be re-delineated, if needed. The back of each animal will be covered with aluminum foil. An approximate 2.5 × 2.5 cm square section will be cut in the aluminum foil to expose each test site on the right side. Treatment sites on the right side will then be exposed to a target dose of 10 J/cm2. UVA light (320 to 400 nm) will be emitted from a bank of eight Sylvania F-40/350 BL blacklight fluorescent tubes. The peak emission of the light source will be 360 nm. After the completion of the UVA light exposure period, the foil will be removed. Any residual test article (from right test sites) will be removed with gauze moistened in deionized water (or appropriate solvent) followed by dry gauze. Animals will remain in restrainers and an approximate 2.5 × 2.5 cm window will be cut in the dental dam on the left side. The test article (or positive control material, if used) will be applied to an area of intact skin on the left side of each animal. The test material will be applied to an approximate 2.5 × 2.5 cm test site on the left side of the animals. The test site will remain unoccluded. Approximately 2 h after test article application on the second test site (on the left side), the remaining dental dam will be removed. Any residual test article (from the left test site) will be removed with gauze moistened in deionized water (or appropriate solvent) followed by dry gauze. The test sites may be redelineated, if needed. The animals will be returned to their cages. c. Occluded Procedure On the day of dose administration (day 0), the test article will be applied to two small areas of intact skin on the left and right side of each appropriate animal as follows: a 0.3 ml or 0.3 g (or maximum dose, powders to be moistened with the appropriate vehicle) dose of the test article (or positive control material as described previously, if used) will be applied to two 25 mm Hilltop chambers just prior to applying the chambers to the back. The Hilltop chambers will be applied to the back as quickly as possible. The test sites will be delineated with a marker. Immediately following application, the animals will be placed in individual restrainers and the chamber will be held at the designated site using rubber dental dam. The dental dam will be pulled taut over the


back and secured to the bottom of the restrainer. The restrainers will be adjusted as necessary to minimize any discomfort of the animals. Animals will remain in restrainers until the completion of the UVA light exposure period. Approximately 2 h after chamber application, an approximate 2.5 × 2.5 cm square will be cut into the dental dam and the chamber located on the right side of each animal will be removed. Excess test article may be removed using dry gauze to have adequate UVA light exposure. The test site may be re-delineated (if needed). The chamber on the left side of the animal will remain undisturbed. The back of each animal will be covered with aluminum foil. An approximate 2.5 × 2.5 cm square section will be cut in the aluminum foil to expose each test site on the right side. Treatment sites on the right side will then be exposed to a target dose of 10 J/cm2. UVA light (320 to 400 nm) will be emitted from a bank of eight Sylvania F-40/350 BL blacklight fluorescent tubes. The peak emission of the light source will be 360 nm. After the completion of the UVA light exposure period, the foil, the remaining dental dam, and chamber (from the left side) will be removed. Any residual test article (from all sites) will be removed with gauze moistened in deionized water (or appropriate solvent) followed by dry gauze.

SECTION 4. MATERIALS AND PROCEDURES FOR PERFORMING DERMAL SENSITIZATION STUDIES33–50 A. THE MODIFIED BUEHLER SENSITIZATION TEST 1.

IN

GUINEA PIGS

Materials

a. Occlusive Materials Type: 25 mm Hilltop® chamber (Hilltop Research, Inc., Cincinnati, OH), 2 × 2 cm Webril® patch; (Professional Medical Products, Greenwood, SC) b. Binding Materials Elastic wrap Type: Expandover®, Coban® c. Securing Materials Type: Conform® (Kendall Health Care Products Co. Mansfield, MA) Size: 1 inch wide d. Depilatory Materials Neet® (Reckitt & Coleman Inc., Wayne, NJ) hair remover cream e. Animal Species Hartley albino guinea pig 2.

Procedures

A topical range-finding irritation screen should generally be performed before initiating the sensitization study. Four graded levels (generally 25% w/v, 50% w/v, 75% w/v, and 100%) are used for this procedure. Optimally, the topical range-finding study should produce no systemic toxicity and a spectrum of dermal responses that includes grades 0 ±, 1 and 2 unless the test substance is not dermally irritating at 100%. Based on the range-finding results, the test substance concentration used for induction should produce no systemic toxicity and a mild to moderate dermal response (grades ±, 1 or 2) unless the test substance is not dermally irritating at 100%. The test substance concentration used for challenge/rechallenge should produce no systemic toxicity and dermal responses generally consisting of grades 0 to ± unless the test substance is not dermally irritating at 100%. Copyright © 2002 by Taylor & Francis

3.

Topical Range-Finding Study

On the day before dose administration, four topical range-finding guinea pigs should be weighed and the hair removed from the right and left side of the animals with a small animal clipper. Care should be taken to avoid abrading the skin during clipping procedures. On the following day, up to four closed patches/chambers at four different concentrations of test substance can be applied to the clipped area of each animal (one patch/chamber for each level of test substance). For liquids, gels, and pastes, a dose of 0.3 or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25 mm Hilltop Chamber (with cotton pad removed) should be utilized. Before chamber application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The patches/chambers should then be applied to the clipped surface as quickly as possible. The trunk of the animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 6 hours after patch/chamber application, the elastic wrap, tape, and patches/chambers should be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). The test sites of the topical range-finding animals should be graded for irritation at approximately 24 and 48 hours after patch/chamber application using the Buehler dermal grading system. 4.

Induction

On the day before the first induction dose administration (day –1), all sensitization study animals should be weighed. The hair should then be removed from the left side of the test animals with a small animal clipper. Care should be taken to avoid abrading the skin during clipping procedures. On the following day (day 0), patches/chambers containing the test substance should be applied to the clipped area of 10 to 20 test animals. For liquids, gels, and pastes, a dose of 0.3 ml or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25-mm Hilltop Chamber (with cotton pad removed) should be utilized. Before chamber application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The patch/chamber should then be applied to the clipped surface as quickly as possible. The trunk of each animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 6 hours after dosing, the elastic wrap, tape, and patch/chamber will be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). The induction clipping, patch application, and grading procedure should be repeated on study day 7 (± 1 day) and study day 14 (± 1 day) so that a total of three consecutive induction exposures should be administered to the test animals. Test sites should be graded for dermal irritation at approximately 24 and 48 h after patch application using the Buehler dermal grading system. The application site may be moved if irritation persists from a previous induction exposure but will remain on the left side of the animal. If a positive control group is necessary, 2, 4-dinitrochlorobenzene (DNCB) and α-hexylcinnamaldehyde is an acceptable positive control substance and a positive control group consisting of 10 DNCB or HCA test animals and 10 DNCB or HCA control animals may be used. The DNCB or HCA test and DNCB or HCA control animals should be treated in the same manner as the sensitization study test and challenge control animals throughout the study. The DNCB concentrations standardly used for induction and challenge are 0.1 to 0.5% w/v and 0.05 to 0.1% w/v, respectively. The HCA concentrations standardly used for induction and challenge are 3.0 to 5.0% and 1.0 to 2.5%, respectively. A response of at least 15% in a nonadjuvant test should be expected for a mild to moderate sensitizer. 5.

Challenge

On the day before challenge dose administration, the hair should be removed from the right side of the test and challenge control animals with a small animal clipper. Care will be taken to avoid Copyright © 2002 by Taylor & Francis

abrading the skin during clipping procedures. On the next day (day 28 ± 1 day), patches/chambers containing the test substance should be applied to a naive site within the clipped area of the test and challenge control animals. For liquids, gels, and pastes, a dose of 0.3 ml or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25 mm Hilltop Chamber (with cotton pad removed) should be used. Before chamber application, the test substance should be moistened with an appropriate vehicle (e.g., distilled water). The patch/chamber should then be applied to the clipped surface as quickly as possible. The trunk of each animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 6 h after dosing, the elastic wrap, tape, and patch/chamber should be removed. The test substance should then be removed with a suitable vehicle (e.g., distilled water). Approximately 20 hours after patch/chamber removal, the test sites may be depilated (optional) as follows: 1. Neet Hair Remover cream should be placed on the test sites and surrounding areas and left on for no more than 15 minutes. 2. The depilatory should then be thoroughly removed with a stream of warm water. The animals should be dried with a towel and returned to their cages. Note: The depilatory process has an advantage of being able to view test sites without hair, however, from time to time suspected test article/depilatory reactions may be observed producing unanticipated dermal responses in the test and control animals. Test sites should be graded for dermal irritation at approximately 24 and 48 hours after patch removal using the Buehler dermal grading system. 6.

Rechallenge

If a rechallenge phase is required, the procedure should be performed on day 35 (± 1 day). The animal’s haircoat should again be clipped on the right side of the animal on the day before dosing. The exposure period, dosing, wrapping, and depilation procedures should be the same as used in the challenge procedure except that the 10 to 20 test and 10 naive rechallenge control animals and a naive skin site is utilized.

B. THE STANDARD BUEHLER SENSITIZATION TEST 1.

IN

Materials

a. Occlusive Materials Type: 25 mm Hilltop® Chamber, 2 × 2 cm Webril® Patch b. Binding Materials Elastic wrap Type: Expandover®, Coban® c. Securing Materials Type: Conform® Size: 1 inch wide d. Depilatory Materials Neet®Hair Remover Cream e. Animal Species Hartley albino guinea pig


GUINEA PIGS

2.

Procedures

A topical range-finding irritation screen generally should be performed before initiating the sensitization study. Four graded levels (generally 25% w/v, 50% w/v, 75% w/v, and 100%) are utilized for this procedure. Optimally, the topical range-finding study should produce no systemic toxicity and a spectrum of dermal responses that includes grades 0, ±, 1, and 2 unless the test substance is not dermally irritating at 100%. Based on the range-finding results, the test substance concentration used for induction should produce no systemic toxicity and a mild to moderate dermal response (grades ±, 1 or 2) unless the test substance is not dermally irritating at 100%. The test substance concentration used for challenge/rechallenge should produce no systemic toxicity and dermal responses generally consisting of grades 0 to ± unless the test substance is not dermally irritating at 100%. 3.


On the day before dose administration, four topical range-finding guinea pigs should be weighed and the hair removed from the right and left side of the animals with a small animal clipper. Care should be taken to avoid abrading the skin during clipping procedures. On the next day, up to four closed patches/chambers at four different concentrations of test substance can be applied to the clipped area of each animal (one patch/chamber for each level of test substance). For liquids, gels, and pastes, a dose of 0.3 ml or 0.4 ml will be placed on a 25-mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25-mm Hilltop Chamber (with cotton pad removed) should be utilized. Before chamber application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The patches/chambers should then be applied to the clipped surface as quickly as possible. The trunk of the animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 6 h after patch/chamber application, the elastic wrap, tape, and patches/chambers should be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). The test sites of the topical range-finding animals should be graded for irritation at approximately 24 and 48 h after patch/chamber application using the Buehler dermal grading system. 4.

Induction

On the day before the first induction dose administration (day –1), all sensitization study animals should be weighed. The hair should then be removed from the left side of the test animals with a small animal clipper. Care will be taken to avoid abrading the skin during clipping procedures. On the next day (day 0), patches/chambers containing the test substance should be applied to the clipped area of 10 to 20 test animals. For liquids, gels, and pastes, a dose of 0.3 ml or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25 mm Hilltop Chamber (with cotton pad removed) should be used. Before chamber application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The patch/chamber should then be applied to the clipped surface as quickly as possible. The trunk of each animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 6 h after dosing, the elastic wrap, tape, and patch/chamber will be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). The induction clipping, patch application, and grading procedure should be repeated three times a week (i.e., Monday-Wednesday-Friday) for 3 consecutive weeks so that a total of nine consecutive induction exposures are administered to the test animals. Test sites should be graded for dermal irritation at approximately 24 and 48 h after patch application using the Buehler dermal grading system. The Copyright © 2002 by Taylor & Francis

application site may be moved if irritation persists from a previous induction exposure but will remain on the left side of the animal. If a positive control group is necessary, DNCB or HCA is an acceptable positive control substance, and a positive control group consisting of 10 DNCB or HCA test animals and 10 DNCB or HCA control animals may be used. The DNCB or HCA test and DNCB or HCA control animals should be treated in the same manner as the sensitization study test and challenge control animals throughout the study. The DNCB concentrations standardly used for induction and challenge are 0.1 to 0.5% w/v and 0.05 to 0.1% w/v, respectively. The HCA concentrations standardly used for induction and challenge are 3.0 to 5.0% and 1.0 to 2.5%, respectively. A response of at least 15% in a nonadjuvant test should be expected for a mild to moderate sensitizer. 5.

Challenge

On the day before challenge dose administration, the hair should be removed from the right side of the test and challenge control animals with a small animal clipper. Care will be taken to avoid abrading of the skin during clipping procedures. On the next day (day 32 ± 1 day), patches/ chambers containing the test substance should be applied to a naive site within the clipped area of the test and challenge control animals. For liquids, gels, and pastes, a dose of 0.3 ml or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25 mm Hilltop Chamber (with cotton pad removed) should be utilized. Before chamber application, the test substance should be moistened with an appropriate vehicle (e.g., distilled water). The patch/chamber should then be applied to the clipped surface as quickly as possible. The trunk of each animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 6 h after dosing, the elastic wrap, tape, and patch/chamber should be removed. The test substance should then be removed with a suitable vehicle (e.g., distilled water). Approximately 20 h after patch/chamber removal, the test sites may be depilated (optional) as follows: 1. Neet Hair Remover cream should be placed on the test sites and surrounding areas and left on for no more than 15 min. 2. The depilatory should then be removed thoroughly with a stream of warm water. The animals should then be dried with a towel and returned to their cages. Note: The depilatory process has an advantage of being able to view test sites without hair, however, from time to time, suspected test article/depilatory reactions may be observed producing unanticipated dermal responses in the test and control animals. Test sites should be graded for dermal irritation at approximately 24 and 48 h after patch removal using the Buehler dermal grading system. 6.

Rechallenge

If a rechallenge phase is required, the procedure should be performed on day 39 (± 1 day). The animal’s haircoat should again be clipped on the right side of the animal on the day before dosing. The exposure period, dosing, wrapping, and depilation procedures should be the same as used in the challenge procedure except that the 10 to 20 test and 10 naive rechallenge control animals and a naive skin site is utilized.

C. THE GUINEA PIG MAXIMIZATION TEST 1.

Materials

a. Injection Materials Monoject® (Sherwood Medical, St. Louis, MO) or equivalent 1 cc Tuberculin syringe with 25 to 27 gauge–5/8-inch needle Copyright © 2002 by Taylor & Francis

b. Occlusive Materials Type: 2 × 2 cm Webril® patch, 2 × 4 cm Modified Webril® Patch, 25 mm Hilltop® Chamber c. Binding Materials Elastic wrap Type: Coban® d. Securing Materials Type: Conform® Zones® Athletic Tape Size: 1-in. wide e. Animal Species Hartley albino guinea pig 2.

Procedures

For the topical screen, four graded levels (generally 25% w/v, 50% w/v, 75% w/v, and 100%) are used for this procedure. Optimally, the topical range-finding study should produce no systemic toxicity and a spectrum of dermal responses that includes grades 0, ±, 1 and 2 unless the test substance is not dermally irritating at 100%. For the intradermal screen, four graded levels (generally 0.1% w/v, 1.0% w/v, 3.0% w/v, and 5.0% w/v) are used for this procedure. Optimally, the intradermal range-finding study should produce no systemic toxicity and only localized reactions at the injection site (responses that do not notably extend beyond the site of injection). Based on this information, the test substance concentration used for intradermal induction should produce no systemic toxicity and only localized reactions at the injection site (responses that do not notably extend beyond the site of injection). For the topical induction, the test substance concentration used should produce a mild to moderate dermal response (grades ±, 1 or 2) unless the test substance is not dermally irritating at 100%. The test substance concentration used for challenge/rechallenge should produce no systemic toxicity and dermal responses generally consisting of grades 0 to ± unless the test substance is not dermally irritating at 100%. 3.


On the day before dose administration, four topical range-finding guinea pigs should be weighed and the hair removed from the right and left side of the animals with a small animal clipper. Care should be taken to avoid abrading the skin during clipping procedures. On the next day, up to four closed patches/chambers at four different concentrations of test substance can be applied to the clipped area of each animal (one patch/chamber for each level of test substance). For liquids, gels and pastes, a dose of 0.3 ml or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25 mm Hilltop Chamber (with cotton pad removed) should be used. Before chamber application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The patches/chambers should then be applied to the clipped surface as quickly as possible. The trunk of the animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber. Approximately 24 h after patch/chamber application, the elastic wrap, tape, and patches/chambers should be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). The test sites of the topical range-finding animals should be graded for irritation at approximately 24 and 48 h after patch/chamber removal using the Buehler dermal grading system.


4.

Intradermal Range-Finding Study

On the day before dose administration, four intradermal range-finding guinea pigs should be weighed and the hair removed from the right and left side of the animals with a small animal clipper. Care should be taken to avoid abrading the skin during clipping procedures. On the next day, up to four intradermal injections at four different concentrations of test substance can be injected into the clipped area of each animal (one injection for each level of test substance). A dose of 0.1 ml should be injected for each concentration using a syringe attached to a hypodermic needle. The test sites of the intradermal range-finding animals should be graded for irritation at approximately 24 and 48 h after intradermal injections using the Buehler dermal grading system. 5.

Induction

On the day before intradermal dosing (day – 1), all sensitization study animals should be weighed. The hair should then be removed from the scapular area of 10 to 20 test and 10 challenge control animals with a small animal clipper. Care should be taken to avoid abrading the skin during clipping procedures. On the next day (day 0), three pairs of intradermal injections should be made in the clipped area of all sensitization study animals. The injections should be kept within an approximate 2 × 4 cm area with one row of three injections on each side of the back bone as indicated below:

2 cm

•

•

-Injection Pair A

2 cm

•

•

-Injection Pair B

2 cm

•

•

-Injection Pair C

4 cm Injections for the test animals should be as indicated: 1) Injection Pair A , 0.1 ml of a 1:1 v/v Freund’s Complete Adjuvant in sterile water emulsion (FCA emulsion); 2) Injection Pair B, 0.1 ml of the test substance preparation; 3) Injection Pair C, 0.1 ml of the test substance in the FCA emulsion. Injections for the challenge control animals should be as indicated: 1) Injection Pair A, 0.1 ml of the FCA emulsion; 2) Injection Pair B, 0.1 ml of the vehicle; 3) Injection Pair C, 0.1 ml of the vehicle/ FCA emulsion. On the day before topical induction, the hair should be clipped from the scapular area of the test and challenge control animals using a small animal clipper. Care should be taken to avoid abrading the skin during the clipping procedures. A 10% w/w sodium lauryl sulfate preparation in petrolatum should then be applied to the 2 × 4 cm intradermal injection area so that the injection area is sufficiently covered with the preparation (0.5 ml). On the next day (day 7 ± 1 day), any residual sodium lauryl sulfate preparation should be removed with a dry gauze, and the test animals should receive a topical dose of the test substance. The challenge control animals should receive a topical dose of the vehicle. Each animal’s dose first should be applied to a modified Webril patch and the patch applied over the intradermal injection sites as quickly as possible. For liquids, gels, and pastes, a dose of 0.8 ml should be placed on the modified Webril patch. For solids and powders, the maximum volume of solid/powder that can be maintained on the modified Webril patch should be used. Before solid/powder application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The trunk of each animal should then be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch and the animal returned to its cage. Approximately 48 h after dosing, the elastic wrap, tape,


and patch should be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). If a positive control group is necessary, DNCB or HCA are acceptable positive control substances, and a positive control group consisting of 10 DNCB or HCA test animals and 10 DNCB or HCA control animals may be used. The DNCB or HCA test and control animals should be treated in the same manner as the sensitization study test and control animals throughout the study. The DNCB concentrations standardly used for induction and challenge are 0.1 to 0.5% w/v and 0.05 to 0.1% w/v, respectively. The HCA concentrations standardly used for induction and challenge are 3.0 to 5.0% w/v and 0.5 to 1.0% w/v, respectively. A response of at least 30% in an adjuvant test should be expected for a mild to moderate sensitizer. 6.

Challenge

On the day before challenge dose administration, all test and challenge control animals should be weighed. The hair should then be removed from the right side of the test and challenge control animals with a small animal clipper. Care should be taken to avoid abrading of the skin during clipping procedures. On the next day (day 21 ± 1 day), the test and challenge control animals should receive a topical dose of the test substance. For liquids, gels, and pastes, a dose of 0.3 ml or 0.4 ml should be placed on a 25 mm Hilltop Chamber or Webril patch. For solids and powders, the maximum volume of solid/powder that can be contained in a 25 mm Hilltop Chamber (with cotton pad removed) should be used. The weight of the solid/powder placed in the chamber should be recorded. Before chamber application, the test substance should be moistened with a suitable vehicle (e.g., distilled water). The patch/chamber should then be applied to the clipped surface as quickly as possible. The trunk of each animal should be wrapped with elastic wrap which is secured with adhesive tape (if necessary) to prevent removal of the patch/chamber and the animal returned to its cage. Approximately 24 h after dosing, the elastic wrap, tape, and patch/chamber should be removed. The test substance should be removed with a suitable vehicle (e.g., distilled water). The test sites should be graded for dermal irritation at approximately 24 and 48 hours after patch removal using the Buehler dermal grading system. 7.

Rechallenge

If a rechallenge phase is required, the rechallenge procedure should be performed on day 28 (±1 day). The animal’s haircoat should be clipped on the left side of the animal on the day before dosing. The exposure period, dosing, and the wrapping procedures are the same as used in the challenge procedure except that 10 naive rechallenge control animals and a naive skin site are utilized.

D. THE MURINE LOCAL LYMPH NODE ASSAY 1.

Materials

a.

Dosing Materials 1. Calibrated pipette or syringe 2. 1-cc disposable syringe, 25 to 27 gauge needle

b.

Lymph Node Collection/Cell Suspension Materials 1. Tissue culture dish (e.g., 60 mm) 2. Tissue culture tube (e.g., 12 × 75 mm) 3. Centrifuge tube (e.g., 15 ml) 4. Nylon or stainless steel screen, ∼100 to 200 μm mesh opening, ∼85 μm thick


5. Pasteur pipet 6. Forceps 7. Scintillation cocktail (e.g., Ecovolume®) c. Animal Species Female CBA mice. 2.

Procedures

The test material is applied directly to the ears for assessing the contact hypersensitization. These procedures evaluate the ability of the test article to cause lymphocyte proliferation as determined by incorporation of 3H-thymidine by lymphocytes within the appropriate draining lymph nodes of topically treated mice, which is then compared to appropriate control mice. Generally, no rangefinding animals are utilized unless there is a concern for dermal trauma (corrosion/severe irritation) or systemic toxicity. Instead, at least three consecutive concentrations from the following range are utilized: 100, 50, 25, 10, 5, 2.5, 1, 0.5, 0.25, and 0.1% (w/v). The selection is made to provide the highest possible test concentration, which is generally limited by compatibility with the vehicle chosen and the suitability of the resulting preparation for unoccluded dermal application. The following vehicles are recommended, in order of preference: acetone–olive oil (4:1), acetone, dimethylformamide, methyl ethyl ketone, propylene glycol, and dimethysulfoxide. Aqueous vehicles are not normally recommended because of insufficient absorption during the dosing procedure; however, aqueous-organic mixtures such as 3:1 acetone–water or 80% ethanol have been used successfully. Materals for positive control include 2,4-dinitrochlorobenzene (DNCB), α-hexylcinnamaldehyde (HCA), 2-mercaptobenzothiazole, and benzocaine. A threefold or greater increase in proliferative activity of the test animals compared with concurrent vehicle treated control animals is the criterion for a classification of skin-sensitizing activity. 3.

Topical Induction

On day 0, 5 females per test article concentration and control group (and positive control test and control group, if utilized) will be weighed and 25 μl of test article will be applied to the dorsal surface of the left and right ear. Care will be taken to ensure that the test article will not run off of the ear during application. Approximately 24 h later (day 1) and 48 h later (day 2), each animal will receive additional applications as described previously. The animals will then be rested for 2 days. The vehicle control animals (and positive control animals, if used) are treated the same as above. 4.

Injection of 3H-thymidine for Lymphocyte Incorporation

On day 5 (approximately 72 h after the final application), the five females per group will receive an intravenous injection of 3H-thymidine for lymphocyte incorporation. The injection will consist of 0.250 ml of phosphate-buffered saline (PBS) containing 20 μCi of 3H-thymidine (specific activity of 5.0 or 6.7 Ci/mmol). An animal will be excluded from the study if the full 0.250 ml of 3Hthymidine/PBS is not properly injected intravenously. 5.

Lymph Node Collection

Approximately 5 h after the 3H-thymidine injections, the animals will be euthanized with carbon dioxide and the appropriate draining (auricular) lymph nodes will be removed and pooled for each individual animal. Care will be taken to assure that the lymph nodes are removed intact and placed in a capped tissue culture tube (e.g. 12 × 75 mm) containing 4 ml of PBS.


6.

Cell Suspension

The lymph nodes will be transferred to a tissue culture dish (e.g., 60 mm) by pouring the PBS tube containing the lymph nodes. The lymph nodes will be mechanically passed through a nylon or stainless steel screen. A pasteur pipet and a small pair of forceps will be used to rinse the screen with PBS into the tissue culture dish, which will then be rinsed with the PBS back into the culture tube to allow the capsule debris to settle to the bottom. The PBS will then be carefully drawn off with a pasteur pipet and will be placed in a centrifuge tube containing 6 ml of PBS (approximately 10 ml total tube volume). The cell suspensions will then be centrifuged and then resuspended in 20 ml of PBS and a second wash will be performed. After completion of the second wash, the cells will be suspended in 3 ml of 5% trichloroacetic acid (w/v TCA in deionized water) and left at approximately 4°C for approximately 18 h. 7.

Scintillation Counting

The cell suspensions will be centrifuged and resuspended in 1 ml of 5% TCA. The individual cell suspensions will be transferred into the appropriate scintillation vials containing 10 ml of scintillation cocktail along with an additional 1 ml of TCA, which has been used to rinse the bottom portion of the tube. The TCA and scintillation fluid will be thoroughly mixed by gently swirling the contents of the vial until the solution becomes clear. The sample will then be counted and recorded in disintergrations per minute (DPM).

E. THE PHOTOSENSITIZATION TEST 1.

IN

MICE

Materials

a. UVA Bulbs Four Sylvania® F-40/350 BL blacklight fluorescent or equivalent. b. UVB Bulbs Phillips® (Phillips Lighting Co. Somerset, NJ) F-40 UVB fluorescent sunlamps or equivalent. c. UVA/UVB Photometer IL 1350 radiometer/photometer. d. Dyer® (Dyer Company, Lancaster, PA) Micrometer Model D-1000. e. Irradiation Deflector 3-mm thick sheet of plate glass (large enough to cover UVA-exposed animals). f. Micropipettor Eppendorf® (Brinkman Instruments, Inc., Westbury, NY) 1 to 50 μL micropipetter. g. Animal Species BALB/c mice. 2.

Procedures

A preliminary irritation screen should generally be included in this test to observe the degree of primary irritation the test substance may produce. Twenty female mice, separated into 4 dose groups (5 mice/ group), are used for this procedure. Up to four different concentrations of the test substance


can be utilized. Each mouse should be placed in a restrainer and two measurements of ear thickness should be performed using an engineer’s micrometer. Two different concentrations may be utilized on each mouse, one concentration for each ear. Eight microliters of the test substance should be used for animals in groups 3 and 4 and 8 μl of the vehicle for groups 1 and 2. The appropriate material should be applied to both sides of the ear. After a 60-min waiting period, the ears should be wiped with gauze moistened with an appropriate vehicle (e.g., distilled water). Mice from groups 2 and 4 should then be exposed to 10 J/cm2 UVA and 25 to 60 mJ/cm2 UVB. Mice in groups 1 and 3 are not irradiated. At approximately 3, 24, and 48 h postirradiation, the mice should be placed back into the restraining device and ear measurements should once again be performed and recorded. 3.

Induction

An induction phase of the photosensitization study should be initiated with an intraperitoneal injection of cyclophosphamide (CP). A dose of 200 mg/kg CP in sterile phosphate-buffered saline at a dose volume of 10 ml/kg should be injected approximately 3 days before the first induction. Because the CP injections may induce toxicity and/or mortality, additional mice should be used for this procedure to allow for a sufficient number of animals on study. On day 0, the backs of all mice should be clipped using a small animal clipper and appropriately sized clipper blade. Standard study designs are set up with four groups of five female mice each. The first two groups are designated as test substance groups with the remaining two groups set up as vehicle control and ultraviolet control groups, respectively. The mice designated for groups 1, 2, and 4 are induced on days 0, 1, and 2. Group 3 animals are not treated during the induction phase. Mice in groups 1 and 2 should receive 50 μl of the appropriate test substance gently rubbed into the skin on the dorsal back of each animal on each of the induction days. Mice in group 4 should receive 50 μl of the designated vehicle in the same manner. Each mouse is then placed in an individual compartment of an irradiation box with a wire lid restraining device. Approximately 60 min after application of the appropriate material, the treated area of each animal should be gently wiped against the grain of hair growth with a gauze patch moistened with an appropriate vehicle. After the wiping procedure, animals from group 2 should be returned to their respective cages and should not receive irradiation. Mice in groups 1 and 4 should be exposed to 10 J/cm2 UVA and 25 mJ/cm2 UVB from a distance of approximately 20 ± 1.0 cm. For the UVA exposure, a 3-mm-thick sheet of plate glass should be placed over the UVA radiometer detector during irradiation measurements to filter out any UVB wavelengths that may be emitted. The mice from groups 1 and 4 are then exposed to UVA light (320 to 400 nm) emitted from a bank of four Sylvania F-40/350 BL blacklight fluorescent tubes. The bank of lights are positioned approximately 20 ± 1.0 cm above the irradiation boxes containing the mice for a target dose of 10 J/cm2. A peak emission of the UVA lights should be at 360 nm. For the UVB exposure, the mice should be positioned under a bank of eight Phillips F40 UVB fluorescent sunlamps for an exposure of 25 mJ/cm2 UVB light. It is preferable to rotate the animals’ positions for each induction exposure so that no one group is irradiated in the same location. 4.

Challenge

A challenge phase of the photosensitization study should be performed 7 days after the first induction phase. Before challenge, each mouse should have the ear thickness measured on both ears using an engineer’s micrometer (Model D-1000). Measurements should be read and recorded as millimeters × 10–2. These measurements should take place while the animal is in a restraining device. While the animal is still restrained, 8 μl of the test substance will be administered to each side of one ear. The vehicle is then applied to both sides of the opposite ear. After approximately 60 min, the ears should be wiped with the appropriate vehicle and the animals in groups 1, 3, and 4 exposed to 10 J/cm2 UVA and 25 mJ/cm2 UVB as indicated previously. The group 2 mice should


not be irradiated. Ear thickness measurements as described previously should be performed at approximately 24 and 48 h after the challenge procedure. 5.

Rechallenge

If a rechallenge phase is required, the procedure should be performed 7 days after the challenge exposure. The exposure period, irradiation, and ear measurement procedures should be the same as used in the challenge procedure.

F. THE PHOTOSENSITIZATION TEST 1.

IN

GUINEA PIGS

Materials

a. UVA Bulbs Four Sylvania® F-40/350 BL blacklight fluorescent or equivalent. b. UVA/UVB Photometer IL 1350 radiometer/photometer. c. Irradiation Deflector Aluminum foil. d. Patching Materials Webril® patch 2 × 2 cm or 25 mm Hilltop® Chamber; rubber dental dam; Blenderm® tape. e. Animal Species Hartley albino guinea pig. 2.

Procedures

Unless the irritation potential of the test substance is known, the study should begin with a topical range-finding study. The range-finding study should include eight Hartley-derived albino guinea pigs (4 males and 4 females). Up to four graded concentrations of the test substance may be used in this procedure. On the day before dose administration, the eight guinea pigs should be weighed and the hair removed from the left and right side of each animal using a small animal clipper. Care should be taken to avoid abrading the skin during the clipping procedure. On the day of dose administration, a 0.3 ml or 0.4-ml dose of the appropriate concentration of the test substance should be administered to a 25 mm Hilltop Chamber or Webril patch and the patch immediately placed on the right or left side of the guinea pig (one patch on either side of the back bone). Four patches (two patches per concentration) may be applied. Immediately after application, the animals should be placed in a Buehler restrainer and the patches occluded using rubber dental dam. The dental dam should be pulled taut over the back of the animal and fastened to the bottom of the restrainer. After an exposure period of 2 hours, a 2 × 2 cm square should be cut into the dental dam and the patch removed from the right side of each animal. The patch on the left side of the animal should remain intact. The back of each animal should then be covered with aluminum foil. An approximately 2 × 2 cm square section should be cut in the aluminum foil on each animal to expose the test site on the right side. The treated sites on the right side of the animal should then be exposed to UVA light (320 to 400 nm) at a target dose of 10 J/cm2. Any heavy residual test substance is removed with dry gauze before irradiation to fully expose the test site. After the exposure, the foil, dental dam, and remaining patches from the left side should be removed and any residual test substance removed with an appropriate vehicle. The dermal test sites should be graded at approximately 24 and 48 h after the initiation of the UVA light exposure.


3.

Induction

The induction phase of the study is initiated by weighing the animals and clipping the hair from the scapular area of the 10 test and 10 challenge control animals. Care is taken to avoid abrading the skin during the clipping procedure. On the day of dose administration (day 0), four 0.1-ml intradermal injections of a 1:1 v/v Freund’s Complete Adjuvant in sterile water emulsion are administered to the previously prepared animals. The injections should be made on each side of the back bone. The center portion of the skin between the injection sites is then tape-stripped using an adhesive tape to remove the outer layers of the epidermis. A single 25 mm Hilltop® Chamber or Webril patch containing 0.3 ml or 0.4 ml of the test substance for the test animals and 0.3 ml or 0.4 ml of vehicle for the challenge control animals should be applied immediately to the center of the tape-stripped area. A piece of rubber dental dam should be placed over the application site and secured to the bottom of the restrainer to provide an occlusive binding. After approximately 2 hours of exposure a 2 × 2 cm square will be cut in the rubber dam and the patch removed. Aluminum foil is then placed over the entire back of each guinea pig and an approximately 2 × 2 cm square window over the test area is cut to allow exposure to the UVA treatment. Any heavy residual test substance is removed with dry gauze before irradiation to fully expose the test site. The test sites should then be exposed to UVA light (320 to 400 nm) at a target dose of 10 J/cm2. After the completion of the exposure period, the aluminum foil and dental dam should be removed and the test substances removed with an appropriate vehicle. The induction procedure should be repeated three times a week (e.g., Monday-Wednesday-Friday) for 2 consecutive weeks for a total of six induction exposures. If a positive control group is necessary, Musk Ambrette is an acceptable positive control substance, and a positive control group consisting of 10 Musk Ambrette test animals and 10 Musk Ambrette control animals should be treated in the same manner as the photosensitization study test and challenge control animals throughout the study. The Musk Ambrette concentrations standardly used for induction and challenge are 15% w/v and 0.5% w/v, respectively. 4.

Challenge

A challenge procedure should be performed on study day 25 (± 1 day). On the day before challenge dose administration, the test and challenge control animals should be weighed and the hair removed from the left and right side of the animal using a small animal clipper. On the next day, a 0.3 ml or 0.4 ml volume of the test substance should be applied to each of 2 25 mm Hilltop Chamber or Webril patches. One patch will be applied to each side of each animal. Immediately after the patching procedure, the animals should once again be placed into restrainers and the test sites immediately occluded with a piece of rubber dental dam. After approximately 2 h of exposure, a 2 × 2 cm square should be cut into the dental dam and the patch from the right side of each animal removed. Aluminum foil should then be placed over the back of each animal and a 2 × 2 cm square window cut in the foil to allow the test area to be exposed to the UVA light. Any heavy residual test substance is removed with dry gauze before irradiation to fully expose the test site. The test sites on the right side should then be exposed to UVA light (320 to 400 nm) at a target dose of 10 J/cm2. After the target exposure, the foil, dental dam, and patches should be removed and any residual test substance removed with an appropriate vehicle. The test sites should be graded at approximately 24 and 48 h after the initiation of the UVA exposure using a Draize grading system. 5.

Rechallenge

If the results of the challenge phase are not conclusive, a rechallenge procedure can be performed on study day 32 (± 1 day). The rechallenge phase should be similar in design to the challenge phase except that 10 naive rechallenge control animals and a naive skin site should be utilized for this phase.


SECTION 5. COMPARISON OF SCORING SYSTEMS TABLE 3.8 Draize Dermal Irritation Scoring System24 Erythema and Eschar Formation

Value

Edema Formation

Value

No erythema Very slight erythema (barely perceptible) Well-defined erythema

0 1 2

0 1 2

Moderate to severe erythema Severe erythema (beet-redness) to slight, eschar formation (injuries in depth)

3 4

No edema Very slight edema (barely perceptible) Slight edema (edges of area well defined by definite raising) Moderate edema (raised approximately 1 mm) Severe edema (raised more than 1 mm and extending beyond the area of exposure)

TABLE 3.9 Human Patch Test Dermal Irritation Scoring System51 Skin Reaction

Value

No sign of inflammation; normal skin Glazed appearance of the sites, or barely perceptible erythema Slight erythema Moderate erythema, possible with barely perceptible edema at the margin; papules may be present Moderate erythema, with generalized edema Severe erythema with severe edema, with or without vesicles Severe reaction spread beyond the area of the patch

0 ± (0.5) 1 2 3 4 5

TABLE 3.10 Chamber Scarification Dermal Irritation Scoring System51 Skin Reaction

Value

Scratch marks barely visible Erythema confined to scratches perceptible erythema Broader bands of increased erythema, with or without rows of vesicles, pustules, or erosions Severe erythema with partial confluency, with or without other lesions Confluent, severe erythema sometimes associated with edema, necrosis, or bullae

0 1 2

TABLE 3.11 Magnusson Sensitization Scoring System38 Skin Reaction No reaction Scattered reaction Moderate and diffuse reaction Intense reddening and swelling


Value 0 1 2 3

3 4

3 4

TABLE 3.12 Split Adjuvant Sensitization Scoring System52 Skin Reaction Normal skin Very faint, nonconfluent pink Faint pink Pale pink to pink, slight edema Pink, moderate edema Pink and thickened Bright pink, markedly thickened

Value 0 ± + ++ +++ ++++ +++++

TABLE 3.13 Buehler Sensitization Scoring System34 Skin Reaction No reaction Very faint erythema, usually confluent Faint erythema, usually confluent Moderate erythema Strong erythema, with or without edema

Value 0 ±(0.5) 1 2 3

TABLE 3.14 Contact Photosensitization Scoring System48 Skin Reaction No erythema Minimal but definite erythera confluent Moderate erythema Considerable erythema Maximal erythema

Value 0 1 2 3 4

TABLE 3.15 Human Patch Test Sensitization Scoring System53 Skin Reaction

Value

Doubtful reaction; faint erythema only Weak positive reaction; erythema, infiltration, discrete papules Strong positive reaction: erythema, infiltration, papules, vesicles Extreme positive reaction; intense erythema, infiltration and coalescing vesicles Negative reaction Irritant reaction of different types Not tested

? or + ? + ++ +++


– IR NT

SECTION 6. COMPARISON OF CLASSIFICATION SYSTEMS TABLE 3.16 Environmental Protection Agency (EPA) Method of Calculating the Primary Irritation Index (PII) for Dermal Irritation Studies54,55 Option 1 Separately add up each animal’s erythema and edema scores for the 1-, 24-, 48-, and 72-h scoring intervals. Add all six values together and divide by the (number of test sites × 4 scoring intervals). Option 2 Add the 1-, 24-, 48-, and 72-h erythema and edema scores for all animals and divide by the (number of test sites × 4 scoring intervals).

TABLE 3.17 Federal Hazardous Substances Act (CPSC-FHSA) Method of Calculating the Primary Irritation Index (PII) for Dermal Irritation Studies56 Option 1 Separately add up each animal’s intact and abraded erythema and edema scores for the 25- and 72-hr scoring intervals. Add all six values together and divide by the (number of test sites × 2 scoring intervals). Option 2 Add the 25- and 72-h erythema and edema scores for all animals (intact and abraded sites) and divide by the (number of test sites × 2 scoring intervals).

TABLE 3.18 European Economic Community’s (EEC) Method of Calculating the Primary Irritation Index (PII) for Dermal Irritation Studies5 For six animals 1. Erythema: Add all 24-, 48-, and 72-h erythema scores for each animal together and divide by the (number of test sites × 3 scoring intervals). 2. Edema: Add all 24-, 48-, and 72-h edema scores for each animal together and divide by the (number of test sites × 3 scoring intervals), For three animals 1. Erythema: Add all 24-, 48-, and 72-h erythema scores of each animal individually and divide by the number of scoring intervals. 2. Edema: Add all 24-, 48-, and 72-h edema scores of each animal individually and divide by the number of scoring intervals.

TABLE 3.19 Environmental Protection Agency (EPA) Dermal Classification System57 Primary Irritation Index 0.00 0.01–1.99 2.00–5.00 5.01–8.00


Irritation Rating Nonirritant Slight irritant Moderate irritant Severe irritant

TABLE 3.20 Environmental Protection Agency (EPA) Standard Evaluation Procedure Dermal Classification System58 Mean Score (Primary Irritation Index)

Response Category

0–0.4 0.5–1.9 2–4.9 5–8.0

Negligible Slight Moderate Strong (primary irritant)

TABLE 3.21 Federal Fungicide, Insecticide, and Rodenticide Act (EPA-FIFRA) Dermal Classification System59 Toxicity Category

Warning Label

I

Corrosive. Causes eye and skin damage (or irritation). Do not get in eyes, on skin, or on clothing. Wear goggles or face shield and gloves when handling. Harmful or fatal if swallowed. (Appropriate first aid statement required.) Severe Irritation at 72 h. Causes eye (and skin) irritation. Do not get on skin or on clothing. Harmful if swallowed. (Appropriate first aid statement required.) Moderate Irritation at 72 h. Avoid contact with skin, eyes, or clothing. In case of contact immediately flush eyes or skin with plenty of water. Get medical attention if irritation persists. Mild or slight irritation at 72 h. (No precautionary statements required.)

II III IV

TABLE 3.22 European Economic Community (EEC) Dermal Classification System60 Mean Erythema Score 0.00–1.99 ≥ 2.00 Mean Edema Score 0.00–1.99 ≥ 2.00

Irritation Rating Nonirritant Irritant Irritation Rating Nonirritant Irritant

TABLE 3.23 Federal Hazardous Substances Act (CPSC-FHSA) Dermal Classification System56 Primary Irritation Score

Irritation Rating

0.00–4.99 ≥ 5.00

Nonirritant Irritant


TABLE 3.24 Draize Dermal Classification System51 Primary Irritation Index

Irritation Rating

5

Mildly irritating Moderately irritating Severely irritating

TABLE 3.25 Department of Transportation (DOT), Occupational Safety and Health Administration (OSHA), and International Maritime Organization (IMO) Packing Group Classification System14,17,61 Packing Group

Definition

I

Materials that cause full-thickness destruction of intact skin tissue within an observation period of up to 60 min starting after the exposure time of 3 min or less. Materials other than those meeting Packing Group I criteria that cause full-thickness destruction of intact skin tissue within an observation period of up to 14 days starting after the exposure time of more than 3 min but not more than 60 min. Materials, other than those meeting Packing Group I or II criteria — 1. That cause full-thickness destruction of intact skin tissue within an observation period of up to 14 days starting after the exposure time of more than 60 min but not more than 4 h; or 2. That do not cause full-thickness tissue destruction of intact skin tissue but exhibit a corrosion rate on steel or aluminum surfaces exceeding 6.25 mm (0.25 in.)/year at a test temperature of 55ºC (130ºF).

II

III

TABLE 3.26 Maximization Sensitization Classification System38 Sensitization Rate, %

Grade

0 > 0–8 9–28 29–64 65–80 81–100

— I II III IV V

Classification Nonsensitizer Weak sensitizer Mild sensitizer Moderate sensitizer Strong sensitizer Extreme sensitizer

TABLE 3.27 Optimization Sensitization Classification System51 Intradermal Positive Animals % s, > 75 s, 50–75 s, 30–50 n.s., 0–30

Epidermal Positive Animals % And/or s, > 50 And/or s, 30–50 n.s., 0–30 n.s., 0

Classification Strong sensitizer Moderate sensitizer Weak sensitizer No sensitizer

s, significant; n.s., not significant (using Fisher’s Exact Test).



SECTION 7. MATERIALS THAT PRODUCE DERMAL IRRITATION AND/OR SENSITIZATION TABLE 3.28 Common Materials Used as Positive Controls10,11,25,27,29–32,44,46,47,50 Material

CAS No.

Suggested Concentrations

Category

Sodium lauryl sulfate Hexyl cinnamic aldehyde Mercaptobenzothiazole Benzocaine p-Phenylenediamine 2,4-Dinitrochlorobenzene (DNCB)

151-21-3 101-86-0 149-30-4 94-09-7 106-50-3 97-00-7

Irritant Mild to moderate sensitizer Mild to moderate sensitizer Mild to moderate sensitizer Sensitizer Sensitizer

Potassium dichromate Neomycin sulfate Nickel sulfate 8-Methoxypsoralen (Oxsoralen Lotion®) 5-Methoxypsoralen (Bergapten) 2,4-Dinitro-3-methyl-6-tertiary-butylanisole (musk ambrette)

7778-50-9 1405-10-3 7786-81-4 298-81-7 298-81-7 83-66-9

2-Chloro- 10-[3-dimethylaminopropyl] phenothiazine hydrochloride (chloropromazine) 3,3,4,5-Tetrachlorosalicylandide (TCSA)

50-53-3

1.0% — — — — Induction: 0.1 to 0.5%, 0.25% w/v in echanol/acetone Challenge: 0.1 to 0.3%, w/v in ethanol/acetone — — — 1.0% 1.0% Induction: 10.0% w/v in ethanol/acetone Challenge: 0.5% w/v in ethanol/acetone Induction: 1.0% w/v in methanol Challenge: 0.1% w/v in methanol Induction: 1.0% w/v in acetone Challenge: 1.0% w/v in acetone

Photosensitizer (in mice and guinea pigs), possible sensitizer in guinea pigs

1154-59-2

Sensitizer Sensitizer Sensitizer Photoirritant Photoirritant Photosensitizer

Photosensitizer

TABLE 3.29 Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material

Irritants

Sensitizer

Photoirritant

Clothing and Textiles Dyes Aminoazotoluene Anthraquinones dyes (Disperse blue 35) Azo dyes Chromium dyes Disperse yellow 39 (methene dye) Naphthol AS (azo dye) p-Phenylenediamine (PPD) Resins Dimethyl oldihydroxyethylene Dimethyl olethylene urea Dimethyl urea (urea formaldehyde) Formaldehyde


Photosensitizer


TABLE 3.29 (Continued) Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material

Irritants

Rubber boots lsopropylaminodiphenylamine (IPPD) Mercaptobenzothiazole (MBT) Fiberglass

Aldehyde citronellal Aluminum palls (in deodorants) Ammonium persulfate Balsam Peru Benzoyl salicylate Celaltronium chloride Chloro-3,5-xylenol 4-(chloroxylenol) Cinnamic acid Cinnamic aldehyde di-tert-butyl hydrogunone dl-α−tocopherol Henna Lanolin Lemongrass oil Perfume Propellants in deodorant Sorbitan monostearate Sorbitan monoleate Triethanolamine Zerconium

Artichoke Asparagus Carrot Cheese Chives Cucumber Endive Fish Flour Garlic Horseradish Leek Lemon peel Lettuce Meats


Photoirritant

Photosensitizer

Melamine formaldehyde Adhesives Dodecyl mercaptan p-tertiary-butylphenol formaldehyde (PTBP resin)

Sensitizer

Cosmetics

Foods



Irritants

Ampicillin Antihistamines Benadryl Benzoic acid Benzophenone Benzoyl peroxide Coumarin Dimercaprol Estrogen cream Fluorouracil Gentian violet Hydrocortisone Mafenide acetate Monoamyl amine Neomycin sulfate Oxyphenbutazone p-Chlorobenzenesulfonylglycolic acid nitrile Penicillin Pristinamycin Promethazene hydrochloride Quinoderm Retinoic acid Salicylic acid Streptomycin Sulisobenzone Sulfonamides Tetracycline (also phototoxic)


Photoirritant

Photosensitizer

Onion Poultry skin and flesh Shellfish Wheat flour Vanilla

4-Hydroxybenzoic acid Ammonium and potassium persulfates Butylated hydroxyanisole Butylated hydroxytoluene Dodecyl gallate Ethoxyquin Hydroquinone Monosodium glutamate Octyl gallate Propyl gallate Sodium benzoate Sorbic acid Sulfur dioxide

Sensitizer

Food Additives

Medicants



Irritants

Sensitizer

Photoirritant

Photosensitizer

Metals Arsenic Beryllium salts Cadmium sulfide Chromate Nickel Selenium

Dichlorphene Dinitrochlorobenzene Dinobuton Diothiocarbarnates Lindane Malathion Maneb Omite Randox Zineb

Angelica Anise Boneset Burdock Caraway Celeriac Celery Chamomile Cocklebur Coriander Cow parsley Daffodil, narcissus Dill Fennel Feverfew Giant hogweed Hogweed, cow parsnip Ivy Marshelder Masterwort Poison ivy and oak Poverty weed Primula Pyrethrum, tansy Ragweed Ragweed of florists and species (alantolactone and parthenium) Sage brush/wormwood

Pesticides Plants




Irritants

Fruits and Vegetables Woods

Abura African blackwood African mahogany American mahogany Australian blackwood Ayan Camphor Cassia oil Ceylon satinwood Cocobolo Cocus Common alder Douglas fir English elm Gaboon Grevillea lpe Iroko Limba Louro Macassar ebony Makore Mansonia Opepe Peroba rosa Pine oil Ramin Teak Toporite Western red cedar


Photoirritant

Photosensitizer

Sneezeweed (alantolactone and parthenium) Tansy Tulip

Artichoke Brussels sprouts, cabbage Carrot Celery Chicory, endive Chive, leek, onion, garlic Horseradish Lettuce Orange, lemon, lime Parsley Parsnip Pineapple

Sensitizer


TABLE 3.29 (Continued) Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material White peroba White poplar West Indian satinwood Yew Acrylamide Acrylonitrile Cyanoacrylic acids and esters Diacrylates (delayed) Methacrylonitrile (poison) Methyl, ethyl, and n-butyl methacrylates

Irritants

Sensitizer

Plastics Epoxy Resin Systems

Allyl resin — diallylglycol carbonate Diallylphthalate Dimethylaniline (poison) Diphenylmethane diisocyanate Epoxy monomer Hardener Hexamethylene diisocyanate Maleic acid anhydride Napththalene diisocyanate Naphthoquinone (poison) Peroxides (catalyst) Phthalic acid anhydride p-tert-Butyl phenol formaldehyde (PTBP) Reactive diluent Toluene diisocyanate Isophoronediamine N-Aminoethylpiperazine Polyether alcohol Polyurethane laquar Triethylamine Cellophane Celluloid Cellulose nitrate Collodion Gun cotton Pyroxylin Rayon Regenerated cellulose Cellulose Acetate Antioxidant: p-tert-butyl phenol Colors: azo dyes solvent yellow 3, solvent red 26, pigment red 481


Hardeners Cellulose Polymers

Photoirritant

Photosensitizer



Irritants

Sensitizer

Photoirritant

Components: plastizer, triphenyl phosphate Polish turpentine Solvent: ethylene glycol monomethyl ether acetate Ultraviolet light stabilizer: resorcinol monobenzoate

Preservatives and Antibacterials Preparations Containing Lanolins (I)/Parabins (P)/Chlorocresol (c) Adcortyl cream (p) Betnovate cream (c) Betnovate lotion (p) Cortenema (p) Dermovate cream (c) Efcortelan cream (c) Efcortelan lotion (p) Hydrocortistab eye ointment (1) Hydromycin-Dornluent ear/eye (1) Medrone acne lotion (p) Medrone cream (p) Motivate cream (c) Myciguent ointment (1) Myciguent opthalmic ointment (1) Neo-Cortef eye and ear ointment (1) Neo-Cortef ointment (p) Neo-Cortef lotion (p) Neo-Medione acne lotion (p) Nerisone cream (p) Nystadermal cream (p) Nystadermal gel (c) Propaderm cream (c) Propaderm lotion (c) Remiderm cream (p) Schericur ointment (l) Synolar creams except for Synalar Forti cream (p) Synolar combination creams (p) Synolar ointments (l) Topilar ointment (l) Topisone (l) Triadcortyl cream (p) Ultradil cream plain (p) Ultradil ointment plain (l) Ultralanum cream plain (l) Ultralanum lotion (p) Ultralanum ointment (l) Ultralanum ointment plain (l) Phenolic Compounds Hexachlorophane


Photosensitizer



Irritants

Sensitizer

Photoirritant

Mercury Ammoniated mercury Mercuric chloride Mercurochrome Mercury fulminate (mercuric cyanate) Merthiolate Phenylmercuric acetate Phenylmercuric borate Phenylmercuric nitrate Phenylmercuric proprimate Quaternary Ammonium Compounds Benzalkonium chloride Bronopol Cetalkonium chloride Cetrimide Chlorhexidine Chloroacetamide Ethylene oxide

?

Rubber/Latex Components

Thiurams Dipentamethylenethiuram disulfide Tetraethylthiuram disulfide Tetramethylthiuram disulfide Tetramethylthiuram monosulfide

Mercapto Group Cyclohexylbenzothiazylsulfenamide Dibenzothiazyldisulfide Mercaptobenzothiazole Morpholinylmercaptobenzothiazole

PPD Group Diaminodiphenylmethane Diphenyl-PPD Isopropylphenyl-PPD (isopropylamino diphenylamine) Phenylcyclohexyl-PPD

Naphthyl Group Phenyl-β−naphthylamine sym-Di-β−napthyl-PPD

Carbamates Zinc diethyldithiocarbamate Zinc dibutyldithiocarbamate

Miscellaneous Dioxydiphenyl Diphenylguanidine Dithiodimorpholine


Photosensitizer



Irritants

Sensitizer

Miscellaneous Compounds Acetaldehyde (10%) Acetyl-1,1,2,3,3,6-hexamethylindan Acetylacetone (slightly) Acridine Acriflavine Alcohol, anhydrous Allyl butyrate (4%) Allyl cinnamate (0.1%) Allyl cyclohexylacetate Allyl epoxypropoxybenzene Allyl phenylacetate Aminobenzoic acid derivatives Aminobenzoic acid p− Aminophenol o− and p− Aminosalicylic acid p− Aminothiazole Aminodarone Ammonia Amyl dimethylamino benzoate, mixed ortho and para isomers Amyl dimethyl PABA Amyl nitrite Amyl phenylacetate Anthracene-acridine Atranorin Benzaldehyde Benzydamine hydrochloride Bergamot oil Bergapten (5-methoxypsoralen) Bromomethyl-4-nitrobenzene Buclosamide Butylphenol Cadmium chloride Cadmium sulfate Caraway oil Carbimazole Carotene βCephalosporins Cetyl alcohol Chlor-2-phenylphenol Chloramine-T Chlormercaptodicarboximide Chloro-6-fluorobenzaldehyde-α-chlorooxime2– Chlorodiazepoxide Chlorothalonil Chlorothiazides Chlorpromazine


Photoirritant

Photosensitizer


TABLE 3.29 (Continued) Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material Cinoxate Cinnamon bark oil Ceylon Citral Clobetasol-17-proprionate Coal tar Cobalt chlorate Cobaltous chloride Cobaltous chlorate Cobaltous nitrate Cobaltous sulfate Cocamide DEA Cocamphocarboxyglycinate Coniferyl benzoate Cu(II)-acetyl acetonate Cumin oil Cyanamide Dacarbazine Decylaminoethanethiol 2-n− Deneclocycline Dexpanthenol Diamine N− Diaminodiphenylmethane Dibucain hydrochloride Dichloro-2-phenylphenol Dichloroquinoline Dicyclohexylcarbodiimide Diethazine Diethylaminopropylamine Diethyl fumarate Diethylstilbestrol Digalloyl trioleate Diglycidyl ether Dihydrocoumarin Dimethoxane Dimethyl antranilate Dimethyl sulfoxide Dioctyl-p-phenylenediamine Diphenhydramine hydrochloride Diphenylcyclopropenone Diphenyl-p-phenylenediamine Dipyrone Docusate sodium Erythrosine Ethacridine lactate monohydrate Ethyl aminobenzoate Ethyl ether Ethylparaben Fig leaf absolute Furocoumarins


Irritants

Sensitizer

Photoirritant

Photosensitizer


TABLE 3.29 (Continued) Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material Geraniol Geranyl formate Ginger oil Griseofulvin Gylceryl p-aminobenzoate Halogenated Phenols Bithionol Bromochlorosalicylanilide (Multifungin) Chlorophenylphenol Dibromosalicylanilide (DBS) Fentichlor Hexachlorophene Buclosamide (Jadit) Tetrachlorosalicylanilide 3, 3′, 4′, 5 (TCSA) Tribomosalicylanilide (TBSA) Trichlorcarbanilide (TCCA) Hexanediol diacrylate Hexantriol Hydratropic aldehyde Iothion Isoamyl alcohol Isocamphyl cyclohexanol Isopropyl alcohol Isostearoamphopropionate Ketoprofen Kynuremic acid Lauroamphocarboxyglycinate Lauroamphoglycinate Lauroamphopropionate Lauryl isoquinolinium bromide Lavender oil Mannide monooleate Mechlorethamine hydrochloride (nitrogen mustard) Menthol 1Mepazine Metamizole Methoxyethylepoxypropoxybenzene Methylanisalacetone αMethylcoumarin 6− Methylcoumarin 7− Methylene blue Methylisothiazolinone Methylparaben Methyl salicylate Minoxidil Musk ambrette Musk xylol Mycanodin Neosilversalvarsan


Irritants

Sensitizer

Photoirritant

Photosensitizer


TABLE 3.29 (Continued) Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material Neroli oil Neutral Red Nicotinyl alcohol Nitrofuroxime Nitrose dimethyl aniline Nonoxynol-9 Oak moss Octoxynol-9 Oleamide Oxybenzone Padimate A or Escalol 506 (amyl p-dimethylaminobenzoate) Papain PBA-1 Pelargonic acid Pentadecylcatechol 3– Pentamethyl-4,6-dinitroindane 1, 1, 3, 3, 5− Pentanol Pentylidenecyclohexanone Perphenazine Petitgrain oil Paraguay Phenanthrene Phenol Phenothiazine Phenylacetaldehyde Phenylbenzimidazol sulfate 2− Phenyl butazone Phenyl gylcidyl ether Phenylphenol Phosphorus sesquisulfide Picryl chloride Pigment orange 5 Pigment orange red 49, calcium lake Pinus pumilio oil Pitch Platinum salts Polysorbate 20 Polysorbate 60 Polysorbate 80 Primin Prochlorperazine Promazine Propionaldehyde Propyl alcohol n– Propylphenbutazone Psoralens Pyridine Pyridoxine hydrochloride Pyrilamine maleate


Irritants

Sensitizer

Photoirritant

Photosensitizer


TABLE 3.29 (Continued) Materials Categorized by Their Ability to Produce Dermal Irritation or Sensitization45,49,53,62,63 Material Quinine Quinine hydrochloride Quinine sulfate Quinoline methanol Rhodamine B Ricinoleic acid Rose Bengal Rue oil Silver bromide Silver fulminate Silver nitrate Sodium hypochlorite Sodium monoglyceride sulfide Sodium monoglyceride sulfonate Sodium octoxynol-2 ethane sulfonate Sodium oleyl laurate Sodium stearate Sodium sulfide Sodium thiosulfate Sorbitan laurate Squaric acid-diethylester Stearalkonium chloride Stearoamphoglycinate Stearyl alcohol Stictic acid Stilbene triazine Sulbentine Sulfadiazine Sulfamerazine Sulfamethazine Sulfanilamide Sulfathiazole Sulfur Sulisobenzone Thioridazine Thiourea Thurfyl nicotinate Toluidine red Tribomsalan Tributyltin oxide Trichlorosalicylanilide 3’,4’,5− Triclocarban Triethylenemelamine Trimeprazine Trinitrobenzene sym− Tropicamide Turkey-red oil Umbelliferone Undecylenic aldehyde digeranyl acetal


Irritants

Sensitizer

Photoirritant

Photosensitizer



Irritants

Sensitizer

Photoirritant

Photosensitizer

Valeraldehyde Vetiverol Vinyl pyridine 4− Xanthotoxin (8-methoxypsoralen) Zinc pyrithione

TABLE 3.30 Dermal Irritants and Sensitizers Listed by Occupation45,49,53,62,63 Occupation

Irritant

Agricultural workers

Artificial fertilizers Disinfectants and cleansers for milking machines Petrol and diesel oil

Artists and sculptors

Solvents Clay Plaster

Automobile and aircraft mechanics

Solvents Cutting oils Paints Hand cleansers

Bakers and confectioners

Flour Detergents

Bartenders

Detergents Citrus fruit


Sensitizer Rubber (clothing and milking equipment) Oats Barley Animal feed (antibiotics, preservatives, additives, and cobalt) Veterinary medicaments Cement Plants Pesticides Wood Preservatives Turpentine Pigments (cobalt, nickel, and chromium) Azo dyes Anthraquinone dyes Aminoazotoluene Colophony Epoxy resin Chromate (primers, passivators, anticorrosives, welding fumes, oils) Nickel Cobalt Rubber Epoxy resins Dimethacrylate resins Dipentene in thinners Flavors and spices (cinnamon, eugenol, vanilla, cardamom) Orange Lemon Lime Pineapple Essential oils Dyes Ammonium persulfate Benzoyl peroxide (improvers in flour) Orange Lemon


TABLE 3.30 (Continued) Dermal Irritants and Sensitizers Listed by Occupation45,49,53,62,63 Occupation

Irritant Wet work

Bookbinders

Solvents Glues

Butchers

Detergents Meat Offal

Cabinetmakers, French polishers, carpenters

Detergents Solvents Thinners for cleaning metal (as a cause of koilonychia, Ancona- Alayon, 1975) Wood and wood preservatives

Cablejointers

Solvents

Cleaners

Detergents Solvents Wet work Dust (coal, stone) Cement Wet conditions Cement

Coal miners

Construction workers

Cooks and catering

Detergents Food juices Wet work Parsley Parsnip Carrots


Sensitizer Lime Flavors ortho-Phenylphenol (in some detergents) Glues Resins Leather Meat (contact urticaria) Teak (knife handles) Nickel Sawdust Stains (including dichromate) Glues (urea, phenol, PTBP-formaldehyde resins) Woods Turpentine Varnishes Colophony Epoxy resin Fluxes (aminoethylethanolamine) Rubber gloves Chromates (bleaches in some countries) Rubber boots Masks Chromate Cobalt Gloves (rubber, leather) Resins (epoxy and formaldehyde) Woods Foods (contact urticaria) Onion Garlic Lettuce Carrots Celery Parsley Parsnip Brussels sprouts Cabbage Spices Flavors Rubber gloves Sodium metabisulfite Lauryl Octyl gallate Formaldehyde (deodorizing solution, fishmongers)



Irritant

Dentists and dental technicians

Detergents Hand cleaners Wet work

Dry cleaners Electricians

Solvents Soldering fluxes

Electroplaters

Acids Alkalis

Embalmers and morticians

Disinfectants Detergents Solvents

Floor layers

Florists

Foundry workers

Manure Fertilizers Pesticides Wet work Cleansers

Funeral directors Garage workers

Gardeners

Hairdressers

Petroleum products Diesel fuel Cleansers Detergents Solvents Artificial fertilizers

Shampoos Perming solutions Bleaching solutions


Sensitizer Local anesthetics (amethocaine, procaine) Methacrylates Eugenol (eugenol and colophony gingivectomy dressing) Mercury Disinfectants Rubber Dental impression material (Impregum and Scutan: the sensitizers are the catalysts methyldichlorobenzene sulfonate and methyl-p-toluoylsulfonate Rubber gloves Fluxes (colophony, hydrazine) Insulating tape (colophony) Resins (epoxy and formaldehyde) Rubber Nickel Chromium Other metals Rubber gloves Formaldehyde Cement Resins (epoxy and formaldehyde) Woods Varnish Linoleum (colophony) Plants (alantolactone and parthenium) Pesticides (DNCB, dichlorphene, lindane) Rubber gloves Phenol and urea formaldehyde (resin-coated sand) Colophony (nitrogen-free sand) Gloves (rubber, chromium) Floral tributes (alantolactone and parthenium) Rubber gloves Chromate Epoxy resin Antifreeze (MBT) Pesticides Plants/flowers Rubber gloves Boots Dyes (p-phenylenediamine, p-toluoylenediamine, o-nitro-pphenylenediamine, p-aminiphenol, henna)



Irritant Wet work

Hospital workers

Detergents Disinfectants Foods Wet work

Housework

Detergents Cleaners Foods Disinfectants Wet work

Jeweler

Detergents Solvents

Metal workers

Cutting and drilling oils Solvents Hand cleansers Disinfectants Detergents Wet work

Nurses

Office workers

Photocopying (ammonia)

Painters

Solvents Thinners Wallpaper adhesives


Sensitizer Persulfates Rubber gloves Lanolin Perfumes Lemongrass oil Formaldehyde (shampoos) Resorcinol Pyrogallol Nickel Rubber gloves Disinfectants Flowers Foods Polishes Hand creams Rubber gloves Foods (onions, garlic, citrus fruit; contact urticaria) Spices Flavors Hand creams Nickel Chromate (bleaches) Flowers Polishes Epoxy resin Metals (nickel, chromium) Sawdust (used for drying jewelry) Chromates Additives in cutting oils (antibacterials and antioxidants) Rubber gloves Formaldehyde Glutaraldehyde Dettol Disinfectants Medicaments (including antibiotics, antihistamines, hydrocortisone, retinoic acid, chlorpromazine) Flowers Rubber (finger stalls) Nickel (clips, photocopying solutions) Copy papers Carbon papers Correction paper fluids Turpentine Dipentene Cobalt (driers, colors)



Irritant Hand cleansers

Photograph developers (X-ray technicians)

Wet work Solvents

Plastic industry

Solvents Acids Styrene Oxidizing agents Hardeners (Polyurethane lacquer, triethylamine)

Plating industry

Acids Alkalis Solvents

Plumbers Printers

Wet work Cleaners Solvents

Radio and television workers

Fluxes

Rubber workers

Solvents Talc Zinc stearate

Secretaries


Sensitizer Chromate (colors) Wallpaper adhesives (formaldehyde, chloroacetamide, and fungicides) Paints (preservatives, e.g., mercurials) Rubber gloves p-Aminophenol (Metol) Color developers Hydroquinone Phenindone Sodium metabisulfite EDTA Glutaraldehyde Pyrogallol Amidol Ethylenediamine Resorcinol Triazine Salicylaldoxime Monomers Hardeners (isophoronediamine, polyether alcohol) Additives Cellulose polymers Cellulose acetate Epoxy resin systems Nickel Chromate Cobalt Mercury Chromate (cement) Rubber (gloves, packing) Chromate UV-cured inks Colophony (paper) Turpentine Rubber gloves Rubber blanket in offset printing Formaldehyde (gum arabic) Resins (epoxy) Fluxes (colophony and hydrazine) Chromate Rubber chemicals Dyes Colophony Carbon paper Photocopy paper (azo compound, thiourea-photosensitizer) Correcting paper Rubber (fingerstall and rubber bands)



Irritant

Shoemakers and cobblers

Solvents

Tannery workers

Acids Alkalis Reducing and oxidizing agents Wet work

Textile workers

Fibers Bleaching agents Solvents

Veterinarians (and slaughterhouse workers)

Disinfectants Wet work Entrails Animal secretions

Woodworkers

Woods

Sensitizer Glues (PTBP resin, colophony) Leather Rubber Turpentine Tanning agents (chromium, vegetable tans, glutaraldehyde, formaldehyde) Rubber (gloves and boots) Fungicides Dyes Formaldehyde resins Dyes Chromate (mordant) Nickel Rubber gloves Medicaments used to treat animals and which contaminate their fur Medicaments Tuberculin Benethamate Benzylpenicillin Spiramycin Tylosin Penethamete Neomycin in a calf drench Mercaptobenzothiazole in a medication Benzisothiazolone fungicide Topical pesticides; malathion Contact urticaria from animal tissues Cow hair and dander in bacon factories, workers eviscerating or cleaning the guts develop an eczema of the fingers, known as “gut” or “fat” eczema; its cause is unknown Lichens (atranorin) Glues Varnishes Colophony Turpentine Balsams

SECTION 8. GLOSSARY OF COMMON TERMINOLOGY 64–68 Acanthosis Hypertrophy of the stratum spinosum and granulosum. Blanching To take color from, to bleach. Characterized by a white or pale discoloration of the exposure area due to decreased blood flow to the skin (ischemia). Challenge exposure A dermal exposure to a test substance after one or more previous induction exposures, to determine whether the subject will react in a hypersensitive manner.


Concomitant sensitization When an individual is sensitized to different substances in different products at the same time. Contact dermatitis A delayed type of induced sensitivity (allergy) of the skin with varying degrees of erythema, edema, and vesiculation, resulting from cutaneous contact with a specific allergen. Contact urticaria Wheal-and-flare response generally elicited within 30 to 60 minutes after cutaneous exposure to a test substance. May be IgE mediated or nonimmunologically mediated. Corrosion Direct chemical action on normal living skin that results in its disintegration or irreversible alteration at the site of contact. Corrosion is generally manifested by ulceration and necrosis with subsequent scar tissue formation. Cross-sensitization An individual that is sensitized to a primary allergen acquires sensitivity to a chemically related molecule, which is called a secondary allergen. Cumulative irritation Irritation resulting from repeated exposures to materials at the same skin site. Dermatitis Inflammation of the skin. Desquamation The shedding of the cuticle in scales or the outer layer of any surface. To shred, peel, or scale off, as the casting off of the epidermis in scales or shreds, or the shedding of the outer layer of any surface. Diagnostic patch testing Utilized to confirm the existence of allergic contact dermatitis. A concentration of the test substance that is known to be nonirritating is applied to the skin in a suitable vehicle. Eczema Inflammatory condition in which the skin becomes red and small vesicles, crusts, and scales develop. Edema An excessive accumulation of serous fluid or water in cells, tissues, or serous cavities. Erythema An inflammatory redness of the skin, as caused by chemical poisoning or sunburn, usually a result of congestion of the capillaries. Eschar A dry scab, thick coagulated crust or slough formed on the skin as a result of a thermal burn or by the action of a corrosive or caustic substance. Exfoliation To remove in flakes, scales or to peel. To cast off in scales, flakes, or the like. To come off or separate, as scales, flakes, sheets, or layers. Detachment and shedding of superficial cells of an epithelium or from any tissue surface. Scaling or desquamation of the horny layer of epidermis, which varies in amount from minute quantities to shedding the entire integument. False cross-sensitivity Occurs when the same antigen is present in different products (e.g., eugenol in perfumes, soft drinks, and underarm deodorants). Fissuring Characterized by a crack or cleft in the skin. Hyperkeratosis Hypertrophy and thickening of the stratum corneum. Index of sensitivity The prevalence of sensitivity to a substance in a given population at a given time. Induction exposure An experimental exposure to a test substance with the intention of inducing a hypersensitive state. Induction period A period of at least 1 week after a dermal exposure during which a hypersensitive state is developed. Irritant A substance that causes inflammation and other evidence of irritation, particularly of the skin, on first contact or exposure; a reaction of irritation not dependent on a mechanism of sensitization. Latent sensitization Subsequent exposure of the skin of a sensitized individual to a lower concentration of a sensitizer can elicit a more intense response than the initial exposure. This response may take hours or even days to develop, and hence it is delayed.


Necrosis Pathologic death of one or more cells, or of a portion of tissue or organ, resulting from irreversible damage. Nonocclusive Site of application of test substance to the skin is not covered with any material and movement of the air to the site is not restricted. Occlusive A bandage or dressing that covers the skin and excludes it from air. Prevents loss of a test substance by evaporation. Photoallergy An increased reactivity of the skin to UV and/or visible radiation produced by a chemical agent on a immunologic basis. Previous allergy sensitized by exposure to the chemical agent and appropriate radiation necessary. The main role of light in photoallergy seems to be in the conversion of the hapten to a complete allergen. Photoirritation Irritation resulting from light-induced molecular changes in the structure of chemicals applied to the skin. Photosensitization The processes whereby foreign substances, either absorbed locally into the skin or systemically, may be subjected to photochemical reactions within the skin, leading to either chemically induced photosensitivity reactions or altering the “normal” pathologic effects of light. UV-A is usually responsible for most photosensitivity reactions. Semiocclusive Site of application of test substance is covered; however, movement of air through covering is only partially restricted. Sensitization (allergic contact dermatitis) An immunologically mediated cutaneous reaction to a substance. Sensitizing potential The relative capacity of a given agent to induce sensitization in a group of humans or animals. Superficial sloughing Characterized by dead tissue separated from a living structure. Any outer layer or covering that is shed. Necrosed tissue separated from the living structure. Ulceration The development of an inflammatory, often suppurating lesion, on the skin or an internal mucous surface of the body caused by superficial loss of tissue, resulting in necrosis of the tissue.

REFERENCES 1. U.S. Environmental Protection Agency, Health Effects Guidelines, OPPTS 870.2500, Acute Dermal Irritation, August, 1998. 2. Japan Agricultural Chemicals Laws and Regulations, Testing Guidelines for Toxicology Studies, 25, 1985. 3. Japan Ministry of Agriculture, Forestry and Fisheries, Agricultural Chemicals Inspection Station, Guidelines on the Compiling of Test Results on Toxicity, Skin Irritation Test, 9–11, December, 1998 (Draft Proposal). 4. Organization for Economic Co-operation and Development, OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Subsection 404: Acute Dermal Irritation/Corrosion, 1, 1992. 5. The Commission of the European Communities, Official Journal of the European Communities, Part B: Methods for the Determination of Toxicity, No. L 383 A/124, B.4. Acute Toxicity (Skin Irritation), 1992. 6. U.S. Environmental Protection Agency, Federal Insecticide, Fungicide, Rodenticide Act, Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Series 81-6: Dermal Sensitization Study, 59, 1984. 7. Japan New Drugs Division Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, 1990 Guidelines for Toxicity Studies of Drugs Manual, 1991, Chap. 7. 8. Japan Agricultural Chemicals Laws and Regulations, Testing Guidelines for Toxicology Studies, 27, 1985. 9. Japan Ministry of Agriculture, Forestry and Fisheries, Agricultural Chemicals Inspection Station, Guidelines on the Compiling of Test Results on Toxicity, Skin Sensitization Test, 16–19, December, 1998 (Draft Proposal).


10. Organization for Economic Co-operation and Development, OECD Guidelines for Testing of Chemicals, Section 4: Health Effects, Subsection 406: Skin Sensitization, 1, 1992. 11. The Commission of the European Communities, Official Journal of the European Communities, Part B: Methods for the Determination of Toxicity, No. L 383 A/131, B.6: Skin Sensitization, 1992. 12. U.S. Consumer Products Safety Commission, 16 CFR Chapter II, Subchapter C: Federal Hazardous Substances Act Regulation, Part 1500, Subsection 1500.41: Method of Testing Primary Irritant Substances, 401, 1993. 13. U.S. Consumer Products Safety Commission, 16 CFR Chapter II, Subchapter C: Federal Hazardous Substances Act Regulation, Part 1500, Subsection 1500.3: Definitions, 382, 1993. 14. U.S. Research and Special Programs Administration, Department of Transportation, 49 CFR, Part 173. 136 and 173. 137, 1998. 15. U.S. Pharmacopeia, National Formulary, USP 24 NF 19 Biological Reactivity Test, IN-VIVO, 1832, 2000, Chap. 88. 16. Canadian Environmental Protection Act, Guidelines for the Notification and Testing of New Substances: Chemicals and Polymers, Section 5.1: Test Procedures and Practices, 50, 1993. 17. International Maritime Dangerous Goods Code, Class 8 Corrosives, International Maritime Organization, London, 1994. 18. Occupational Safety and Health Administration, Labor, 29 CFR Chapter XVII, Part 1910, Appendix A to Section 1900.1200: Health Hazard Definitions (Mandatory), 364, 1991. 19. American Society for Testing and Materials, 1991 Annual Book of ASTM Standards, F719–81 (1986), 13.01: Practice for Testing Biomaterials in Rabbits for Primary Skin Irritation, 976, 1991. 20. American Society for Testing and Materials, 1991 Annual Book of ASTM Standards, F720–81 (1986), 13.01, Practice for Testing Guinea Pigs for Contact Allergens: Guinea Pig Maximization Test, 976, 1991. 21. American Society for Testing and Materials, 1991 Annual Book of ASTM Standards, E 993–88, 11.01, Test Method for Evaluation of Delayed Contact Hypersensitivity, 964, 1991. 22. The Cosmetic, Toiletry and Fragrance Association, Inc., CTFA Safety Testing Guidelines, Section II: Guidelines for Evaluating Primary Skin Irritation Potential, 2, 1991. 23. The Cosmetic, Toiletry and Fragrance Association, Inc., CTFA Safety Testing Guidelines, Section IV, Guidelines for Evaluating Contact Sensitization Potential, 7, 1991. 24. Draize, J. H., Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, The Association of Food and Drug Officials of the United States, 49, 1959. 25. Springborn Laboratories, Inc., Protocol for a Primary Irritation Study in Rabbits, EPA/PSI 1 — 10/2000, Spencerville, OH, 2000. 26. Springborn Laboratories, Inc., Protocol for a Dermal Corrosivity Study in Rabbits, DOT/COR-1 — 5/2000, Spencerville, OH, 2000. 27. Hakim, R.E., Freeman, R.G., Griffin, A.C., and Knox, J.M., Experimental toxicologic studies on 8-methoxypsoralen in animals exposed to the long ultraviolet, J. Pharmacol. Exp. Ther., 131:394, 1961. 28. OECD Guidelines for the Testing of Chemicals, Acute Dermal Photoirritation Screening Test, February, 1995 (Draft Proposal). 29. Springborn Laboratories, Inc., Protocol for a Photoirritation Study in Rabbits with Non-Occlusive Conditions, SLI No. OECD/PHIRBU-1-8/2000, Spencerville, OH, 2000. 30. Springborn Laboratories, Inc., Protocol for a Photoirritation Study in Rabbits with Occlusive Conditions, SLI No. OECD/PHIRBO-1-8/2000, Spencerville, OH, 2000. 31. Springborn Laboratories, Inc., Protocol for a Photoirritation Study in Guinea Pigs with Non-Occlusive Conditions, SLI No. OECD/PHIGPU-1-8/2000, Spencerville, OH, 2000. 32. Springborn Laboratories, Inc., Protocol for a Photoirritation Study in Guinea Pigs with Occlusive Conditions, SLI No. OECD/PHIGPO-1-8/2000, Spencerville, OH, 2000. 33. Gad, S.C. and Chengelis, C.P., Photosensitization and phototoxicity, Acute Toxicol. Testing, 117, 1990. 34. Buehler, E.V. and Griffin, F., Experimental skin sensitization in the guinea pig and man, Animal Models Dermatol., 55, 1975. 35. Springborn Laboratories, Inc., Protocol for a Dermal Sensitization Study in Guinea Pigs — Modified Buehler Design, EPA/MB-1–10/2000, Spencerville, OH, 2000. 36. Buehler, E.V., Delayed contact hypersensitivity in the guinea pig, Arch. Dermatol, 91, 171, 1965. 37. Springborn Laboratories, Inc., Protocal for a Dermal Sensitization Study in Guinea Pigs — Standard Buehler Design, EPA/MB-1–10/2000, Spencerville, OH, 2000.


38. Magnusson, B. and Kligman, A., Allergic Contact Dermatitis in the Guinea Pigs, C.C. Thomas, Springfield, IL, 1970. 39. Springborn Laboratories, Inc., Protocol for a Dermal Sensitization Study in Guinea Pigs — Maximization Design, EPA/MAX-1–10/2000 Spencerville, OH, 2000. 40. Gerberick, G.F., Kimber, I., and Basketter, D., The local lymph node assay ICCVAM test method submission, April, 1998, in The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds, NIH Publication No. 99-4494, February 1999. 41. Kimber, I., Hilton, J., Dearman, R., Gerberick, G.F., Ryan, C., Basketter, D.A., Lea, L., House, R.V., Ladics, G.S., Loveless, S.E., and Hastings, K.L., Assessment of the skin sensitization potential of topical medicaments using the local lymph node assay: an interlaboratory evaluation, J. Toxicol. Environ. Health, A, 52, 563–579, 1998. 42. Loveless, S.E., Ladics, G.S., Gerberick, B.F., Ryan, C.A., Basketter, D.A., Scholes, E.W., House, R.V., Hilton, J., Dearman, R.J., and Kimber, I., Further evaluation of the local lymph node assay in the final phase of an international collaborative trial, Toxicol., 108, 141–152, 1996. 43. Dearman, R.J., Hilton, J., Evans, P., Harvey, P., Basketter, D.A., and Kimber, I., Temporal stability of local lymph nodes assay response to hexylcinnamic aldehyde, J. Appl. Toxicol., 18, 281–284, 1998. 44. Siglin, J.C., Jenkins, P.K., Smith, P.S., Ryan, C.A., and Gerberick, G.F., Evaluation of a New Murine Model for the Predictive Assessment of Contact Photoallergy (CPA), American College of Toxicology Annual Meeting, Savannah, GA, 1991. 45. Gerberick, G.F. and Ryan, C.A., A predictive mouse ear-swelling model for investigating topical phototoxicity, Fd. Chem. Toxicol., 27, 813, 1989. 46. Springborn Laboratories, Inc., Protocol for a Photoallergy Study in Mice, FDA/PHS-2–2/94, Spencerville, OH, 1994. 47. Ichikawa, H., Armstrong, R.B., and Harber, L.C., Photoallergic contact dermatitis in guinea pigs: Improved induction technique using Freund’s complete adjuvant, J. Invest. Dermatol., 76, 498, 1981. 48. Harber, L.C., Shalita, A.R., and Armstrong, R.B., Immunologically mediated contact photosensitivity in guinea pigs, in Dermatotoxicology, 2nd ed., Marzulli, F.N. and Maibach, H.I., Eds., Hemisphere Publishing, Washington, 1983. 49. Maurer, T., Predictive animal test methods for allergenicity, in Contact and Photocontact Allergens, a Manual of Predictive Test Methods, Vol. 3, Calnan, C.D. and Maibach, H.I., Eds., Marcel Dekker, New York, 1983. 50. Springborn Laboratories, Inc., Protocol for a Photosensitization Study in Guinea Pigs, FDA/PHS OECD-1–1/2000 Spencerville, OH, 2000. 51. Patrick, E. and Maibach, H.I., Dermatotoxicology, in Principles and Methods of Toxicology, 2nd ed., Hayes, A.W., Ed., Raven Press, New York, 1989. 52. Klecak, G., Identification of contact allergies: predictive tests in animals, in Dermatotoxicology, 2nd ed., Marzulli, F.N. and Maibach, H.I., Eds., Hemisphere Publishing, Washington, 1983. 53. Fischer, T. and Maibach, H.I., Patch testing in allergic contact dermatitis, in Exogenous Dermatoses: Environmental Dermatitis, Menné, T. and Maibach, H.I., Eds., CRC Press, Boca Raton, FL, 1991. 54. U.S. Environmental Protection Agency, Federal Insecticide, Fungicide, Rodenticide Act, Pesticide Assessment Guidelines, Subdivision F, Hazard Evaluation: Human and Domestic Animals, Series 81-5 Dermal Irritation, 55e, 1984. 55. U.S. Environmental Protection Agency, Toxic Substances Control Act, Test Guidelines, 40 CFR Part 798, Subpart E — Specific Organ/Tissue Toxicity, Section 798.4470 Primary Dermal Irritation, 491, 1992. 56. U.S. Consumer Products Safety Commission, 16 CFR Chapter II, Subchapter C: Federal Hazardous Substances Act Regulation, Part 1500, Subsection 1500.3: Definitions, 381, 1993. 57. U.S. Environmental Protection Agency, Federal Insecticide, Fungicide, Rodenticide Act, Pesticide Assessment Guidelines, Subdivision F: Hazard Evaluation: Humans and Domestic Animals — Addendum 3 on Data Reporting, 1988. 58. U.S. Environmental Protection Agency, Federal Insecticide, Fungicide, Rodenticide Act, Pesticide Assessment Guidelines, Hazard Evaluation Division, Standard Evaluation Procedure, Guidance for Evaluation of Dermal Irritation Testing, 1, 1984. 59. U.S. Environmental Protection Agency, Toxic Substances Control Act, Test Guidelines, 40 CFR Chap. 1 (7-1-93), Part 156: Labeling Requirements for Pesticides and Devices, Section 156.10, 75, 1993.


60. The Commission of the European Communities, Official Journal of the European Communities, Annex VI, General Classification and Labelling Requirements for Dangerous Substances, No. L 257/11, 1983. 61. U.S. Occupational Safety and Health Administration, Labor, 29 CFR Chapter XVII, Part 1910, Appendix A to Section 1900.1200 — Health Hazard Definitions (Mandatory), 364, 1991. 62. DeGroot, A.C., Patch Testing, Test Concentrations and Vehicles for 2800 Allergens, Elsevier Science, Amsterdam, 1986. 63. Cronin, E., Contact Dermatitis, Churchill Livingstone, New York, 1980. 64. Casarett and Doull’s Toxicology, The Basic Science of Poisons, 4th ed., Klaassen, C.D., Amdur, M.O., and Doull, J., Eds., Pergamon Press, New York, 1991. 65. Marzulli, F.N. and Maibach, H.I., Eds., Dermatotoxicology, 2nd ed., Hemisphere Publishing, Washington, 1977. 66. Stedman’s Medical Dictionary, 25th ed., Williams & Wilkins, Baltimore, 1990. 67. The American Heritage Dictionary of the English Language, New College Edition, Morris, W., Ed., Houghton Mifflin, Boston, 1978. 68. U.S. Environmental Protection Agency, Federal Insecticide, Fungicide, Rodenticide Act, Pesticide Assessment Guidelines, Hazard Evaluation Division, Standard Evaluation Procedure, Guidance for Evaluation of Dermal Sensitization, 1, 1984.


ADDITIONAL RELATED INFORMATION TABLE 3.31 Relative Ranking of the Skin Permeability in Different Animal Species Ranking

Animal Species

Most permeable

Least permeable

Thickness of Stratum Corneum

Epidermis (µm)

Whole Skin (mm)

5.8

12.6

0.84

26.4 16.8

65.8 46.9

3.43 2.97

Mouse Guinea pig Goat Rabbit Horse Cat Dog Monkey Pig Human Chimpanzee

From Leung, H-W. and Paustenbach, D.J., Percutaneous toxicity, in: General and Applied Toxicology, Ballantyne, B., Marrs, T.C., and Syversen, T., Eds., Groves’s Dictionaries, New York, 1999, chap. 29, pp. 577–586. With permission. © Nature Publishing Group Reference.

TABLE 3.32 In Vivo Human Percutaneous Absorption Rates of Some Neat Chemical Liquids Chemical

Percutaneous Absorption Rate (mg cm–2 h–1)

Aniline Benzene 2-Butoxyethanol 2-(2-Butoxyethoxy) ethanol Carbon disulfide Dimethylformamide Ethylbenzene 2-Ethoxyethanol 2-(2-Ethoxyethoxy) ethanol Methanol 2-Methoxyethanol Methyl n-butyl ketone Nitrobenzene Styrene Toluene Xylenes (mixed) m-Xylene

0.2–0.7 0.24–0.4 0.05–0.68 0.035 9.7 9.4 22–33 0.796 0.125 11.5 2.82 0.25–0.48 2 9–15 14–23 4.5–9.6 0.12–0.15

From Leung, H-W. and Paustenbach, D.J., Percutaneous toxicity, in General and Applied Toxicology, Ballantyne, B., Marrs, T.C., and Syversen, T., Eds., Groves’s Dictionaries, New York, 1999, chap. 29, pp. 577–586. With permission. © Nature Publishing Group Reference.


TABLE 3.33 In Vitro Human Percutaneous Permeability Coefficients of Aqueous Solutions of Some Industrial Chemicals Organic Chemical

Kp (cm h–1)

2-Amino-4-nitrophenol 4-Amino-2-nitrophenol Benzene p-Bromophenol Butane-2,3-diol n-Butanol Butan-2-one Chlorocresol o-Chlorophenol p-Chlorophenol Chloroxylenol m-Cresol o-Cresol p-Cresol Decanol 2,4-Dichlorophenol Diethanolamine Diethyl ether 1,4-Dioxane Ethanol Ethanolamine

0.00066 0.0028 0.11 0.036 75% Fluorescein staining ≤ 25% of total corneal surface > 25% but ≤ 50% > 50% but ≤ 75% > 75% Neovascularization and pigment migration ≤ 25% of total corneal surface > 25% but ≤ 50% > 50% but ≤ 75% > 75% Perforation Maximal corneal score Iridal Observations Iritis is quantitated by the cells and flare in the anterior chamber, iris, hyperemia, and capillary light reflex Cells in aqueous chamber A few A moderate number Many Aqueous flare (Tyndall effect) Slight Moderate Marked Iris hyperemia Slight Moderate Marked Pupillary reflex Sluggish Absent Maximal iridal score Conjunctival Observations Hyperemia Slight Moderate Marked Chemosis Slight


Grades

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 4 20

1 2 3 1 2 3 1 2 3 1 2 11

1 2 3 1


Location of Observations

Grades

Moderate Marked Fluorescein staining Slight Moderate Marked Ulceration Slight Moderate Marked Scarring Slight Moderate Marked Maximal conjunctival score

2 3 1 2 3 1 2 3 1 2 3 15

From Chan, P-K. and Hayes, A.W., in Toxicology of the Eye, Ear, and Other Special Senses, Hayes, A.W., Ed., Raven Press, New York, 1985. With permission.

E. OCULAR SCORING SYSTEM

FOR

RABBITS BASED

ON

SLIT LAMP EXAMINATION

From Dermatotoxicology, 4th ed., pp. 780–785, Marzulli, F.N. and Maibach, H.I., Eds., Hemisphere, New York, 1991. Reproduced with permission. All rights reserved.26 1. Conjunctiva Conjunctival changes can be divided clinically into congestion, swelling (chemosis), and discharge. Generally, the sequence of events for these changes is congestion, discharge, and swelling. a.

Conjunctival Congestion +0 = Normal. May seem blanched to reddish pink without perilimbal injection (except at 12 and 6 o’clock positions) with vessels of the palpebral and bulbar conjunctivae easily observed. +1 = A flushed, reddish color predominantly confined to the palpebral conjunctiva with some perilimbal injection but primarily confined to the lower and upper parts of the eye from the 4, 7, 11, and 1 o’clock positions. +2 = Bright red color of the palpebral conjunctiva with accompanying perilimbal injection covering at least 75% of the circumference of the perilimbal region. +3 = Dark, beefy red color with congestion of both the bulbar and the palpebral conjunctivae along with pronounced perilimbal injection and the presence of petechia on the conjunctiva. The petechiae generally predominate along the nictitating membrane and the upper palpebral conjunctiva.

b. Conjunctival Swelling There are five divisions from 0 to +4. +0 = Normal or no swelling of the conjunctival tissue. +1 = Swelling above normal without eversion of the lids (can be ascertained easily by noting that the upper and lower eyelids are positioned as in the normal eye); swelling generally starts in the lower cul-de-sac near the inner canthus, which requires slit lamp examination. +2 = Swelling with misalignment of the normal approximation of the lower and upper eyelids; primarily confined to the upper eyelid so that in the initial stages the misapproximation



of the eyelids begins by partial eversion of the upper eyelid. In this stage, swelling is confined generally to the upper eyelid, although it exists in the lower cul-de-sac (observed best with the slit lamp). +3 = Swelling definite with partial eversion of the upper and lower eyelids essentially equivalent. This can be easily ascertained by looking at the animal head-on and noticing the positioning of the eyelids; if the eye margins do not meet, eversion has occurred. +4 = Eversion of the upper eyelid is pronounced with less pronounced eversion of the lower eyelid. It is difficult to retract the lids and observe the perilimbal region. c. Conjunctival Discharge Discharge is defined as a whitish-gray precipitate, which should not be confused with the small amount of clear, inspissated, mucoid material that can be formed in the medial canthus of a substantial number of rabbit eyes. This material can be removed with a cotton swab before the animals are used. +0 = Normal. No discharge. +1 = Discharge above normal and present on the inner portion of the eye but not on the lids or hairs of the eyelids. The small amount that is in the inner and outer canthus can be ignored if it has not been removed before starting the study. +2 = Discharge is abundant, easily observed, and has collected on the lids and around the hairs of the eyelids. +3 = Discharge has been flowing over the eyelids, wetting the hairs substantially on the skin around the eye. d. Aqueous Flare The intensity of the Tyndall phenomenon is scored by comparing the normal Tyndall effect observed when the slit lamp beam passes through the lens with that seen in the anterior chamber. The presence of aqueous flare is presumptive evidence of breakdown of the blood-aqueous barrier +0 = Absence of visible light beam in the anterior chamber (no Tyndall effect). +1 = The Tyndall effect is barely discernible. The intensity of the light beam in the anterior chamber is less than the intensity of the slit beam as it passes through the lens. +2 = The Tyndall beam in the anterior chamber is easily discernible and is equal in intensity to the slit beam as it passes through the lens. +3 = The Tyndall beam in the anterior chamber is easily discernible; its intensity is greater than the intensity of the slit beam as it passes through the lens. 2. Light Reflex The pupillary diameter of the iris is controlled by the radial and sphincter muscles. Contraction of the radial muscle due to adrenergic stimulation results in mydriasis, whereas contraction of the sphincter muscle due to cholinergic stimulation results in miosis. Because an ophthalmic drug can exert potential effects on these neural pathways, it is important to assess the light reflex of an animal as part of the ophthalmic examination. Using full illumination with the slit lamp, the following scale is used: 0 = Normal pupillary response. 1 = Sluggish pupillary response. 2 = Maximally impaired (i.e., fixed) pupillary response.



3. Iris Involvement In the following definitions the primary, secondary, and tertiary vessels are used as an aid to determine a subjective ocular score for iris involvement. The assumption is made that the greater the hyperemia of the vessels and the more the secondary and tertiary vessels are involved, the greater the intensity of iris involvement. The scores range from 0 to +4. +0 = Normal iris without any hyperemia of the iris vessels. Occasionally around the 12 to 1 o’clock position near the pupillary border and the 6 and 7 o’clock position near the pupillary border there is a small area around 1 to 3 mm in diameter in which both the secondary and tertiary vessels are slightly hyperemic. +1 = Minimal injection of secondary vessels but not tertiary. Generally, it is uniform, but may be of greater intensity at the 1 or 6 o’clock position. If it is confined to the 1 or 6 o’clock position, the tertiary vessels must be substantially hyperemic. +2 = Minimal injection of tertiary vessels and minimal to moderate injection of the secondary vessels. +3 = Moderate injection of the secondary and tertiary vessels with slight swelling of the iris stroma. This gives the iris surface a slightly rugose appearance, which is usually most prominent near the 3 and 9 o’clock positions. +4 = Marked injection of the secondary and tertiary vessels with marked swelling of the iris stroma. The iris seems rugose; may be accompanied by hemorrhage (hyphema) in the anterior chamber. 4. Cornea The scoring scheme measures the severity of corneal cloudiness and the area of the cornea involved. Severity of corneal cloudiness is graded as follows: +0 = Normal cornea. Appears with the slit lamp adjusted to a narrow slit image as having a bright gray line on the epithelial surface and a bright gray line on the endothelial surface with a marble-like gray appearance of the stroma. +1 = Some loss of transparency. Only the anterior half of the stroma is involved as observed with an optical section of the slit lamp. The underlying structures are clearly visible with diffuse illumination, although some cloudiness can be readily apparent with diffuse illumination. +2 = Moderate loss of transparency. In addition to involving the anterior stroma, the cloudiness extends all the way to the endothelium. The stroma has lost its marble-like appearance and is homogeneously white. With diffuse illumination, underlying structures are clearly visible. +3 = Involvement of the entire thickness of the stroma. With optical section, the endothelial surface is still visible. However, with diffuse illumination the underlying structures are just barely visible (to the extent that the observer is still able to grade flare and iritis, observe for pupillary response, and note lenticular changes). +4 = Involvement of the entire thickness of the stroma. With the optical section, cannot clearly visualize the endothelium. With diffuse illumination, the underlying structures cannot be seen. Cloudiness removes the capability for judging and grading flare, iritis, lenticular changes, and pupillary response. The surface area of the cornea relative to the area of cloudiness is divided into five grades from 0 to +4.



+0 +1 +2 +3 +4

= Normal cornea with no area of cloudiness. = 1 to 25% area of stromal cloudiness. = 25 to 50% area of stromal cloudiness. = 51 to 75% area of stromal cloudiness. = 76 to 100% area of stromal cloudiness.

Pannus is vascularization or the penetration of new blood vessels into the corneal stroma. The vessels are derived from the limbal vascular loops. Pannus is divided into three grades. +0 = No pannus. +1 = Vascularization is present but vessels have not invaded the entire corneal circumference. Where localized vessel invasion has occurred, vessels have not penetrated beyond 2 mm. +2 = Vessels have invaded 2 mm or more around the entire corneal circumference. The use of fluorescein is a valuable aid in defining epithelial damage. For fluorescein staining, the area can be judged on a 0 to +4 scale. +0 = Absence of fluorescein staining. +1 = Slight fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are easily visible. (The outline of the pupillary margin is as if there were no fluorescein staining). +2 = Moderate fluorescein staining confined to a small focus. With diffuse illumination the underlying structures are clearly visible, although there is some loss of detail. +3 = Marked fluorescein staining. Staining may involve a larger portion of the cornea. With diffuse illumination, underlying structures are barely visible but are not completely obliterated. +4 = Extreme fluorescein staining. With diffuse illumination, the underlying structures cannot be observed. Interpretation is facilitated by rinsing the eye with an isotonic irrigating solution to remove excess and nonabsorbed fluorescein. Slit lamps are equipped with cobalt blue filters, which can be placed in front of the light from the slit illuminator to excite fluorescence of the fluorescein. Photographs using fluorescein staining require the use of this filter, and fluorescence will be enhanced by a yellow filter placed in front of the objectives of the corneal microscope. 5. Lens The crystalline lens is readily observed with the aid of the slit lamp biomicroscope, and the location of lenticular opacity can be discerned readily by direct and retroillumination. The location of lenticular opacities can be divided arbitrarily into the following lenticular regions beginning with the anterior capsule: anterior capsular, anterior subcapsular, anterior cortical, posterior cortical, posterior subcapsular, posterior capsular. The lens should be evaluated routinely during ocular evaluations and graded as either N (normal) or A (abnormal). The presence of lenticular opacities should be described and the location noted as defined above.

SECTION 5. CLASSIFICATION SCHEMES A. CLASSIFICATION

OF

COMPOUNDS BASED

ON

EYE IRRITATION PROPERTIES

This classification scheme developed by Kay and Calandra27 utilizes the Draize scoring system to rate the irritating potential of substances.



1. Step 1

Using the Draize eye irritation scoring system, find the maximum mean total score for all three tissues (cornea, iris, and conjunctivae) occurring within the first 96 h after instillation for which the incidence of this score plus or minus 5 points is at least 40%. 2. Step 2 Choose an initial or “tentative rating” on the basis of the score found in Step 1 as follows: Score from Step 1

Tentative Eye Irritation Rating

0.0–0.5 points Nonirritating 0.5–2.5 points Practically nonirritating 2.5–15 points Minimally irritating 15–25 points Mildly irritating 25–50 points Moderately irritating 50–80 points Severely irritating 80–100 points Extremely irritating 100–110 points Maximally irritating For borderline scores, choose the higher rating

Symbol N PN M1 M2 M3 S E Mx

3. Step 3 Tentative Rating

Requirement for Maintenance

N PN M1 M2 M3

MTS24 = 0; for MTS24 >0, raise one level As for N MTS48 = 0; for MTS48 >0, raise one level MTS96 = 0; for MTS96 >0, raise one level 1. MTS ≤ 20; for MTSf >20, raise one level 2. ITSf = 10 (60%); if not true then no rabbit may show ITSf 30; otherwise raise one level 1. As for M3 except use MTSf ≤40 2. As for M3 except use ITSf ≤30 (60%) and 60 for high 1. As for M3 except use MTSf ≤80 2. As for M3 except use ITSf ≤60 (60%) and 100 for high 1. MTSf >80 (60%); for MTSf ≤80, lower one level 2. ITSf >60 (60%); otherwise lower one level

S E Mx

Symbols: MTS = mean total score; ITS = individual rabbit total score. Subscripts denote scoring interval: 24, 48, or 96 hr; f = final score (7 days).

Two requirements must be met before a tentative rating may become final. First, the mean total score for the 7-day scoring interval may not exceed 20 points if the rating is to be maintained. Second, individual total scores for at least 60% of the rabbits should be 10 points or less and in no case may any individual rabbit’s total score exceed 30. If either or both of these requirements are not met, then the “tentative rating” must be raised one level and the higher level becomes the “final rating.”

B. NATIONAL ACADEMY OF SCIENCES (NAS) METHOD BASED ON SEVERITY AND PERSISTENCE28 This descriptive scale, adapted from work conducted by Green et al.,29 attaches significance to the persistence and reversibility of responses. It is based on the most severe response observed in a group of animals rather than the average response.



1. Inconsequential or Complete Lack of Irritation Exposure of the eye to a material under the specified conditions causes no significant ocular changes. No staining with fluorescein can be observed. Any changes that occur clear within 24 h and are no greater than those caused by isotonic saline under the same conditions. 2. Moderate Irritation Exposure of the eye to the material under the specified conditions causes minor, superficial, and transient changes of the cornea, iris, or conjunctiva as determined by external or slit lamp examination with fluorescein staining. The appearance at the 24-hour or subsequent grading interval of any of the following changes is sufficient to characterize a response as moderate irritation: opacity of the cornea (other than a slight dulling of the normal luster), hyperemia of the iris, or swelling of the conjunctiva. Any changes that are seen clear up within 7 days. 3. Substantial Irritation Exposure of the eye to the material under the specified conditions causes significant injury to the eye, such as loss of the corneal epithelium, corneal opacity, iritis (other than a slight injection), conjunctivitis, pannus, or bullae. The effects clear up within 21 days. 4. Severe Irritation or Corrosion Exposure of the eye to the material under the specified conditions results in the same types of injury as in the previous category and in significant necrosis or other injuries that adversely affect the visual process. Injuries persist for 21 days or more.

C. MODIFIED NAS METHOD DEVELOPED

BY

ALLIEDSIGNAL, INC.30

This classification scheme helps distinguish mildly irritating substances from moderately irritating substances, as well as identifying strongly and severely irritating substances. It is based on the most severe ocular response observed in a group of animals, rather than the average response, and on the persistence of the response. 1. Nonirritation Exposure of the eye to the material under the specified conditions causes no ocular changes. No tissue staining with fluorescein is observed. Slight conjunctival injection (grade 1, some vessels definitely injected) that does not clear within 24 h is not considered a significant change. This level of change is inconsequential as far as representing physical damage to the eye and can be seen to occur naturally for unexplained reasons in otherwise normal rabbits. 2. Mild Irritation Exposure of the eye to the material under the specified conditions causes minor and/or transient changes as determined by external or slit lamp examination or fluorescein staining. No opacity, ulceration, or fluorescein staining of the cornea (except for staining that is characteristic of normal epithelial desquamation) are observed at any grading interval. The appearance of any of the following changes is sufficient to characterize a response as mild irritation: • Grade 1 hyperemia of the iris that is observed at 1 hour, but resolves by 24 h. • Grade 2 conjunctival hyperemia (redness) that is observed at 1, 24, and/or 48 h, but resolves by 72 h.



• Grade 2 conjunctival chemosis (swelling) that is observed at 1 hour, but diminishes to grade 1 or 0 by 24 h; or Grade 1 conjunctival chemosis that is observed at 1 and/or 24 and/or 48 h, but resolves by 72 h. 3. Moderate Irritation Exposure of the eye to the material under the specified conditions causes major ocular changes as determined by external or slit lamp examination or fluorescein staining. The appearance of any of the following changes is sufficient to characterize a response as moderate irritation: • Opacity of the cornea (other than slight dulling of the normal luster) is observed at any observation period, but resolves by day 7. • Ulceration of the cornea (absence of a confluent patch of corneal epithelium) is observed at any observation period, but resolves by day 7. • Fluorescein staining of the cornea (greater than that which is characteristic of normal epithelial desquamation) is observed at 1, 2, 3, and/or 4 days, but no staining is found by day 7. • Grade 1 or 2 hyperemia of the iris (circumcorneal injection, congestion) is observed and persists to 24 h or longer, but resolves by day 7. • Grade 2 conjunctival hyperemia is observed and persists to at least 72 h, but resolves by day 7; or Grade 3 conjunctival hyperemia is observed at any observation period, but resolves by day 7. • Grade 1 or greater conjunctival chemosis is observed and persists to 72 h or longer, but resolves by day 7. 4. Strong Irritation (Clearing within 21 Days) Exposure of the eye to the material under the specified conditions results in the type of injury described in the former category, but the effects (possibly including pannus or bullae) heal or clear within 21 days. 5. Severe Irritation (Persisting for 21 Days) or Corrosion Exposure of the eye to the material under the specified conditions results in the type of injury described in the two former categories, but causes significant tissue destruction (necrosis) or injuries that probably adversely affect the visual process. The effects of the injuries persist for at least 21 days.

D. CATEGORIZATION OF SUBSTANCES USING AND FLUORESCEIN

Site

“Accept”

Conjunctiva

Hyperemia without chemosis

Cornea

Staining, corneal stipplinga without confluence at 24 h 0

Anterior chamber a b c

THE

SLIT LAMP BIOMICROSCOPE

“Accept with Caution” Chemosis, less than 1 mm at the limbus Confluenceb of staining at 24 to 48 h 0

“Probably Injurious to Human Eyes” Chemosis, greater than 1 mm at the limbus Staining with infiltration or edema Flarec (visibility of slit beam; rubeosis of iris)

Corneal stippling: multiple discrete punctate irregularities in the corneal epithelial layer which retain fluorescein. Confluence: uniform zones for fluorescein retention larger than 1 mm in diameter. Flare: Tyndall effect in a beam traversing the aqueous humor.

From Beckley, J.H. et al.31 Source: Environmental Protection Agency, 1988. Guidance for Evaluation of Eye Irritation Testing, Hazard Evaluation Division, Standard Evaluation Procedures, EPA-540/09-88-105, Washington.32



E. CATEGORIZATION

AND

LABELING

OF

PESTICIDES33

Label Statements Regarding Eye Irritation Hazards Due to Pesticides.

Toxicity Category

Signal Word

Skull and Crossbones and “Poison” Required

I. Corrosive (irreversible destruction of ocular tissue), corneal involvement, or irritation persisting for more than 21 days.

Danger

No

II. Corneal involvement or irritation clearing in 21 days or less.

Warning

No

III. Corneal involvement or irritation clearing in 7 days or less.

Caution

No

IV. Minimal effects clearing in less than 24 h.

Caution

No

Precautionary Statement

Practical Treatment

a

Corrosive. Causes If in eyes: Flush with plenty irreversible eye damage. of water. Get medical Harmful if swallowed. Do attention. not get in eyes or on If swallowed: drink clothing. Wear (goggles, promptly a large quantity face shield, or safety of milk, egg whites, gelatin glasses)b. Wash thoroughly solution, or, if these are not with soap and water after available, drink large handling. Remove quantities of water. Avoid contaminated clothing and alcohol. wash before reuse. Note to physician: Probable mucosal damage may contraindicate the use of gastric lavage. Causes substantial but Same as above; omit note to temporary eye injury. Do physician statement. not get into eyes or on clothing. Wear (goggles, face shield, or safety glasses).b Harmful if swallowed. Wash thoroughly with soap and water after handling. Remove contaminated clothing and wash before reuse. Causes (moderate) eye If in eyes: Flush with plenty injury (irritation). Avoid of water. Get medical contact with eyes or attention if irritation clothing. Wash thoroughly persists. with soap and water after handling. None required. None required.

a

The term “corrosive” may be omitted if the product is not actually corrosive. Choose appropriate form of eye protection. Recommendation for goggles or face shield is more appropriate for industrial, commercial, or nondomestic uses. Safety glasses may be recommended for domestic or residential use. b

F. CONSUMER PRODUCT SAFETY COMMISSION, FEDERAL HAZARDOUS SUBSTANCES ACT (FHSA) REGULATIONS FOR CLASSIFYING AN EYE IRRITANT (16 CFR 1500.42) Ocular reactions to a test substance are examined and scored in six test rabbits. An animal is considered as exhibiting a positive reaction if the test substance produces any of the following ocular tissue responses 24, 48, or 72 h after instillation: Copyright © 2002 by Taylor & Francis


Ocular Tissue Cornea Iris Conjunctivae

Positive Response Ulceration of the cornea (other than a fine stippling) or opacity (other than slight dulling of normal luster) Inflammation (other than a slight deepening of the folds or rugae, or a slight circumcorneal injection of the blood vessels) An obvious swelling with partial eversion of the lids or diffuse crimson-red with individual vessels not easily discernible

Classification of a test substance is based on the number of animals exhibiting a positive reaction.

Test Group Result (n = 6) 4–6 animals exhibit a positive reaction 2–3 animals exhibit a positive reaction 0–1 animals exhibit a positive reaction

FHSA Classification Eye Irritant Inconclusivea Nonirritant

a

If two to three animals exhibit a positive reaction, the test is repeated using six new animals. If three or more animals in the second test exhibit a positive reaction, the test substance is classified as an “eye irritant.” If one to two animals in the second test exhibit a positive reaction, a third test is conducted using new animals. If one or more animals in the third test exhibit a positive reaction, the test substance is classified as an “eye irritant.”

SECTION 6. SPECIALIZED TECHNIQUES USED TO EXAMINE THE EYE FOR TOXIC EFFECTS

Technique

Description

Refs.

Slit lamp biomicroscopy

Used as a visual aid to evaluate the external features of the eye and the anterior portion of the globe (conjunctiva, cornea, iris, lens, anterior portion of the vitreous) Used as a monocular visual aid to evaluate the ocular media and fundus Used to measure the degree of corneal swelling Used to determine diffuse retinal damage and to evaluate the functional integrity of the retina when fundoscopic viewing is impaired due to lens opacification (the technique measures the normal change in electrical potential of the eye caused by a diffuse flash of light) Used to evaluate the integrity of the corneal endothelium Used to analyze and document changes in lens transparency (cataract development) Used to measure intraocular pressure of the eye (both contact and noncontact techniques are available)

25, 26, 34, 35

Direct ophthalmoscopy Pachymetry Electroretinography

Specular microscopy Scheimpflug photography Tonometry


36 37–41 36

42 43 44–47


SECTION 7. PROPOSED TIER APPROACHES TO EYE IRRITATION TESTING Test Substance

pH < _ or > _ 11.5

pH MEASUREMENT pH > 2 and < 11.5

Positive VALIDATED IN VITRO TEST

NO FURTHER TESTING

Negative or Equivocal DERMAL IRRITATION TEST Severely Irritating (PDII > _ 5.0) to Corrosive

Nonirritating to Moderately Irritating RABBIT EYE IRRITATION TEST

FIGURE 4.5 Schematic screening procedure to assess a test substance’s irritation potential before conducting an eye irritation test. New Material Needs Ocular Safety Assessment

Historical Data Analysis Physical/Chemical Characterization

In Vitro Test Battery

Innocuous-Slight

Slight-Moderate

Moderate-Severe

Assessment of all Available Data

Limited In Vivo Confirmation 1-3 Rabbits

Safety Assessment

Marketplace Surveillance

FIGURE 4.6 Diagrammatical representation of how in vitro alternatives may be incorporated into the ocular saftey assessment process. Initially, all previous testing data and physical chemical characterstics are evaluated. If necessary, the materials are then evaluated in a battery of in vitro assays. After the in vitro testing, all data are assessed again. Then either a saftey assessment would be made or an in vivo test would be performed in a limited number of animals before making the final saftey assessment. (From Hobson, D.W., Dermal and Ocular Toxicology, Fundamentals and Methods, CRC Press, Boca Raton, FL, 1991. With permission.)



SECTION 8. OCULAR ADVERSE EFFECTS OF CHEMICAL SUBSTANCES Tables 4.9 through 4.20 present lists of chemicals that are capable of producing certain ocular adverse effects. The tables were developed from information found in Grant’s Toxicology of the Eye,49 an excellent reference on chemicals, drugs, plants, toxins, and venoms, and their effects on the eyes or vision.

TABLE 4.9 Possible Adverse Corneal Effects Chemical/Drug

Adverse Effects

Beryllium poisoning Calcium hydroxide burn Phenylmercuric nitrate Polyethylene sulfonic acid Vitamin D poisoning

Corneal calcification (band keratopathy)

Allyl alcohol Diethylamine Diethyl diglycolate Diisopropylamine Dimethylamine Dimethylaminopropylamine Dimethyl diglycolate Ethylenediamine N-Ethylmorpholine N-Ethylpiperidine N-Methylmorpholine Morpholine tert-Octylamine Tetramethylbutanediamine Tetramethylethylenediamine Triethylenediamine

Corneal epithelial edema (painless) with delayed onset of haloes from local action

Allyl alcohol p-Anisyl chloride Butyl amine Cardiac glycosides Colchicine Diazomethane Dichlorobutenes Diethylamine Digitalis glycosides Diisopropylamine Dimethylaminopropylamine Dimethylphosphorochloridothionate Dimethyl sulfate Diphenylcyanoarsine Diving mask defogger Dyes (cationic) Emetine Erythrophleine Ethylene oxide Ethylenimine Euphorbias Fish (decomposing) Formaldehyde

Corneal epithelial injury (painful) with delayed onset from local action



TABLE 4.9 (Continued) Possible Adverse Corneal Effects Chemical/Drug

Adverse Effects

Hydrogen sulfide Hypochlorite-ammonia mixtures Ipecac Manchineel Methyl bromide Methyl chloroacrylate Methyl dichloropropionate Methyl fluorolsulfate Methyl silicate Mustard gas Mustard oil Nitrosomethyl urethane Osmic acid Oxalyl chloride Podophyllum Poison ivy Squill Sulfur Surfactants Triacetoxyanthracene Trimethyl siloxane Vinblastine Amiodarone Amodiaquine Bismuth subnitrate Chloroquine Chlorpromazine Clofazimine Fluphenazine Gold Hydroxychloroquine Isotretinoin Mepacrine Monobenzone Perhexiline Tilorone Triparanol

Corneal epithelial deposits from systemic drugs (humans)

BA 6650 Carbutamide Chlorpropamide 1,2-Dibromoethane 1,2-Dichloroethane Dichloronitroaniline Dimethylhydrazine Epinephrine Iminodipropionitrile Isoretinoin Phthalofyne Practolol Tobutamide

Corneal opacities from systemic drugs (animals)

Aniline Hydroquinone Mustard gas

Corneal scarring, late (humans)



TABLE 4.10 Possible Adverse Corneal and Conjunctival Effects Chemical/Drug Acetone Alcohol Ammonia Benzalkonium chloride Benzene Brake fluid Brilliant green Calcium hydroxide Castor beans (ricin) a-Chloroacetophenone o-Chlorobenzylidene malononitrile Chlorobromomethane Chlorobutanol 2-Chloroethanol Chloroform Chrysarobin (chrysophanic acid) Clove oil Croton oil Crystal violet 2-Cyanoacrylic acid esters Cytarabine Dibenzoxazepine Dibutyl phthalate Dieffenbachia juice Digitoxin Digoxin Dimethyl phthalate Dimethyl sulfate Emetine hydrochloride Euphorbia latex Formaldehyde Gentian violet Hydrochloric acid Hydrogen peroxide Hydrogen sulfide Hydroquinone-benzoquinone Iodine vapor Isopropyl alcohol Lewisite Mustard gas Mustard oil Nitrogen mustards Osmium tetroxide Podophyllum Potassium permanganate Propylene imine Silver nitrate Spitting Cobra venom Soap Sodium hydroxide Styrene


Adverse Effects Burns (humans)


TABLE 4.10 (Continued) Possible Adverse Corneal and Conjunctival Effects Chemical/Drug

Adverse Effects

Sulfur dioxide Trichloroacetic acid Trichloroethylene Urea Vinblastine Zinc chloride

TABLE 4.11 Possible Adverse Conjunctival Effects Chemical/Drug

Adverse Effects

Allyl cyanide (rats) Aminosalicylic acid Arsenic (inorganic) Arsphenamine Barbiturates Bromide Chloral hydrate Chlorambucil Chlorpropamide Cyclophosphamide Cytarabine Dixyrazine Ethylphenylhydratoin Gold Hexachlorobenzene Hypericum Isotretinoin Lantana (animals) Methotrexate Methyldopa Noramidopyrine Novobiocin Oxprenolol Penicillamine Phenazone Phenolphthalein Phenazopyridine (dogs) Phensuximide Phenylbutazone Phenytoin Phthalofyne Practolol Sulfadiazine Sulfamerazine Sulfarsphenamine Sulfathiozole

Inflammation from systemic substances



TABLE 4.12 Possible Adverse Lens Effects Chemical/Drug Acetaminophen (A) Alloxan (A) Allyl cyanide (A) Aminotriazole (A) Arabinose (A) 5-Aziridino-2, 4-dinitrobenzamide (A) Bis-(phenylisopropyl)-piperidine & UVA (H) Bleomycin (A) Boron hydride disulfide (A) Bromodeoxuridine (reversible) (A) Busulfan (A)(H) Carbutamide (A) Chlorphentermine (A) Chlorophenylalanine (A) Chlorpropamide (A) Clomiphene (A) Cobalt chloride (A) Corticosteroids (H) Decahydronaphthalene (A) Diazacholesterol (A) Diazoxide (reversible) (A) Dichlorisone (H) Dichloroacetate (reversible) (A) Diethylaminoethoxyandrostenone (A) 4-(p-Dimethylaminostyryl)quinoline (A) Dimethylnitroquinoline (A) Dimethyl terephthalate (A) Dinitrocresol (A,H) Dinitrophenol (A,H) Diquat (A) Disophenol (A) Dithizone (A) Epinephrine (A) Galactose (A,H) Hematoporphyrin (A) Hygromycin B (A) ICI 33828 (A) Iodoacetate (A) Methoxsalen and UVA (A) Methyl dichlorisone (H) Mimosine (A) Miotics (H) Mirex (A) Mitotane (H) Nafoxidine (A) Naphthalene (A) 2-Naphthol (A) 1,2-Naphthoquinone (A) Nitrogen mustard (A) Nitroquinolones (A)


Adverse Effects Opacity (cataract) from systemic administration or exposure


TABLE 4.12 (Continued) Possible Adverse Lens Effects Chemical/Drug

Adverse Effects

Opiates (A) Petroleum fraction (A) Phenelzine and serotonin (A) 3-(2-Phenyl)-hydrazopropionitrile (A) 2-(4-Phenyl-l-piperazinylmethyl)-cyclohexanone (A) (4′-Pyridyl)l-piperazine derivatives (A) Pyrithione (A) Selenium (A) SQ 11290 (A) Streptomycin (A) Streptozotocin (A) Sulfaethoxypyridazine (A) Tetrahydronaphthalene (A) Thallium (A) Tolbutamide (A) Tretamine (A) Triaziquone (A) Trinitrotoluene (H) Triparanol (A,H) Verapamil (A) Xylose (A) Amiodarone (H) Chlorpromazine (A) (H) Copper (H) Fluphenazine (H) Iprindole (A) Iron (H) Mercury (H) Phenylmercuric salts (H) Silver (H)

Lens deposits or discoloration

Note: A = animals; H = humans.

TABLE 4.13 Possible Adverse Eyelid Effects Chemical/Drug

Adverse Effects

Amphetamine Cocaine Levodopa Methoxamine Phenylephrine

Lid retraction

Barbiturates Botulism Bretylium Chloral hydrate Chloralose

Ptosis of eyelids (topical or systemic substances)



TABLE 4.13 (Continued) Possible Adverse Eyelid Effects Chemical/Drug

Adverse Effects

Conhydrine Coniine Corticosteroids Curare Gelsemium sepervirens Guanethidine Levodopa Mephenesin Methylpentynol Pelletierine Penicillamine Phenoxybenzamine Primidone Reserpine Snake venoms Spider venoms Sulfonal Tetraethylammonium Thallium Trichloroethylene decomposition Trimethadione Vincristine

TABLE 4.14 Possible Adverse Retinal Effects Chemical/Drug Acridine (A) Ammi majus seeds (H) Chloramphenicol (H) Cobalt (A) Cyanide (A) Dithizone (A) Ergotamine (H) Ethyl hydrocuprein (H) Fluoride (A) Glue sniffing (H) Glutamate (A) Helichrysum (A) Iminodipropionitrile (A) Iodate (A) (H) lodoquinol (H) Methanol (H) Naphthalene (A) Optochin (H) Phosphorus (A) Quinine (H) Pyrithione (A)


Adverse Effects Retinal edema from systemic administration


TABLE 4.14 (Continued) Possible Adverse Retinal Effects Chemical/Drug

Adverse Effects

Radiopaque media (H) Streptomycin (A) Triaziquone (A) Acetylphenylhydrazine (H) Alloxan (A) Arsphenamine (H) Aspirin (H) Benzene (H) Bicycloheptadiene dibromide (H) Carbenoxolone (H) Carbon disulfide (H) Carbon monoxide (H) Chloramphenicol (H) Dapsone (H) Desoxycortone acetate (A) Dextran (A) Dicumarol (H) Diodone (H) Diquat (A) Dithizone (A) Epinephrine (A) Ethambutol (H) Hexachlorophene (H) Iodoform (H) Isotretinoin (H) Lead (H) Licorice (H) Methaqualone (H) Methyl bromide (H) Miotane (H) Naphthalene (A) 2-(2-Naphthyloxy) ethanol (A) Phenprocoumon (H) Phenylbutazone (H) Phosphorus (A) Potato leaf smoking (H) Pyrithione (A) Radiopaque media (H) Snake venoms (H) Sulfanilamide (H) Sulfathiozole (H) Trichloroethylene decomposition (H) Triethyl tin (H) Vitamin A (H) Warfarin (H)

Retinal hemorrhages from systemic administration

Amiodarone (A) (H) AY 9944 (A) Chlorcyclizine (A) 1-Chloroamitriptyline (A) Chloroquine (A)

Retinal lipidosis (phospholipidosis) from systemic administration




Adverse Effects

Clomipramine (A) Diethylaminoethoxyhexestrol (A) Dithiozone (A) Imipramine (A) Iprindole (A) Perhexilene (A) Triparanol (A) Ammeline (A) Ammi majus seeds (A) Aramite (A) Benzoic acid (A) Bracken fern (A) Bromoacetate (A) Cardiac glycosides (H) Colchicine (A) Diaminodiphenylmethane (A) Digitalis (H) Digitoxin (H) Ethylenimine Ethylhydrocupreine (A) Fluorescein (with light) (A) Fluoride (A) Furmethonol (H) Halothane (A) Helichrysum (A) Hematoporphyrin (with light) (A) Hexachlorophene (A) Iodate (A) Iodoacetate (A) Methylazoxymethanol acetate (A) N-Methyl-N-nitrosourea (A) 2-Naphthol (A) P-1727 (A) Quinine (A) (H) Quinoline (A) Sodium azide (A) Stypandra imbricata (A) Sucrose (A) Urethane (A)

Retinal photoreceptor damage by systemic administration

Aspidium (A) Diaminodiphenoxypentane (A) Ergotamine (H) Ethylenimine (A) Ethylhydroxycupreine (H) Eucupine (H) Iron (H) Lead (A) Oxygen (A) (H) P-1727 (A) Quinine (H)

Retinal vessel narrowing from systemic administration




Adverse Effects

Ammi majus seeds (A) Arsanilic acid (A) (H) Aspidium (A) Carbon dioxide (H) Carbon disulfide (A) Chloramphenicol (H) Cinchona derivatives (A) Cysteine (A) Ergot (A) Ethylhydrocupreine (A) (H) Glutamate (A) Locoweed (A) Methanol (H) Quinine (A) (H) Quinoline (A) Swainsona plants (A) Tellurium (A) Thallium (H) Vincristine (H)

Retinal ganglion cell damage by systemic administration

Alloxan (A) Aminophenoxyalkanes (A) Ammeline (A) Ammi majus seeds (A) Amopyroquin (A) Aspartate (A) Bilirubin (A) Bromoacetate (A) Cephaloridine (A) Chloroquine (A) (H) Cobalt (A) Colchicine (A) Deferoxamine (H) Diaminodiphenoxyheptane (A) Diaminodiphenoxypentane (A) Diaminodiphenylmethane (A) Dibutyl oxalate (A) Dihydro-dihydroxynaphthalene (A) Dithizone (A) Epinephrine (A) Ethambutol (high dose) (H) Ethylenimine (A) Ethylhydrocupreine (H) Fluoride (A) Glutamate (A) Hydroxychloroquine (H) Iminodipropionitrile (A) Iodate (A) (H) Iodoacetate (A) Isopropylhydroxybenzylpyrazolopyrimidine (A) Lead (A)

Retinal pigment epithelial changes from systemic administration




Adverse Effects

Mesidine (A) N-Methyl-N-nitrosourea (A) Naphthalene (A) Naphthoquinone (A) Naphthyl benzoate (A) Nitrofurazone (A) Ouabain (A) Oxygen (A) (H) Penicillamine (H) Pheniprazine (A) Phenylhydrazine (A) Phlorizin (A) Phosphorus (A) Piperidychlorophenothiazine (A) (H) Quinine (H) Quinoline (A) Sodium azide (A) Sparsomycin (H) Tetrahydronaphthalene (A) Thioridazine (A) (H) Toxotoxin (A) Trenimon (A) Triaziquone (A) Trifluoromethylphenylisopropylamine (A) Urethane (A) Vinblastine (A) Vincristine (A) Vitamin A (A) Acetazolamide (H) Aldrin (A) 4-Aminobutyric acid (A) Aminophoxyalkanes (A) Ammeline (A) Ammonium poisoning (A) Amodiaquine (H) Amyl acetate (A) Aramite (A) Aspartate (A) Barbiturates (A) Befunolol (A) Carbaryl (A) Carbon disulfide (A) Carbon monoxide (A) Cardiac glycosides (A) Chloramphenicol (H) Deferoxamine (H) 2-Deoxyglucose (A) Desipramine (A) Diaminodiphenoxypentane (A) Digitalis (H)


Electroretinogram altered by systemic administration



Adverse Effects

Digoxin (A) (H) Dithizone (A) Epinephrine (A) Ethambutol (A) Fluoride (A) Formaldehyde (A) Glucose 6-phosphate (A) Glutamate (A) Halothane (H) Hydroxychloroquine (A) (H) Iminodipropionitrile (A) Iodate (A) Iodoacetate (A) Methanol (H) Mitomycin C (A) Nitrofurazone (A) Ouabain (A) Oxygen (A) (H) Oxypertine (A) Piperidylchlorophenothiazine (A) Quinine (A) (H) Rifampin (A) Sodium azide (A) Styrene (A) Sucrose (A) Thallium (A) Trimethadione (A) (H) Urethane (A) Vincristine (H) Vitamin A (A) Amoproxan Caramiphene Carbon disulfide Chloramphenicol Chloroquine Chlorpropamide Clomiphene Digitalis Diogitoxin Digoxin Dinitrobenzene Dinitrochlorobenzene Dinitrotoluene Disulfiram Ergotamine Emetine Ethambutol Ethchlorvynol Ethyl alcohol Ethylene glycol


Central (or cecocentral) scotomas from systemic administration (humans)



Adverse Effects

Flumequine Ibuprofen Iodate Iodoform Isoniazid Lead Methanol Methyl bromide Minoxidil Octamoxin Pheniprazine Plasmocid Streptomycin Sulfonamides Tetraethyl lead Thallium Thiacetazone Tobacco smoking Trichloroethylene decomposition Wasp sting Amodiaquine Arsacetin Arsanilic acid Bee sting Botulism toxin Carbon dioxide Carbon monoxide Chloramphenicol Cortex granati Dionitrochlorobenzene Emetine Ethambutol Ethylhydrocupreine Ethylmercuritoluenesulfonanilide Eucupine Iodate Methylmercury compounds Methanol Naphthalene Orsudan Oxygen Pheniprazine Piperidylchlorophenothiazine Quinine Trichloroethylene decomposition Tryparsamide Note: A = animals; H = humans.


Peripheral visual field constriction from systemic administration (humans)


TABLE 4.15 Possible Adverse Optic Nerve Effects Chemical/Drug

Adverse Effects

Acetarsone (H) Acetylarsone (H) Antimony potassium tartrate (H) Arsacetin (H) Arsanilic acid (A) (H) Aspidium (A) (H) Bee sting Brayera (H) Broxyquinoline (H) Carbon dioxide (H) Carbon disulfide (H) Caster beans (H) Chloramphenicol (H) Clioquinol (H) Cortex granati (H) Dapsone (H) Dinitrobenzene (H) Dinitrochlorobenzene (H) Ethambutol (H) Ethyl hydrocuprein (H) Ethylmercuritoluenesulfonanilide (H) Eucupine (H) Finger cherries (H) Formic acid (A) Halquinols (H) Hexachlorophene (A) (H) Hexamethonium (H) Iodoform (H) lodoquinol (H) Isoniazid (H) Lead (H) Methanol (A) (H) Octamoxin (H) Pheniprazine (H) Plasmocid (H) Quinine (H) Solvent sniffing (H) Thallium (H) Trichloroethylene decomposition (H) Triethyl tin (H) Tryparsamide (H) Vincristine (H)

Optic nerve atrophy from systemic administration

Acetarsone (H) Acetylarsan (H) Acrylamide (A) Antirabies vaccine (H) Arsacetin (H) Arsanilic acid (A) (H) Aspidium (H) Bee sting (H) Botulism (H)

Optic neuropathy from systemic administration



TABLE 4.15 (Continued) Possible Adverse Optic Nerve Effects Chemical/Drug

Adverse Effects

Carbon disulfide (H) Cassava (H) Chloramphenicol (H) Clioquinol (A) (H) Cyanoacetic acid (A) Deferoxamine (H) Dinitrobenzene (H) Dinitrochlorobenzene (H) Dinitrotoluene (H) Disulfiram (H) Ethambutol (A) (H) Ethchlorvynol (H) Ethylene glycol (H) Filicin (A) Glutamate (A) Helichrysum (A) Hexachlorophene (H) Iminodipropionitrile (A) Indarsol (A) Iodoform (H) Isoniazid (H) Lead (A) (H) Methanol (H) Octamoxin (H) Penicillamine (H) Perhexiline maleate (H) Phosphorus (H) Plasmocid (H) Sodium azide (A) Streptomycin (H) Stypandra imbricata (A) Sulfonamides (H) Tellurium (A) Thallium (H) Tolbutamide (H) Trichloroethylene decomposition (H) Triethyl tin (A) Trinitrotoluene (H) Tryparsamide (H) Vincristine (H) Antimony potassium tartrate (H) Arsphenamine (H) Aspirin (H) Bee sting (H) Carbenoxolone (H) Cephaloridine (H) Chlorambucil (H) Chloramphenicol (H) Chlordecone (H) Cisplatin (H) Contraceptive hormones (H) Corticosteroids (H)


Papilledema (swelling of the optic disc) from systemic administration



Adverse Effects

p-Dichlorobenzene (A) Dynamite (H) Ergotamine (H) Ethylene glycol (H) Helichrysum (A) Hexachlorophene (A) (H) Isoniazid (H) Isotretinoin (H) Ketoprofen (H) Lead (H) Levothyroxine (H) Minocycline (H) Minoxidil (H) Mitotane (H) Nalidixic acid (H) Nitrofurantoin (H) DL-Penicillamine (H) Penicillin (H) Perhexilene maleate (H) m-Phenylenediamine (H) p-Phenylenediamine (H) Phosphorus (H) Sulfonamides (H) Tetracycline (H) Triethyl tin (H) Vitamin A (H) Aspidium Carbon disulfide Cassava Chloramphenicol Deferoxamine Dinitrobenzene Dinitrochlorobenzene Dinitrotoluene Disulfiram Ethambutol Iodoform Isoniazid Lead Octamoxin m- or p-Phenylenediamine Thallium Tolbutamide Trichloroethylene decomposition Trinitrotoluene

Retrobulbar neuritis from systemic administration

Chloramphenicol (A) Cyanide (A) Chloramphenicol (A) Cyanide (A) Ethambutol (A) (H) Helichrysum (A)

Optic chiasm injury by systemic administration




Adverse Effects

Hexachlorophene (H) Stypandra imbricata (A) Tellurium (A) Triethyl tin (A) Vincristine (H) Note: A = animals, H = humans.

TABLE 4.16 Possible Adverse Extraocular Muscle Effects Chemical/Drug

Adverse Effects

Alcuronium Amanita phalloides Amitriptyline Anesthesia, spinal Antirabies vaccine Arsphenamine Barbiturates Botulinus toxin Carbamazepine Curare Diazinon Ethyl alcohol Ethylene glycol Furmethonol Gelsemium sempervirens Hexachlorophene Isopentaquine Lead Minocycline Nalidixic acid Pamaquine Penicillamine Pentaquine Piperazine Plasmocid Primidone Scorpion venom Snake venoms Streptomycin Sulfonal Thallium Trichloroethylene decomposition Triethyl tin Vincristine Vitamin A

Weakness or paralysis from systemic administration



TABLE 4.17 Possible Adverse Effects on Intraocular Pressure Chemical/Drug

Adverse Effects

Acetazolamide (A) (H) Alcohol (H) Alcuronium (H) Aminophylline (H) Ascorbic acid (H) BA 6650 (A) Bromocriptine (H) Calcium chloride (A) Cardiac glycosides (H) Catha edulis (H) Chlorpromazine (H) Chlorthalidone (H) Cholera toxin (H) Contraceptive hormones (H) Dextran A (H) Dibenamine (H) Dichlorphenamide (H) Digitoxin (H) Digoxin (H) Dihydroergotoxine (H) Ethoxolamide (H) Glucose (H) Glycerine (H) Iodate (A) Iodoacetate (A) Isosorbide (H) Lanatoside C (A) Mannitol (H) Meprobamate (H) Mercaptomerin (A) Mercuderamide (A) Methazolamide (H) Methyldopa (H) Nialamide (A) Nitroglycerin (H) Ouabain (A) Pargyline (H) Phentolamine (H) Propranolol (H) Propylene glycol (H) Quinine (H) Sodium ascorbate (H) Sodium chloride (H) Sodium lactate (H) Sorbitol (H) Thiopental (H) Timolol (H) Trometamol (H) Urea (H)

Reduction of intraocular pressure by systemic administration

Note: A = animals; H = humans.



TABLE 4.18 Possible Adverse Effects on Vision Chemical/Drug

Adverse Effects

Barbiturates Carbon monoxide Diatrizoate Lead Lomotil Methadone Methylergonovine Methylmercury compounds Vincristine

Cortical blindness (humans)

Acetyl digitoxin Aconite Amodiaquine Barbiturates Cannabis Carbon dioxide Carbon disulfide Chloramphenicol Chlorothiazide Contraceptive hormones Digitalis Digoxin Dihydrostreptomycin Diphenhydramine theoclate Ethambutol Furmethonol Herbatox Ibuprofen Lead Lysergide Nalidixic acid Pentylenetetrazole Salicylate

Color vision alterations from systemic administration

Carbon dioxide Carbon disulfide Carbon monoxide Deferoxamine Digitalis Digitoxin Halothane Indomethacin Piperidylchlorophenothiazine

Altered dark adaption from systemic administration (humans)

Acetazolamide Aminophenazone Arsphenamine Bendrofluazide Chlorothiazide Chlorthalidone Clofenamide

Acute transient myopia from systemic administration without cyclotonia or miosis (humans)



TABLE 4.18 (Continued) Possible Adverse Effects on Vision Chemical/Drug

Adverse Effects

Dichlorphenamide Ethoxolamide Hydrochlorothiazide Isotretinoin Neoarsphenamine Phenformin Polythiazide Prochlorperazine Promethazine Quinine Spironolactone Sulfonamides Tetracycline Trichlormethiazide

TABLE 4.19 Possible Adverse Irritating Effects Chemical/Drug Acrolein Allyl propyl disulfide Bromoacetone Bromoacetophenone Bromobenzylcyanide Bromomethyl ethyl ketone Bromotoluene Bromoxylene Chloroacetone Chloroacetophenone Chlorobenzylidene malononitrile Chlorosulfonic acid esters Cyanic acid Cyanogen chloride Dibenzoxazepine Dibromomethyl ether Dichloroformoxime Dichloronitroethane Diphenylchlorarsine Ethylarsine dichloride Ethyl benzene Ethyl bromoacetate Ethyl ioodoacetate Hexafluoroisopropanol Iodotoluene Lewisite Methyl arsine dichloride


Adverse Effects Lacrimation from direct exposures


TABLE 4.19 (Continued) Possible Adverse Irritating Effects Chemical/Drug

Adverse Effects

Methyl iodoacetate Methyl vinyl ketone Nitrobenzyl chloride Nitroethylene Onion vapor Pelargonic acide morpholide Phenylcarbylamine chloride Trichloroacetronitrile Trichloromethane sulfonyl chloride Trichloromethanethiol Trichloropyrimidine Xylyl bromides Xylyl chlorides Arsenic, inorganic (H) Bethanechol (H) Bismuth subnitrate (A) Chloral hydrate (H) Cyclohexanol (A)(H) Diazoxide (H) Dichlorophenoxy acetic acid (A) Dimercaprol (H) Dimidium bromide (A)(H) Diphenylarsenic acid (H) Emetine (H) Fish (Ciquatera) poisoning (H) Herion (H) Hexachloronaphthalene (A) Hydralazine (H) Iodide (H) Mercury (acrodynia)(H) Methotrexate (H) Morphine withdrawal (H) Nicothiazone (H) Nitrofurantoin (H) Pentazocine withdrawal (H) Phthalofyne (A)(H) Practolol (H) Pyrithione (A) Reserpine (H) Scorpion venom (H) Sulfathiazole (H) Tegafur (H) Thiacetazone (H) Triethyl tin (H) Zoxazolamine (H) Note: A = animals; H = humans.


Lacrimation with burning or itching sensation from systemic administration


TABLE 4.20 Possible Ocular Teratogenesis Chemical/Drug

Adverse Effects

Alloxan (A) Aspidium (A) Azathioprine (A) Busulfan (H) Butylated hydroxytoluene (A) Caffeine (A) Carbutamide (A) Chlorambucil (A) (H) Clomiphene (A) Colchicine (A) Cyclizine (A) Cyclophosphamide (A) 2,4-Dichlorophenyl-p-nitrophenyl ether (A) Felicin (A) Heptachlor (A) Idoxuridine (A) Isotretinoin (A) Lysergide (A) (H) 1-Methyl-3-nitro-1-nitroguanidine (A) 2-Naphthol (A) Nickel carbonyl (A) Quinine (A) (H) Salicylate (A) Thalidomide (A) (H) Trimethadione (H) Trypan blue (A) Urethane (A) Veratrum californicum (A) Vidarabine (A) Vinblastine sulfate (A) Vitamin A (A) Warfarin (H)

Abnormalities of the eyes

Note: A = animals, H = humans.

SECTION 9. GLOSSARY OF TERMS RELATING TO THE EYE Accommodation The adjustment of the eye for seeing at different distances, accomplished by changing the shape of the lens through action of the ciliary muscle, thus focusing a clear image on the retina. Aniridia Congenital absence of the iris. Anophthalmos Absence of a true eyeball. Anterior chamber The aqueous-containing cavity of the eye, bounded by the cornea anteriorly, the chamber angle structures peripherally, and the iris and lens posteriorly. Aphakia Absence of the lens. Biomicroscopy Examination of the eye using a biomicroscope (slit lamp). Blepharitis Inflammation of the eyelids.


Blepharoptosis Drooping of an upper eyelid due to paralysis. Blepharospasm Involuntary spasm of the lids. Bulla A large bleb or blister filled with lymph or serum. Bullous Characterized by bullae. Canal of Schlemm A circular modified venous structure in the anterior chamber angle. Canthus The angle of either end of the eyelid aperture; specified as outer (temporal) and inner (nasal). Cataract An opacity of the lens or its capsule. Chemosis Intense edema of the conjunctiva. The conjunctiva is a loose fibrovascular connective tissue which is relatively rich in lymphatics and responds to noxious stimuli by swelling to the point of prolapse between the lids. Choroid The vascular middle coat between the retina and sclera. Ciliary body Portion of the uveal tract between the iris and the choroid consisting of ciliary processes and the ciliary muscle. Cones Retinal receptor cells concerned with visual acuity and color discrimation. Conjunctiva Mucous membrane that lines the posterior aspect of the eyelids (palpebral conjunctiva) and the anterior sclera (bulbar conjunctiva). Conjunctivitis Inflammation of the conjunctiva. Cornea Transparent portion of the outer coat of the eyeball forming the anterior wall of the anterior chamber. Corneal perforation A hole made through the cornea, resulting from the destruction of corneal tissue (secondary infectious agent(s) may or may not play a contributing role). Corneal vascularization The development of blood vessels in the cornea. If there is sufficient tissue necrosis, then vascularization accompanies wound healing. Uncomplicated healing of corneal wounds occurs without vascularization. Cycloplegic A drug that causes paralysis of the ciliary muscle, thus preventing accommodation. Dacryocystitis Infection of the lacrimal sac. Ectropion Turning out of the eyelid. Edema The presence of an abnormally large amount of fluid in the intercellular tissue spaces (e.g., edema of the cornea is manifested as increased thickness). Endophthalmitis Inflammation of one or more of the intraocular cavities and adjacent structures. Enophthalmos Abnormal retrodisplacement of the eyeball. Entropion A turning inward of the eyelid. Enucleation Complete surgical removal of the eyeball. Exophthalmos Abnormal protrusion of the eyeball. Exudate Material, such as fluid, cells, or cellular debris, which has escaped from blood vessels and has deposited in tissues or on tissue surfaces, usually as a result of inflammation. An exudate, in contrast to a transudate, is characterized by a high content of protein, cells, or solid material derived from cells. Fibrosis The formation of fibrous connective tissue. Flare (aqueous flare) The scattering of light as it passes through a medium that contains particles. It is analogous to the Tyndall effect, and is seen when a thin beam of high intensity light is passed into the anterior chamber (of the eye) containing cellular material or increased amounts of protein. Fluorescein (fluorescein sodium) A fluorescent dye, the simplest of the fluorane dyes and the mother substance of eosin, which is commonly used intravenously to determine the state of adequacy of circulation in the retina and to a lesser degree the chorioid and iris. Another important use is to detect epithelial lesions of the cornea and conjunctiva. Peak excitation occurs with light at a wavelength between 485 and 500 nm and peak emission occurs between 520 and 530 nm. Fornix The junction of the palpebral and bulbar conjunctiva.


Fovea Depression in the macula adapted for most acute vision. Fundus The posterior portion of the eye visible through an ophthalmoscope. Gonioscope An optical instrument used for examination of the anterior chamber angle. Granulation The formation of fibrovascular tissue in wounds or ulcers. Hemorrhage An escape of blood from the vessels; bleeding. Hyperemia Excess of blood in a part due to local or general relaxation of the arterioles. Blood vessels become congested and give the area involved a reddish or red-blue color. Hyphema Hemorrhage within the anterior chamber of the eye. Hypopyon An accumulation of pus in the anterior chamber of the eye. Hypotony Abnormally soft eye from any cause. Injection Congestion of blood vessels. Iris The circular pigmented membrane behind the cornea and immediately in front of the lens; the most anterior portion of the vascular tunic of the eye. It is composed of the dilator and sphincter muscles and the two-layered posterior epithelium, and mesodermal components that form the iris stroma. Iritis Inflammation of the iris, manifested by vascular congestion (hyperemia). An outpouring of serum proteins into the aqueous (flare) may accompany the inflammatory reaction. Keratitis Inflammation of the cornea. Keratoconus Cone-shaped deformity of the cornea. Keratocyte One of the connective tissue cells found between the layers of fibrous tissue in the corneal stroma. Keratometer An instrument for measuring the curvature of the cornea, used in fitting contact lenses. Lens A transparent biconvex structure suspended in the eyeball between the aqueous and the vitreous. Its function is to bring rays of light to a focus on the retina. Accommodation is produced by variations in the magnitude of this effect. Lens remnants Those portions of the lens capsule and variable amounts of cortex and nucleus remaining after discontinuity of the lens capsule. Such remnants are usually opaque and may also be called cataractous lens remnants. Limbal Of or pertaining to the limbus. Limbus Zone of merger between cornea and sclera. In the normal eye, the cornea is transparent, the limbus semitransparent, and the sclera opaque. The limbus may vary from 1 to 3 mm in width. Macula lutea The small avascular area of the retina surrounding the fovea, often having yellow pigment. Megalocornea Abnormally large cornea (> 13 mm in diameter). Microphthalmos Abnormal smallness of the eyeball. Miotic A drug causing pupillary constriction. Mydriatic A drug causing pupillary dilation. Necrosis Death of tissue, usually as individual cells, groups of cells, or in a small localized area. Nystagmus An involuntary, rapid movement of the eyeball that may be horizontal, vertical, rotatory, or mixed. Ophthalmoscope An instrument with a special illumination system for viewing the inner eye, particularly the retina and associated structures. Optic atrophy Optic nerve degeneration. Optic disk Ophthalmoscopically visible portion of the optic nerve. Optic nerve The nerve that carries visual impulses from the retina to the brain. Palpebral Pertaining to the eyelid. Panophthalmitis Inflammation of the entire eyeball. Pannus Vascularization and connective tissue deposition beneath the epithelium of the cornea. Papilledema Swelling of the optic disk.


Phlyctenule Localized lymphocytic infiltration of the conjunctiva. Photophobia Abnormal sensitivity to light. Posterior chamber Space filled with aqueous anterior to the lens and posterior to the iris. Proptosis A forward displacement of the eyeball. Pterygium A triangular growth of tissue that extends from the conjunctiva over the cornea. Ptosis Drooping of the upper eyelid. Pupil The round opening at the center of the iris which allows transmission of light to the posterior of the eyeball. Pupillary light reflex The abrupt narrowing of pupillary aperture occurring when light is cast into the eye. The neuromotor reflex is mediated through the brain stem and involves both eyes. Retina The innermost or nervous tunic of the eye which is derived from the optic cup (the outer layer develops into the pigmented monolayer of epithelium and the inner layer develops into the complex sensory layer). Retinal detachment The condition in which the inner sensory layer of the retina separates from the outer layer of retinal pigment epithelium. Retrocorneal fibrous membrane Formation of fibrous tissue on the posterior surface of the cornea; this tissue may replace the corneal endothelium. Rods Retinal receptor cells concerned with peripheral vision under decreased illumination. Schlemm’s canal A narrow channel in the anterior chamber angle that drains aqueous to the aqueous veins. Sclera The white tough covering of the eye that, with the cornea, forms the external protective coat of the eye. Slit lamp A biomicroscope especially adapted to examine the eye. Symblepharon Adhesions between the bulbar and palpebral conjunctiva. Synechia Adhesion of the iris to the cornea (anterior synechia) or to the lens (posterior synechia). Tonometer An instrument for measuring intraocular pressure. Ulcer A lesion resulting from the loss of substance on a cutaneous or mucosal surface. It may lead to gradual disintegration and necrosis of the tissues. Ulceration The formation or development of an ulcer. Uvea (uveal tract) The iris, ciliary body, and choroid considered together. Uveitis Inflammation of one or all portions of the uvea. Vascular congestion Excessive or abnormal accumulation of blood in a tissue caused by dilatation of its blood vessels. Vascularization The process of becoming vascular, or the development of vessels in a tissue. Vitreous Transparent, colorless mass of soft, gelatinous material filling the space in the eyeball posterior to the lens and anterior to the retina. Zonule A system of fine fibers which extends from the ciliary processes to the equator of the lens and which holds the lens in place.

REFERENCES 1. Beckley, J.H., Comparative eye testing: man vs. animal, Toxicol. Appl. Pharmacol., 7, 93, 1965. 2. Maurice, D.M. and Giardini, A.A., A simple optical apparatus for measuring the corneal thickness, and the average thickness of the human cornea, Br. J. Opthalmol., 48, 61, 1951. 3. Marzulli, F.N. and Simon, M.E., Eye irritation from topically applied drugs and cosmetics: preclinical studies, Am. J. Optom., 48, 61, 1971. 4. Gaasterland, D.E., Barranger, J.A., Rapoport, S.I., Girton, M.E., and Doppman, J.L., Long-term ocular effects of osmotic modification of the blood-brain barrier in monkeys. I. Clinical examinations; aqueous ascorbate and protein, Invest. Ophthalmol. Visual, Sci., 24(2), 153, 1983.


5. Kinsey, V.E., Comparative chemistry of aqueous humor in posterior and anterior chambers of rabbit eye, Arch. Ophthalmol., 50, 401, 1953. 6. DeBarnadinis, E. et al., The chemical composition of the human aqueous humor in normal and pathological conditions, Exp. Eye Res., 4, 179, 1965. 7. Graymore, C.N., Biochemistry of the Eye, Academic Press, New York, 1970. 8. Davson, H. and Graham, L.T., Comparative aspects of the intraocular fluids, in The Eye, vol. 5, Davson, H., Ed., Academic Press, New York, 1974. 9. Bito, L., Intraocular fluid dynamics. I. Steady-state concentration gradients of magnesium, potassium and calcium in relation to the sites and mechanisms of ocular cation transport processes, Exp. Eye Res., 10, 102, 1970. 10. Kinsey, V.E. and Reddy, D.V.N., The Rabbit in Eye Research, Prince, J.H., Ed., Charles C Thomas, Springfield, IL, 1964. 11. laurent, V.B.G., Hyaluronate in aqueous humour, Exp. Eye Res., 33, 147, 1981. 12. Wegener, J.K. and Moller, P.M., Oxygen tension in the anterior chamber of the rabbit eye, Acta Ophthalmol., 49, 577, 1971. 13. Kleifeld, O. and Neumann, H.G., Der sauerstaffgehalt des menschlichen kammerwassers, Klin. Monatsbl. Augenheilkd., 35, 224, 1959. 14. Walker, A.M., Comparison of the chemical composition of aqueous humor, cerebrospinal fluid, lymph and blood from frogs, higher animals and man. Reducing substances, inorganic phosphate, uric acid, urea., J. Biol. Chem., 101, 269, 1933. 15. Duke-Elder, Sir S., Physiology of the Eye, vol. 4 of System of Ophthalmology, C.V. Mosby, St. Louis, 1968. 16. Dernouchamps, J.P., The proteins of the aqueous humour, Doc. Ophthalmol., 53, 193, 1982. 17. Blogg, J.R. and Coles, E.H., Vet. Bull., 40, 347, 1970. 18. Furuichi, C., The influence of various experimental injuries on creatine, creatinene metabolism of aqueous fluid of the rabbit’s eye, Acta Soc. Ophthalmol., 65, 561, 1961. 19. McLaughlin, P.S. and McLaughlin, B.G., Chemical analysis of bovine and porcine vitreous humors: correlation of normal values with serum chemical values and changes with time and temperature, Am. J. Vet. Res., 48, 467, 1987. 20. Nordmann, J., Biologie et Chirurgie due Corps Vitre, Brini, A., Ed., Masson & Cis, Paris, 1968. 21. Goldenthal, E.I., Current views on safety evaluation of drugs, FDA Papers, 2, 13, 1968. 22. Federal Register, Interagency Regulatory Liaison Group recommended guideline for acute eye irritation testing, National Technological Information Service PB82-117557, 1981. 23. Organization for Economic Cooperation and Development, Acute eye irritation and corrosion, Publication 405, OECD Publications and Information Center, Washington, 1987. 24. Draize, J.H., Woodard, G., and Calvery, H.O., Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes, J. Pharmacol., Exp. Ther., 82, 377, 1944. 25. Baldwin, H.A., McDonald, T.O., and Beasley, C.H., Slit-lamp examination of experimental animal eyes. II. Grading scales and photographic evaluation of induced pathological conditions, J. Soc. Cosmet. Chem., 24, 181, 1973. 26. Hackett, R.B. and McDonald, T.O., Eye irritation, in Dermatotoxicology, 4th ed., Marzulli, F.N. and Maibach, H.I., Eds., Hemisphere, New York, 1991. 27. Kay, J.H. and Calandra, J.C., Interpretation of eye irritation tests, J. Soc. Cosmet. Chem., 13, 281, 1962. 28. Committee for the revision of NAS Publication 1138, Principles and Procedures for Evaluating the Toxicity of Household Substances, National Academy of Sciences, Washington, 1977. 29. Green, W.R. et al., A Systematic Comparison of Chemically Induced Eye Injury in the Albino Rabbit and Rhesus Monkey, The Soap and Detergent Association, New York, 1978, 407. 30. Dunn, B., Toxicology of the eye, in CRC Handbook of Toxicology, Derelanko, M.J. and Hollinger, M.A., Eds, CRC Press, Boca Raton, FL, 1995, 186. 31. Beckley, J.H., Russell, T.J., and Rubin, L.F., Use of the Rhesus monkey for predicting human response to eye irritants, Toxicol. Appl. Pharmacol., 15, 1, 1969. 32. Environmental Protection Agency, Guidance for evaluation of eye irritation testing, Hazard Evaluation Division Standard Evaluation Procedures, EPA-540/09-88-105, Washington, 1988. 33. Camp, D.D., Federal Register, 49, 188, 1984.


34. McDonald, T.O., Baldwin, H.A., and Beasley, C.H., Slit-lamp examination of experimental animal eyes. I. Techniques of illumination and the normal animal eye, J. Soc. Cosmet. Chem., 24, 163, 1973. 35. McDonald, T.O., Kasten, K., Hervey, R., Gregg, S., Borgmann, A.R., and Murchison, T., Acute ocular toxicity of ethylene oxide, ethylene glycol, and ethylene chlorohydrin, Bull. Parenter. Drug Assoc., 27, 153, 1973. 36. Chang, D.F., Ophthalmic examination, in General Ophthalmology, 12th ed., Vaughan, D., Asbury, T., Tabbara, K., Eds., Appleton & Lange, Norwalk, CT, San Mateo, CA, 1989. 37. Mishima, S., Corneal thickness, Survey Ophthalmol., 13, 57, 1968. 38. Mishima, S. and Hedbys, B.O., Measurements of corneal thickness with the Haag-Streit pachometer, Arch. Ophthalmol., 80, 710, 1968. 39. Jacobs, G.A. and Martens, M.A., An objective method for the evaluation of eye irritation in vivo, Fd. Chem. Toxicol., 27, 255, 1989. 40. Morgan, R.L., Sorenson, S.S., and Castles, T.R., Prediction of ocular irritation by corneal pachymetry, Food Chem. Toxicol., 25, 609, 1987. 41. Kennah, H.E., Hignet, S., Laux, P.E., Dorko, J.D., and Barrow, C.S., An objective procedure for quantitating eye irritation based on changes of corneal thickness, Fundam. Appl. Toxicol., 12, 258, 1989. 42. Leibowitz, H.M. and Laing, R.A., Specular microscopy, in Corneal Disorders: Clinical Diagnosis and Management, Leibowitz, H. M., Ed., W.B. Saunders, Philadelphia, 1984. 43. Scheimpflug, T., Der photoperspektograph und seine Anweedung, Photor. Korr., 43, 516, 1906. 44. Forbes, M., Pico, G. Jr., and Grolman, B., A noncontact applanation tonometer, Arch. Ophthal., 91, 134, 1974. 45. Callaway, S., Gazzard, M.F., Price Thomas, D., and Swanston, D.W., The calibration and evaluation of a handheld tonometer as a means of measuring the intraocular pressure in the conscious rabbit, Exp. Eye Res., 15, 383, 1973. 46. Pollack, I.P., Viernstein, L.J., and Radius, R.L., An instrument for constant-pressure tonography, Exp. Eye Res., 29, 579, 1979. 47. Hilton, G.F. and Shaffer, R.N., Electronic applanation tonometry, Am. J. Ophthalmol., 62, 838, 1966. 48. Reinhardt, C.A., Pelli, D.A., and Zbinden, G., Interpretation of cell toxicity data for the estimation of potential irritation, Food Chem. Toxicol., 23, 247, 1985. 49. Grant, W.M., Toxicology of the Eye, 3rd ed., Charles C Thomas, Springfield, IL, 1986.

5

Fundamental Inhalation Toxicology Paul E. Newton, Ph.D., D.A.B.T.

CONTENTS Section 1. Introduction Section 2. Respiratory Tract Anatomy Table 5.1 Comparative Lung Biology: Morphologic Features of Pleura, Interlobular and Segmental Septa, and Distal Airways Table 5.2 Interspecies Comparison of Nasal Cavity Characteristics Table 5.3 Comparative Anatomy of the Lung Parenchyma and Air-Blood Tissue Barrier Table 5.4 Allometry of Pulmonary Structural Variables Table 5.5 Total Tissue Volumes, Surface Areas, and Mean Tissue Thickness in the Alveolar Region of Normal Mammalian Lungs Table 5.6 Tracheobronchial and URT Liquid Lining Layer Thicknesses Table 5.7 Alveolar Region Tissue and Blood Compartment Dimensions of Normal Mammalian Lungs Table 5.8 Anatomical Data: Trachea of Various Species Table 5.9 Dimensions of the Cross Sections of the Nasal Cast of the Rat Table 5.10 Dimensions of the Cross Sections of the Nasal Cast of the Guinea Pig Table 5.11 Dimensions of the Cross Sections of the Nasal Cast of the Beagle Table 5.12 Dimensions of the Cross Sections of the Nasal Cast of the Rhesus Monkey Figure 5.1 Relationship Between Body Mass and the Logarithms of Tracheal Diameter and Length for a Variety of Species Figure 5.2 Allometric Plot of Mean Lung Volume to Mean Body Mass for Mammalian Species Figure 5.3 Allometric Plot of Alveolar Surface Area and Harmonic Mean Tissue Thickness to Body Mass for Mammalian Species Figure 5.4 Allometric Plot of Capillary Volume to Body Mass for Mammalian Species Figure 5.5 Allometric Plot of Pulmonary Diffusing Capacity to Body Mass for Mammalian Species Figure 5.6 Pleural Space Thickness vs. Body Weight in Five Mammalian Species Figure 5.7 Mean Alveolar Diameter vs. Mean Ventilatory Unit Diameter


Section 3. Cells in the Respiratory Tract Table 5.13 Comparison of Abundance and Percentage of Cell Types in Tracheas of Seven Mammalian Species Table 5.14 Density of Cells in the Bronchiolar Epithelium of Adults Table 5.15 Height of the Airway Epithelium in Adult Mammals Table 5.16 Population Densities (% Cells) of the Tracheal Surface Epithelium in Adult Mammals and Juvenile Birds Table 5.17 Population Densities (% Cells) of the Bronchial Surface Epithelium in Adult Mammals (Mainstem, Primary Bronchi) Table 5.18 Population Densities (% Cells) of the Bronchial Surface Epithelium in Adult Mammals (Lobar Bronchi, Generations 2–6) Table 5.19 Population Densities (% Cells) of the Bronchial Surface Epithelium in Adult Mammals (Segmental Bronchi, Generations 7–11) Table 5.20 Comparison of Species Differences in Microenvironment of Bronchiolar Cells Table 5.21 Comparison of Species Differences in Cellular Composition of Centriacinar Bronchiolar Epithelium Table 5.22 Comparison of Numerical Density of Bronchiolar Epithelium and Density and Percentage of Clara Cells in Bronchiolar Epithelial Population of Adults Table 5.23 Comparison of Relative Proportions (Percentages) of Cellular Components in Clara Cells Table 5.24 Characteristics of Cells from the Alveolar Region of Normal Mammalian Lungs Table 5.25 Summary of Experimentally Determined Turnover Times for Selected Cells of Respiratory Tracts of Rats and Mice Figure 5.8 Allometric Relationship for Alveolar Cells Section 4. Pulmonary Function Table 5.26 Allometry of Pulmonary Diffusing Capacity Table 5.27 Allometry of O2 Consumption and Flux Table 5.28 Allometry of Blood Variables Table 5.29 Values of Standard (Basal) Oxygen Consumption for Representative Mammalian Species of Different Body Mass Table 5.30 Allometric Equations for Respiratory Variables in Mammals Table 5.31 Maximal Oxygen Consumption for Mammals Table 5.32 Blood Respiratory Variables Table 5.33 Normal Values of Blood Gases and Blood Buffering Table 5.34 Body Weight and Lung Volumes in Fischer-344 Rats at Various Ages Table 5.35 Body Weight and Lung Volumes in Adult and Older Hamsters Table 5.36 Ventilatory Parameters in Fischer-344 Rats of Various Ages Table 5.37 Ventilatory Parameters in Hamsters at Various Ages Table 5.38 The Lung Volumes (% TLC) at Transpulmonary Pressures for Various Young Adult Mammals Table 5.39 Morphometric Values in Sprague-Dawley Rats of Various Ages


Section 5. Bronchoalveolar Table 5.40 Table 5.41 Table 5.42

Lavage Fluid (BALF) Normal Cytology of BALF Normal Biochemical Content of BALF Relative Proportions of Immunocompetent Cell Populations Obtained by Bronchoalveolar Lavage Table 5.43 Lymphocyte Subpopulations Observed in Bronchoalveolar Lavage Fluid of Lung Tissue Section 6. Pulmonary Deposition and Clearance Table 5.44 Lung and Alveolar Macrophage (AM) Parameters as they May Relate to in Vivo Particle Uptake Table 5.45 Tracheal Mucociliary Clearance Table 5.46 Nasal Mucociliary Clearance Table 5.47 Comparative Pulmonary Clearance Data for Relatively Insoluble Particles Inhaled by Laboratory Animals and Humans Figure 5.9 Particle Deposition Efficiency in Experimental Animals Figure 5.10 Alveolar Clearance Rate as a Function of Total Particle Volume in the Lung, with Data from Different Investigators Using Different Insoluble Particles Figure 5.11 Tracheal Mucous Velocities in a Log-Log Plot vs. the Body Weight of a Range of Species Figure 5.12 Tracheal Mucous Velocity for Beagle Dogs vs. Age Section 7. Pulmonary Toxicity Table 5.48 Slopes of Ventilatory Responses to Carbon Dioxide Table 5.49 Species Comparison of Lung Function Response After Exposure to Air Pollutants Table 5.50 Lung Dysfunction After Toxicant Exposure Table 5.51 Agents Causing Lung Tumors in Laboratory Animals After Inhalation Exposure Table 5.52 Carcinogenic Agents Causally Associated with Human Lung or Pleural Cancer Table 5.53 Effects of Inhaled Toxicants on Mucociliary Clearance Table 5.54 Effects of Inhaled Toxicants on Clearance from the Respiratory Region of the Lungs Table 5.55 Effects of Inhaled Toxicants on the Phagocytic Activity of Alveolar Macrophages Figure 5.13 Double-Logarithmic Correlation of Inhalation LC50 and Oral LD50 Figure 5.14 Correlation Between Time-Weighted Average Threshold Limit Values (ACGIH) and RD50 Values Determined in Mice for 26 Irritants Figure 5.15 Ventilatory Response to 12% Inspired O2 (or PI O2 at 90 torr) in 10 Species Over a Wide Range of Body Size Section 8. Inhalation Exposure Generation Table 5.56 Uniformity Test of Different Exposure Chambers Table 5.57 Ammonia Concentrations in an Inhalation Chamber Table 5.58 Conversion Table for Gases and Vapors (ppm:mg/l) Table 5.59 Characteristics of Nebulizers Table 5.60 Operating Characteristics of Compressed Air Nebulizers Table 5.61 Water Droplet Lifetimes Table 5.62 Characteristics of Dry Powder Generators


Table 5.63

Efficiencies in Terms of Residual Water Content of Selected Solid Desiccants Used for Drying 25ºC Nitrogen Gas at 225 cc/min through a Bed 14 mm i.d. and 450 mm Deep Figure 5.16 Spatial Variations of Aerosol Concentrations in Hazelton 2000 Exposure Chambers as a Function of Aerosol Particle Size References Additional Related Information Table 5.64 Some Xenobiotic Metabolizing Enzymes in the Nasal Cavity Table 5.65 Summary of P-450 Isozymes Reported in the Rat and Rabbit Nasal Cavities Table 5.66 Some P-450 Isozymes Reported in Lungs of Various Species

SECTION 1. INTRODUCTION Potential exposure to toxic materials is greater via inhalation than any other route of exposure. The potential is greatest through the lung because more air is inhaled each day than water or food is ingested. The surface area of the lung far exceeds the surface area of the skin and gastrointestinal tract. Some inhalation exposures may be intentional, as with inhaled drugs. However, most exposures are unintentional via environmental pollutants in the industrial setting or ambient air. Inhalation toxicity studies determine the health effects of these materials through exposure to animals, which then allows for human risk assessment. Inhalation studies involve all the standard types of toxicity studies and their various endpoints including acute, subchronic, chronic, oncogenicity, reproductive, developmental, neurotoxicity, as well as in vitro exposures. The following tables and graphs are a compilation that has proved to be useful in the conduct of inhalation studies and the extrapolation of effects among different species. The compilation includes data on respiratory tract anatomy, pulmonary function, bronchoalveolar lavage, metabolism, pulmonary deposition and clearance, pulmonary toxicity, and data associated with exposure chambers and the generation and monitoring of exposure atmospheres.

SECTION 2. RESPIRATORY TRACT ANATOMY TABLE 5.1 Comparative Lung Biology: Morphologic Features of Pleura, Interlobular and Segmental Septa, and Distal Airways

Human Pleura Thick Interlobular and Extensive, segmental interlobular connective tissue partially surrounds many lobules Nonrespiratory Several bronchiole generations (nonalveolarized)

Macaque Monkey

Dog, Cat

Ferret

Mouse, Rat, Gerbil Hamster, Guinea Pig, Rabbit Horse, Sheep

Thin Little

Thin Little, if any

Thin Little

Thin Little, if any

Several generations

Several generations

Fewer Fewer generations, generations commonly only one


Thick Extensivea interlobular partially surrounds many lobules Several generations

Ox, Pig Thick Extensive, interlobular surrounds many lobules completely Several generations

TABLE 5.1 (Continued) Comparative Lung Biology: Morphologic Features of Pleura, Interlobular and Segmental Septa, and Distal Airways

Respiratory bronchiole (alveolarized)

Human

Macaque Monkey

Dog, Cat

Ferret

TB ends in respiratory bronchioles




Several generations

Several generations

Several generations

Several generations

Mouse, Rat, Gerbil Hamster, Guinea Pig, Rabbit Horse, Sheep

Ox, Pig

TB ends in alveolar ducts or very short respiratory bronchioles Absent or a single short generation



Note: TB = terminal nonrespiratory bronchiole. a

The interlobular connective tissue of the sheep appears extensive and lobules appear completely separated in gross preparations, but not in LM, SEM, or HRCT. From Tyler, W.S. and Julian, M.D., in Treatise on Pulmonary Toxicology, vol. 1, Comparative Biology of the Normal Lung, CRC Press, Boca Raton, FL, 1992. With permission.

TABLE 5.2 Interspecies Comparison of Nasal Cavity Characteristics Sprague-Dawley Rat

Guinea Pig

Beagle Dog

Rhesus Monkey

Body weight Naris cross-section Bend in naris Length Greatest vertical diameter Surface area (both sides of nasal cavity) Volume (both sides)

250 g 0.7 mm2 40º 23 cm 9.6 mm 10.4 cm2

600 g 2.5 mm2 40º 3.4 cm 12.8 mm 27.4 cm2

10 kg 16.7 mm2 30º 10 cm 23 mm 220.7 cm2

7 kg 22.9 mm2 30º 5.3 cm 27 mm 61.6 cm2

~70 kg 140 mm2

0.4 cm3

0.9 cm3

20 cm3

8 cm3

Bend in nasopharynx Turbinate complexity

15º Complex scroll

30º Complex scroll

30º Very complex membranous

80º Simple scroll

16–19 cm3 (does not include sinuses) ~90º Simple scroll

Man

7–8 cm 40–45 mm 181 cm2

From Schreider, J.P., in Nasal Tumors in Animals and Man, vol. III, Experimental Nasal Carcinogenesis, CRC Press, Boca Raton, FL, 1983.26 With permission.


Species Shrew (Surcus etrascus) White mouse (Mus musculus) Waltzing mouse (Mus wagneri) Syrian golden hamster (Mesocricetus auratus) White rat (Rattus rattus) White rat (Sprague-Dawley) White rat (Fischer-344) Male: 5 mo Female: 5 mo Male: 26 mo Female: 26 mo Guinea pig (Cavia porcellus) Rabbit (Oryctolagus cuniculus) Dwarf mongoose (Helogalepervula) Genet cat (Genetta ligrino) Dog (Canis familiaris) Dog (C. familiaris) Dog (C. familiaris) Dog (C. familiaris)


N 4 5 5 4 8 6 4 4 4 4 15 6 3 2 3 8 4 6

Body Weight (g) 2.6 23 13 118 140 360

± ± ± ± ± ±

0.2 2 1 7 7 4

289 ± 13 182 ± 5 391 ± 11 298 ± 7 429 ± 11 3,560 52,800 ± 9,800 137,200 ± 4,300 5,400 11,200 ± 400 16,000 ± 3,000 22,800 ± 600

Lung Volume (ml) 0.10 0.74 0.58 2.81 6.34 10.82

± ± ± ± ± ±

Alveolar Surface Area (Both lungs), cm2

0.01 0.07 0.06 0.24 0.25 0.38

170 680 630 2,760 3,880 4,865

± ± ± ± ± ±

10 85 40 250 190 380

8.60 ± 0.31 7.48 ± 0.10 12.67 ± 0.74 9.39 ± 0.40 13.04 ± 3.03 79.2 30.6 ± 5.6 99.0 ± 12.2 284.2 736 ± 25 1,322 ± 64 1,501 ± 74

3,915 3,420 4,630 4,020 9,100 58,600 16,100 56,300 182,000 407,000 510,000 897,000

± ± ± ± ± ± ± ± ± ± ± ±

390 125 440 25 280 12,400 2,600 6,400 135,000 39,000 10,000 69,000

Capillary Surface Area (Both lungs), cm2 130 590 540 2,410 4,070 4,270

± ± ± ± ± ±

15 60 30 190 200 385

3,830 3,260 4,490 3,570 7,400 47,000 14,600 42,300 141,000 329,000 570,000 718,000

± ± ± ± ± ± ± ± ± ± ± ±

395 185 485 165 230 8,800 3,400 1,600 111,000 16,000 20,000 69,000

Capillary Volume (Both lungs), ml 0.0118 0.084 0.065 0.294 0.480 0.63

± ± ± ± ± ±

0.002 0.009 0.008 0.011 0.022 0.07

0.65 ± 0.06 0.46 ± 0.10 0.67 ± 0.10 0.34 ± 0.05 1.50 ± 0.08 7.15 ± 1.88 2.06 ± 0.52 5.04 ± 0.63 26.0 ± 24.9 50.2 ± 5.0 92 ± 5 71.8 ± 4.5

Tissue (µm) 0.27 0.32 0.26 0.39 0.37 0.40

± ± ± ± ± ±

0.02 0.01 0.002 0.10 0.02 0.02

0.38 ± 0.03 0.34 ± 0.01 0.37 ± 0.01 0.37 ± 0.01 0.42 ± 0.01 0.50 ± 0.04 0.39 ± 0.02 0.51 ± 0.02 0.43 ± 0.02 0.46 ± 0.01 0.45 ± 0.01 0.48 ± 0.01


TABLE 5.3 Comparative Anatomy of the Lung Parenchyma and Air-Blood Tissue Barriera

a

5 2 1 2 2 1 2 2 2 4 1 2 6 5 8

46,100 231,700 ± 2,700 383,000 3,300 ± 300 4,200 ± 100 102,000 109,800 ± 16,300 20,900 ± 1,000 21,800 ± 200 192,500 ± 24,000 700,000 510,000 ± 0 3,710 29,000 ± 3,000 74,000 ± 4,000

2,888 15,900 ± 1,400 21,000 209.4 ± 0.6 313.4 ± 1.2 7,678 7,835 ± 1,550 1,370 ± 15 17,055 ± 435 10,145 ± 1,960 22,450 37,650 ± l,050 184.2 2,393 ± 100 4,341 ± 285

1,769,000 ± 456,000 4,305,000 ± 584,000 6,361,000 96,900 ± 5,500 146,000 ± 700 3,908,000 3,829,000 ± 950,000 449,000 ± 12,000 671,000 ± 71,000 3,850,000 ± 420,000 12,830,000 24,560,000 ± 124,000 133,000 ± 12,700 496,000 ± 77,000 1,430,000 ± 120,000

1,319,000 ± 375,000 2,726,000 ± 292,000 5,516,000 81,300 ± 13,000 130,000 ± 6550 2,813,000 3,378,000 ± 460,000 439,000 ± 12,000 645,000 ± 139,000 3,795,000 ± 392,000 11,380,000 16,630,000 ± 1,080,000 116,000 ± 15,400 386,000 ± 95,000 1,260,000 ± 120,000

234 ± 69 378 ± 100 965 12.4 ± 0.7 22.6 ± 3.3 472 584 ± 98 101 ± 8 146 ± 35 700 ± 124 2,770 2,800 ± 300 15.5 ± 2.7 44 ± 17 213 ± 31

0.53 ± 0.08 0.60 ± 0.06 0.60 0.56 ± 0.09 0.43 ± 0.02 0.37 0.46 ± 0.04 0.54 ± 0.03 0.53 ± 0.05 0.50 ± 0.04 0.51 0.60 ± 0.02 0.50 ± 0.03 0.67 ± 0.06 0.62 ± 0.04

All values are mean ± SEM.

From Pinkerton, R.E., Gehr, P., and Crapo, J.D., in Treatise on Pulmonary Toxicology, vol. 1, Comparative Biology of the Normal Lung, CRC Press, Boca Raton, FL. 1991.3 With permission.



Dog (C. familiaris) Camel (Camelus dromedarus) Giraffe (Giraffa camelopordalis) Suni (Nesotragus moschatus) Dik-dik (Madogua kirkii) Wildebeest (Connochaetes tauriras) Waterbuck (Kobus defasso) African goat (Capra hircus) African sheep (Ovis aries) Zebu cattle (Bos indicus) Swiss cow (B. taurus) Horse (Equis cabalbus) Monkey (Macaca irus) Baboon (Papio papio) Man (Homo sapiens)


TABLE 5.4 Allometry of Pulmonary Structural Variables Taxon

Slope

N

Mb Range

VL, ml; Lung Volume 56.7 1.02 40.0 1.021 46.0 1.059 47.5 1.060 54.9 1.15 52.8 1.07 96.4 1.07

21 13 33 47 4 1 5

0.01–2.000 0.003–3.71 0.003–700 0.003–700 4.6–27.6 2.65–57 0.005–0.173

S(A), m2, Alveolar Surface Area Mammals 1.87 0.888 13 Mammals 3.34 0.949 33 Mammals 3.36 0.935 47 Canids 1.12 1.38 4 Dog (C. familiaris) 3.12 1.05 1 Batsb 5.18 1.01 5

0.003–3.71 0.003–700 0.003–700 4.6–27.6 2.65–57 0.005–0.173

Mammals Mammals Mammals Mammals Canids Dog (Canis familiaris) Bats

Intercept

S(c), m2; Pulmonary Capillary Surface Area Mammals 2.73 0.952 33 0.003–700 Mammals 2.72 0.941 47 0.003–700 Canids 1.14 1.25 4 4.6–27.6 Dog (C. familiaris) 2.53 1.05 1 2.65–57 V(c), ml; Pulmonary Capillary Volume Mammals 3.20 1.000 33 0.003–700 Mammals 3.63 1.009 47 0.003–700 Canids 1.92 1.23 4 4.6–27.6 Dog (C. familiaris) 3.28 1.12 1 2.65–57 Bats 3.73 0.954 5 0.005–0.173 τht µm; Harmonic Mean Thickness of Alveolar Membrane Mammals 0.416 0.050 33 0.003–700 Mammals 0.413 0.053 47 0.003–700 Canids 0.476 0.000 4 4.6–27.6 Dog (C. familiaris) 0.39 0.80 1 2.65–57 Bats 0.249 0.021 5 0.005–0.173 Note: Allometry of pulmonary structural variables that contribute to gas conductance of the lung and morphometric estimates of pulmonary diffusing capacity (DL0). Variables are related to body mass (Mb, in kg) by the function: Y = a · Mbb, where Y is the variable. a is the intercept of the log-transformed linear regression, and b is the slope of the logtransformed linear regression. N is the number of species. In some cases, regression equations were calculated from data given in the reference. Units have been converted for equivalence, where necessary. From Jones, J.H. and Longworth, K.E., in Treatise on Pulmonary Toxicology, vol. 1, Comparative Biology of the Normal Lung, CRC Press, Boca Raton, FL, 1992.6 With permission.



TABLE 5.5 Total Tissue Volumes, Surface Areas, and Mean Tissue Thickness in the Alveolar Region of Normal Mammalian Lungs†

Body weight, kg Lung volume, ml Total volumes, cm3/both lungs Air Capillary lumen Tissue Type I epithelium Type II epithelium Cellular interstitium Noncellular interstitium Endothelium Macrophages Surface area, m2 both lungs‡ Alveolar epithelium Type I Type II Capillary endothelium Arithmetic mean, tissue thickness, µm Epithelium Type I Type II Interstitium Endothelium

Fischer-344 Rat (n = 4)

Sprague-Dawley Rat (n = 8)

0.29 ± 0.01a 8.6 ± 0.31a

0.36 ± 0.01 10.55 ± 0.37a

5.978 0.649 0.428 0.082 0.037 0.068 0.128 0.094 0.019

± ± ± ± ± ± ± ± ±

0.197 0.057a 0.046a 0.006a 0.009a 0.005a 0.016a 0.009a 0.007a

0.391 ± 0.39a 0.015 ± 0.005a 0.383 ± 0.39a

0.212 2.758 0.500 0.246

± ± ± ±

0.008b 0.424ab 0.028a 0.011a

7.216 0.659 0.671 0. 144 0.053 0.079 0.214 0.156 0.025

± ± ± ± ± ± ± ± ±

0.278a 0.055a 0.041a 0.010a 0.009a 0.015a 0.016a 0.007a 0.006a

0.387 ± 0.025a 0.015 ± 0.002a 0.452 ± 0.035a

0.384 3.653 0.693 0.358

± ± ± ±

0.038a 0.266a 0.058a 0.031a

Dog (n = 4)

Baboon (n = 5)

16 ± 3 1322 ± 64

29 ± 3 2393 ± 100

74 ± 4 4341 ± 284

914 92 78 16.5 5.6 12.9 22.8 17.6 2.5

1851 44 68 14.4 3.5 9.4 24.6 13.4 2.4

3422 ± 223 169 ± 24 314 ± 41 32.5 ± 3.9 32.1 ± 5.0 54.0 ± 7.0 98.3 ± 12.4 42.6 ± 5.4 54.7 ± 15.7

± ± ± ± ± ± ± ± ±

52 5b 4b 1.9b 0.5b 0.7b 0.7b 1.6 1.1b

51.0 ± 1.0b 1.0 ± 0.2 57.0 ± 2.0

0.327 4.138 0.658 0.308

± ± ± ±

0.043ab 0.340ac 0.033a 0.019a

± ± ± ± ± ± ± ± ±

24 17b 9b 2.3b 0.8b 1.6b 3.7b 2.6 1.1b

47.7 ± 7.7b 1.9 ± 0.3 38.6 ± 9.5

0.308 1.839 0.847 0.361

± ± ± ±

0.021ab 0.141b 0.140a 0.038ab

Human (n = 8)

89.0 ± 8.0 7.0 ± 1.0 91.0 ± 9.0

0.361 5.019 1.634 0.474

± ± ± ±

0.024a 0.551c 0.164 0.052b

†

All data are mean ± SEM. For comparisons between species all data connected by the same letter subscript are not statistically different from each other. ‡ Type I surface area (SA) is the SA of basement membrane under type I cells: type II SA is the air surface of type II cells excluding the extra SA contributed by microvilli; endothelial SA is the luminal surface of the endothelial cells.

From Pinkerton, K.E., Gehr, P., and Crapo, J.D., in Treatise on Pulmonary Toxicology, vol. 1, Comparative Biology of the Normal Lung, CRC Press, Boca Raton, FL, 1992.3 With permission.

TABLE 5.6 Tracheobronchial and URT Liquid Lining Layer Thicknesses Species

Location

Thicknessa (µm)

Comments

Cat Guinea pig

Trachea Trachea Intrapulmonary

≤20, usually pigeon = mouse > rat Squirrel monkey = rat > chimpanzee = pigeon > mouse Squirrel monkey = rhesus monkey > pigeon = rat > baboon > chimpanzee Squirrel monkey > rhesus monkey = pigeon = rat = baboon > chimpanzee Squirrel monkey > chimpanzee = rat > pigeon Pigeon > chimpanzee > rat Rhesus monkey > squirrel monkey > pigeon > mouse

Morphine Chlorpromazine δ-9-THC Phencyclidine

↑ = Rate increase; ↓ = rate decrease; FI = fixed interval schedule of reinforcement; FR = fixed ratio schedule of reinforcement. a

From McMillan (1990).38 With permission.


TABLE 7.20 Comparisons of Endpoints in Developmental Neurotoxicologya Functional Category Sensory

Motivational/arousal

Cognition

Motor

Social

Rodents

Nonhuman Primates

— PI-ASR Sensory-evoked potential Activity Sleep-wake — Seizures — — — — Habituation Short-term memory Long-term memory Pavlovian conditioning SCOB — Reflex dev. Locomotor dev. Motor control EMG Suckling Mother/infant contact Communication Aggression Play Reproductive behavior

Humans

— PI-ASR Sensory-evoked potential

Sensory psychophysics PI-ASR Sensory-evoked potential

Activity Sleep-wake

Activity Sleep-wake Impulsivity Seizures Bayley MDI IQ Visual recognition memory Language development Habituation Short-term memory Long-term memory Pavlovian conditioning SCOB Bayley PDI Reflex dev. Locomotor dev. Motor control EMG Suckling Mother/infant contact Language Aggression Play Reproductive behavior

— Seizures — — Visual recognition memory — Habituation Short-term memory Long-term memory Pavlovian conditioning SCOB — Reflex dev. Locomotor dev. Motor control EMG Suckling Mother/infant contact Communication Aggression Play Reproductive behavior

a

Abbreviations: Dev., development; EMG, electromyograph-; MDI, mental development index; PDI, physical developmental index; PI-ASR, prepulse inhibition of acoustic startle response; SCOB schedule-controlled operant behavior.

From Stanton and Spear (1990).39 With permission.

TABLE 7.21 Comparison of Neuropathological and Neurobehavioral Effects of Developmental Methyl Mercury Exposure Across Species Human

Nonhuman Primate

Small Mammals

High Brain Doses (12–20 ppm) Neuropathology Decrease in size of brain; damage to cortex, basal ganglia, and cerebellum; sparing of diencephalon; ventricular dilation; myelinated fibers; ectopic cells; gliosis; disorganized layers; misoriented cells; loss of cells.


Decrease in size of brain; damage to cortex and basal ganglia; sparing of diencephalon; gliosis; loss of cells (sparing of cerebellum); ventricular dilation; ectopic cells; disorganized layers.

Decrease in size of brain; damage to cortex, basal ganglia, hippocampus; and cerebellum; sparing of diencephalon; ventricular dilation; loss of myelin; misoriented cells; loss of cells.

TABLE 7.21 (Continued) Comparison of Neuropathological and Neurobehavioral Effects of Developmental Methyl Mercury Exposure Across Species Neurobehavior Blindness, deafness, cerebral palsy, spacisticity, mental deficiency, seizures

Blindness, cerebral palsy, spacisticity, seizures.

Blindness, cerebral palsy, spasticity, seizures.

Moderate Brain Doses (3–11 ppm) Neuropathology No data

Neurobehavior Mental deficiency, abnormal reflexes and muscle tone, retarded motor development

No data

Decrease in size of brain, damage to cortex and cerebellum, loss of myelin

Retarded development of object performance, visual recognition memory, and social behavior, visual disturbances, reduced weight at puberty (males)

Abnormal on water maze, auditory startle, visual evoked potentials, escape and avoidance, operant tasks, activity, response to drug challenge

Low Brain Doses (240 6.9 9 5.6 25.8 ± 5.3 min n = 166

0.7 9.6 1 45 36 40

Materials tested neat in vivo unless indicated. All materials tested at 20% of the indicated concentration that was used in vivo.47 Draize modified maximum average scores taken 24 h or more after treatment.


SECTION 3. ASSESSMENT OF DERMAL IRRITATION POTENTIAL Selection of the most appropriate dermal model depends on the question being asked and the cells/tissue available. Many models are used for both irritation and efficacy assessment. Often, the first step in assessing active ingredients may be to use a simple cell-based assay to identify hazard or effect, without regard to actual penetration and exposure in the skin. Most major cell types from human skin are available for cell-based studies. More complex exposure kinetics require ex vivo skin or three-dimensional engineered human skin construct model systems that provide both the barrier properties of skin and the necessary target cells, and offer a measure of the actual response expected in skin. Specialized assays using cell and tissue construct models have been developed for phototoxicity and corrosivity and have received a measure of regulatory approval. Basic skin penetration studies may be performed with living or dead epidermis from humans or animals. Xenobiotic metabolism studies require live skin from the species of interest, although human skin constructs are being used for this purpose because of limited availability of fresh human skin. Although these constructs do not yet duplicate the barrier properties of adult human skin, they do provide reproducible target cells for metabolism studies.

TABLE 20.18 Cell/Tissue-Based Models for the Evaluation of Action on the Skin Screening of individual chemicals/ingredients for toxicity or efficacy in monolayer culture systems: • Models serve to assess the potential for the material to act on the target cells without regard to the actual exposure that might be achieved in vivo. • The measurement is independent of penetration into skin since the cells are exposed directly in aqueous medium. • Model systems composed of monolayer cell cultures of individual cell types (e.g., keratinocytes, dermal fibroblasts, melanocytes, dendritic cells) • Test material prepared in aqueous medium and usually over a series of dilutions • Requires that ingredient be water miscible • Aqueous insoluble ingredients may be tested in a nontoxic solvent on a tissue construct • Assay end points may include: • Direct toxicity (immediate and delayed) • Induction/inhibition of inflammatory mediator expression • Phototoxicity • Inhibition of differentiated function (e.g., collagen synthesis/release) Assessment of ingredients, mixtures, and formulations where the goal is to predict the action on skin in vivo: • Action of the formulation/active ingredients is mediated by their ability to penetrate through the stratum corneum. • Tissue constructs, with a functional stratum corneum, are the models of choice • Epidermal-only or epidermal/dermal “full-thickness” constructs • Allow direct application of the test material to the “dry” stratum corneum • Time to observed action depends on the penetration as well as the “innate” toxicity/potential activity of the material/formulation • Applications • Direct toxicity (immediate and delayed) • Induction/inhibition of inflammatory mediator expression • Phototoxicity • Inhibition of differentiated function • Additional end points from specialized systems with multiple cells types: • Inhibition of melanin deposition in keratinocyte/melanocyte constructs • Epidermal–dermal cell interaction (e.g., balanced cytokine expression or induction of proteases)


TABLE 20.19 Examples of Monolayer Cell-Based Assay Systems for Assessing Action on the Skin Culture Systema

Cell Type Balb/c 3T3 fibroblasts (mouse) Human keratinocytes

Applications

Serum-containing medium Serum-free medium derived from MCDB 153

30

Serum-free medium derived from MCDB 153 without hydrocortisone54

End Point(s)

Phototoxicity

Neutral Red Uptake

Direct cytotoxicity Phototoxicity Stimulation of expression/release of inflammatory mediators Anti-inflammatory action-decreased expression/release of inflammatory mediators

Neutral Red Uptake Neutral Red Uptake ELISA: TNFα, IL-1α, IL-8 ELISA: TNFα, IL-1α, IL-8

Human dermal fibroblasts

Serum-containing medium Ascorbic acid required

Direct cytotoxicity Suppression/stimulation of collagen synthesis

Tetrazolium dye reduction ELISA for procollagen c-peptide

Human melanocytes

Various media including low serum formulations

Melanin synthesis

Changes in tyrosinase activity

Human microvascular endothelial cells

Low serum medium

Proinflammatory responses

Upregulation of adhesion proteins

Human dendritic cells (from bone marrow stem cells)

Proprietary formulations that induce differentiation

Interaction with antigenic/sensitizing agents

Cytokine expression (e.g., IL-1β)

a

See cell supplier literature for the recommended culture medium.


TABLE 20.20 Neutral Red Uptake Phototoxicity Assay in BALB/c 3T3 Mouse Fibroblasts Theory Cells are exposed to serial doses of the test article in the presence or absence of a nontoxic flux of UVA light. A phototoxin transfers light energy to the cells in a deleterious manner, leading to increased cytotoxicity relative to the doses of the test article alone. The uptake of neutral red dye (3-amino-7-dimethylamino-2-methylphenazine hydrochloride) by Balb/c 3T3 mouse fibroblast cell cultures is used to measure changes in cell viability relative to controls.55-57 Experimental Procedure Target Cell Preparation • Stock Balb/c 3T3 cell cultures are maintained at 37 ± 1°C in a humidified atmosphere containing 5 ± 1% CO2. • Cells are subcultured when the stock culture is 50 to 80% confluent. • A cell suspension is prepared to yield 1.0 × 105 cells/ml. • 100 µl (~10,000 cells per well) of the cell suspension are added into the designated wells of the 96-well bioassay plate. • The cultures are incubated for approximately 24 h. • Since UV sensitivity of the cells increases with aging, cells are used at passage numbers 5 is indicative of phototoxic potential of the test material. • The MPE measures the effect of UV exposure over a range of concentrations. A material is considered nonphototoxic if the MPE is 100 58.2 17.8 3.24 0.33 0.60 ± 0.35

>100 >100 >100 74.9 >100 9.57 ± 1.41

none >1.7 >5.62 23.0 >305 15.96

n = 14 1.04 ± 0.33

26.34 ± 5.24

25.3

n = 21 a

The ratio of the IC50 (without UVA)/IC50(with UVA) where a value of 5 or greater is indicative of a phototoxin. b EBSS (Earle’s Balanced Salt Solution), HBSS (Hanks’ Balanced Salt Solution).

TABLE 20.22 Examples of Tissue Models for Skin Studies General Description Human skin: Partial thickness, living or previously frozen59 Full thickness, living

Applications Excised Skin Percutaneous absorption studies; live tissue allows assessment of biotransformation Cytotoxicity/irritancy/corrosion

Regulation of inflammatory mediator (e.g., arachidonic acid products and cytokines) Biotransformation

Porcine skin: Partial thickness, living or previously frozen

Percutaneous absorption studies.

Rodent skin: Living or previously frozen60

Cytotoxicity/irritancy/corrosion

EpiDerm™ Epidermal tissue only with a developed stratum corneum62,63


Tissue Constructs61 Cytotoxicity/irritancy/corrosion Phototoxicity Regulation of inflammatory mediator (e.g., arachidonic acid products and cytokines)

Examples of End Points Measured Quantitative measurements of radiolabeled or nonlabeled materials in the receiver fluid Transepithelial resistance Transepithelial water loss Cell viability measured by vital dye reduction ELISA or HPLC assays for arachidonic acid products RT-PCR for mRNA ELISA for protein products Quantitative measurements of radiolabeled or nonlabeled materials in the receiver fluid Quantitative measurements of radiolabeled or nonlabeled materials in the receiver fluid Transepithelial resistance Transepithelial water loss

Cell viability measured by vital dye reduction ELISA or HPLC assays for arachidonic acid products RT-PCR for mRNA ELISA for protein products

TABLE 20.22 (Continued) Examples of Tissue Models for Skin Studies General Description

Applications

EPISKIN™ Epidermal tissue only with a developed stratum corneum64 (not commercially available)

Cytotoxicity/irritancy/corrosion

SkinEthic™ Epidermal tissue only with a developed stratum corneum65

Cytotoxicity/irritancy

TestSkinII Epidermal with a developed stratum corneum and dermal tissue (66)

Regulation of inflammatory mediator (e.g., arachidonic acid products and cytokines)

Regulation of inflammatory mediator (e.g., arachidonic acid products and cytokines) Cytotoxicity/irritancy Regulation of inflammatory mediator (e.g., arachidonic acid products and cytokines) Interaction of epithelial and stromal cells

Examples of End Points Measured Cell viability measured by vital dye reduction ELISA or HPLC assays for arachidonic acid products RT-PCR for mRNA ELISA for protein products Cell viability measured by vital dye reduction ELISA or HPLC assays for arachidonic acid products RT-PCR for mRNA ELISA for protein products Cell viability measured by vital dye reduction ELISA or HPLC assays for arachidonic acid products RT-PCR for mRNA ELISA for protein products

TABLE 20.23 Sample Protocol for the Time Course Assay with EpiDerm™ Cultures: MTT End Point Theory For many classes of materials, irritancy/corrosivity is manifested in cell cytotoxicity and/or upregulation of inflammatory mediators. This assay evaluates the potential dermal irritancy of a test article as a function of the exposure time of a test article required to reduce cell viability to 50% of control viability to the EpiDerm™ construct. Cell viability is measured by the reduction of MTT and is expressed as a percentage relative to untreated (negative control) cultures. Cytokine expression may be combined with the cytotoxicity end point to refine the assay further. Applications and Use • The EpiDerm model is composed of human keratinocytes stratified into a three-dimensional epidermal structure consisting of several layers, including a functioning stratum corneum. Similar models may also be employed (see Table 20.22). • The EpiDerm construct is suited to address the sensitivity range from very mild to severely aggressive, or corrosive, materials. • Test materials are applied topically at formulation strength. • Suited for both water-soluble and -insoluble formulations. • Suitable for testing creams, pastes, highly viscous materials, and powders. • Cells are of human origin and can be induced to express and generate inflammatory response cytokines. Experimental Procedure Receipt and Preparation of Cultures • Each culture is removed with sterile forceps from the agarose gel in the shipping container, inspected, and transferred to a prelabeled six-well plate containing 0.9 ml of assay medium per well. The EpiDerm cultures are incubated at 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO 2 in air for at least 1 h prior to dosing. Assay Procedure • The test materials are tested neat or at end-use concentrations by topical application to the stratum corneum. Pastes and highly viscous materials may be “creamed” to effect application. • The positive control is 1.0% Triton X-100 and is exposed for 4 and 8 h.


TABLE 20.23 (Continued) Sample Protocol for the Time Course Assay with EpiDerm™ Cultures: MTT End Point • Positive controls for modeling inflammatory responses (e.g., croton oil, PMA) may be included. • The negative control is sterile, deionized water generally exposed concurrently with the longest and shortest exposure times of the test or positive control articles. • 100þμl (liquids) or 30þmg (solids) of the test or control article are applied topically onto the tissue surface. • The cultures are returned to the incubator for the appropriate exposure times. Generally, a minimum of four exposure times, ranging from 30 min to 24 h, are selected. For addressing severely irritating or corrosive materials, exposure times of 3 to 60 min may be selected and the treatment volume reduced to 50 μl/tissue. • After the appropriate exposure time, the test articles are rinsed from the cultures using DPBS without Ca2+ and Mg2+. • The cultures are transferred to wells containing 0.3 ml of MTT reagent (1þmg/ml) and incubated for 3 h. • After incubation, the cultures are blotted on absorbent paper and extracted in 2 ml of isopropanol for 2 hours, while shaking. • 200 μl of each extraction solution is transferred to a 96-well plate and the absorbance at 550 nm (OD550) recorded. • Medium samples may be collected and prepared for a variety of cytokine analyses to assess inflammatory responses. Data Evaluation • The relative survival is determined by comparing the mean corrected OD550 of the test article-treated wells to the mean corrected OD550 of the negative control–treated wells. • Exposure time–response curves may be plotted with the percent of control on the ordinate and the test article exposure times on the abscissa. • The ET50 (the time of exposure to the test article that reduces MTT conversion by 50%) is determined by interpolation from the exposure time–response curves. • Occasionally, a test article may directly reduce the MTT giving erroneous results. A direct MTT reduction test is performed as a prescreen, and “killed tissue” controls may be assayed concurrently. • Interpretation: Exposure response curves and ET50 values are compared between test formulations as well as benchmarks, if applicable.

TABLE 20.24 Sample Data from the Corrosivity and Irritancy Assays Using the Human Skin Construct EpiDerm™ Test Material Corrosivity Assay (50 μl treatment with 3-min exposure)a

Assay End Point Measure Percent viability compared with controls using MTT end point (1440 min 329 ± 42 min 8 = 87

In Vivo Classification Corrosive Corrosive Noncorrosive Noncorrosive

Irritant Irritant Mild irritant Nonirritant

TABLE 20.25 Assays for Skin Corrosion Models

Basis of the Assay

End Point Measured

Corrositex™a

Penetration of the test material through a collagen biomatrix “biobarrier”

Nontissue Methods Color change in the chemical detection solution below the biobarrier

Time required to penetrate biobarrier

Generally applicable only to acids, acid derivatives, and bases; absence of a test article-induced color change in the chemical detection system precludes use of the assay

EpiDerm63

Damage to cells in a three-dimensional artificial human skin construct after topical exposure to test materials

Tissue-Based Methods Number of viable cells as estimated by uptake and reduction of the dye MTT

Time required to reach 50% cell death

Applicable to most chemicals without regard to their physical state (solids or liquids) or water solubility

EPISKIN69 (not commercially available)

Damage to cells in a three-dimensional artificial human skin construct after topical exposure to test materials

Number of viable cells as estimated by uptake and reduction of the dye MTT

Time required to reach 50% cell death


Rat Skin Transcutaneous Electrical Resistanceb

Loss of normal stratum corneum integrity and barrier function after topical exposure to test material

Reduction in transcutaneous electrical resistance

Treatment time required to reduce transcutaneous electrical resistance below a predetermined threshold level


a b

Scoring

Approved for use in the United States after review by ICCVAM and acceptance by several constituent regulatory agencies. Approved for use in the Europe an Union after validation by ECVAM and acceptance into Annex V test guidelines.


Notes

TABLE 20.26 Sample Protocol for the Corrositex Assay Theory Many corrosive materials act rapidly to break down tissue proteins as they penetrate through the skin. This assay evaluates the potential corrosivity of a test article as a function of the time required to penetrate through a calibrated protein biobarrier into a chemical detection system. The penetration time and acid/alkaline reserve potential of the test article are used in the prediction of corrosive potential. Applications and Use • The Corrositex assay is based on the time that is required for the test sample to pass through a biobarrier membrane and produce a change in the Chemical Detection System (CDS). The Corrositex biobarrier membrane consists of a reconstituted collagen matrix. • The Corrositex assay has been approved by the U.S. Department of Transportation for testing potential corrosive materials and assigning United Nations packing group categories for certain chemical classes. • Test materials are applied to the test system at formulation strength. • The Corrositex assay system is limited to testing those materials that cause detectable pH changes in the CDS (i.e., acids, acid derivatives, and bases). Test materials must cause a color change in the CDS to be detected. • The assay system is not suited for testing materials that cannot be detected in the CDS (i.e., neutral organics, some metals, etc.). Experimental Procedure Receipt and Preparation of Corrositex Biobarrier Membrane • A qualification screen with the CDS is performed prior to biobarrier membrane preparation. The test article is added directly into a sample of the CDS solution to determine if a color change can be detected. • A categorization screen is performed to categorize weak acids/bases and strong acids/bases. • Weak acids and bases are assayed according to the 1 h protocol (Category 2); strong acids and bases are assayed according to the 4-h exposure protocol (Category 1). • The biobarrier membrane is used no less than 8 h and no more than 7 days after preparation. Assay Procedure • The biobarriers are placed on the CDS solution tubes. • The biobarrier membrane batch is qualified on each day of use by testing the positive control (NaOH). Results must fall within an acceptable range. A timer is started immediately. • 500þμl (liquids) or 500þmg (solids) of test article are added to the biobarrier. A timer is started immediately. • As soon as a color change is detected in the CDS, the elapsed detection time is recorded. • Four replicate biobarrier membranes are treated with each test article. • As soon as a color change is detected in each vial of the CDS, the elapsed detection times for each vial are recorded. • Packing group assignments are made based upon the time required for the test article to break through the CDS and cause a color change. Data Evaluation Time Required For Breakthrough to the CDS (Minutes)

Category Category 1 Category 2

0 to 3 min 0 to 3 min Packing Group I


>3 to 60 min >3 to 30 min Packing Group II

>60 to 240 min >30 to 60 min Packing Group III

>240 min >60 min Noncorrosive

TABLE 20.27 Sample Data from the Corrositex Assay Corrositex Results Test Materiala

Category

Boron trifluoride-dihydrate Phosphoric acid (85%) Butyric acid Ethanolamine Sodium hydrogen fluoride Calcium carbonate Sodium hypochlorite (5%) 2-tert-Butylphenol Isopropanol Historical positive control range: Sodium hydroxide pellet

1 1 1 1 2 2 2 Not testable Not testable

a

Breakthrough Time (min) 1.53 10.96 40.57 23.88 71.57 >240 >240

In Vitro Classification Packing Group Packing Group Packing Group Packing Group Noncorrosive Noncorrosive Noncorrosive

I II II II

In Vivo Classification Packing Group Packing Group Packing Group Packing Group Noncorrosive Noncorrosive Noncorrosive Packing Group Noncorrosive

I II II II

III

11.97 ± 1.4 n = 113

Data from Reference 68.

REFERENCES 1. Curren, R. D., Southee, J. A., Spielmann, H., Liebsch, M., Fentem, J. H., and Balls, M., The role of prevalidation in the development, validation, and acceptance of alternative methods, ATLA, 23, 211–217, 1995. 2. Bruner, L. H., Carr, G. J., Chamberlain, M., and Curren, R. D., Validation of alternative methods for toxicity testing, Toxicol. In Vitro, 10, 479–501, 1996. 3. Bruner, L. H., Carr, G., Chamberlain, M., and Curren, R., No prediction model, no validation study, ATLA, 24, 139–142, 1996. 4. Cooper-Hannan, R., Harbell, J. W., Coecke, S., Balls, M., Bowe, G., Cervinka, M., Clothier, R., Hermann, F., Klahm, L. K., de Lange, J., Liebsch, M., and Vanparys, P., The principles of good laboratory practice: application to in vitro toxicology studies, ATLA, 27(4), 539–577, 1999. 5. Anonymous, ECVAM News and Views, ATLA, 22, 7–11, 1994. 6. Anonymous, Validation and Regulatory Acceptance of Toxicological Test Methods, National Institute of Health Publication 97-3981, Interagency Coordinating Committee on the Validation of Alternative Methods, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 1997. 7. Ahmad, A. E., Aronson J., and Jacobs, S., Induction of oxidative stress and TNF-alpha secretion by dichloroacetonitrile, a water disinfectant by-product, as possible mediators of apoptosis or necrosis in a murine macrophase cell line (RAW), Toxicol. In Vitro, 14(3), 199–210, 2000. 8. Nieminen, A. L., Gores, G. J., Bond, J. M., Imberti, R., Herman, B., and Lemasters, J. J., A novel cytotoxicity screening assay using a multiwell fluorescence scanner, Toxicol. Appl. Pharmacol., 115(2), 145–155, 1992. 9. Rhoads, L. S., Cook, J. R., Patrone L. M., and van Buskirk R. G., A human epidermal model can be assayed employing a multiple fluorescent endpoint assay and the Cytofluor 2300, J. Toxicol. Cutaneous Ocular Toxicol., 12(2), 87–108, 1993. 10. Magoffin, D. A., Kushell, D. L., and Schlesinger, R. J., The chromium-51 release cytotoxicity test: comparison to the agar overlay and cell-growth inhibition tests, in Cell-Culture Test Methods, ASTM STP 810, Brown, S. A., Ed., American Society for Testing and Materials, Philadelphia, 1983, 94–101. 11. Bockman, C. S., Griffith, M., and Watsky, M. A., Properties of whole-cell ionic currents in cultured human corneal epithelial cells, Invest. Ophthalmol. Vision Sci., 39(7), 1143–1151, 1998. 12. Buckman, S. Y., Gresham, A., Hale, P., Hruza, G., Anast, J., Masferrer, J., and Pentland, A. P., COX2 expression is induced by UVB exposure in human skin: implications for the development of skin cancer, Carcinogenesis, 19(5), 723–729, 1998. Copyright © 2002 by Taylor & Francis

13. McConnell, H. M., Owicki, J. C., Parce, J. W., Miller, D. L., Baxter, G. T., Wada, H. G., and Pitchford, S., The Cytosensor microphysiometer: biological applications of silicon technology, Science, 257, 1906–1912, 1992. 14. Harbell, J. W., Osborne, R., Carr, G. J., and Peterson, A., Assessment of the Cytosensor microphysiometer in the COLIPA in vitro eye irritation validation study, Toxicol. In Vitro, 13, 313–323, 1999. 15. Masters, B. A., Palmoski, M. J., Flint, O. P., Gregg, R. E., Wang-Iverson, D., and Durham, S. K., In vitro myotoxicity of the 3-hydroxy-3-methylgluaryl coenzyme A reductase inhibitors, Parvastatin, Lovastatin, and Simvastatin, using neonatal rat skeletal myocytes, Toxicol. Appl. Pharmacol., 131, 163–174, 1995. 16. Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65, 55–63, 1983. 17. Weislow, O. S., Kiser, R., Fine, D. L., Bader, J., Shoemaker, R. H., and Boyd, M. R., New solubleformazan assay for HIV-1 cytopathic effects: application to high-flux screening of synthetic and natural products for AIDS-antiviral activity, J. Natl. Cancer Inst., 81(8), 577–586, 1989. 18. Filman, D. J., Brawn R. J., and Dandliker, W. B., Intracellular supravital stain delocalization as an assay for antibody-dependent complement-mediated cell damage, J. Immunol. Methods, 6(3), 189–207, 1975. 19. Barstad, R., Cortesi, J., and Janus, J., Use of Clonetics' neutral red bioassay to optimize components of serum-free medium for normal human anchorage-dependent cells, In Vitro Cell. Dev. Biol., 27(3), 160, 1991. 20. Jiang, T., Grant, R. L., and Acosta, D., A digitized fluorescence imaging study of intracellular free calcium, mitochondrial integrity and cytotoxicity in rat renal cells exposed to ionomycin, a calcium ionophore, Toxicology, 85(1), 41–65, 1993. 21. North-Root, H., Yackovich, F., Demetrulias, J., Gacula, M., Jr., and Heinze, J. E., Evaluation of an in vitro cell toxicity test using rabbit corneal cells to predict the eye irritation potential of surfactants, in Safety Evaluation and Regulation of Chemicals, Homburger, F., Ed., Karger, Basel, Switzerland, 1983, 259–269. 22. Pham, X. T. and Huff, J. W., Cytotoxicity evaluation of multipurpose contact lens solutions using an in vitro test battery, CLAO J., 25(1), 28–35, 1999. 23. Livingston, R. B., Titus, G. A., and Heilbrun, L. K., In vitro effects on DNA synthesis as a predictor of biological effect from chemotherapy, Cancer Res., 40, 2209–2212, 1980. 24. Harbell, J. W., Wallace, K. A., Curren, R. D., Naughton, G. K., and Triglia, D., A comparison of four measures of toxicity applied to human dermal fibroblasts grown in three dimensional culture on nylon mesh (Skin2 Dermal Model), in Alternative Methods Toxicol., Goldberg, A. M., Ed., Mary Ann Liebert, New York, 8, 301–309, 1991. 25. Lewis, R. W., McCall, J. C., and Botham, P. A., A comparison of two cytotoxicity tests for predicting the ocular irritancy of surfactants, Toxicol. In Vitro, 7, 155–158, 1993. 26. Berridge, M. V., Tan, A. S., McCoy, K. D., and Wang, R., The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts, Boehringer Mannheim Biochem., 4, 14–23, 1996. 27. Marshall, N. J., Goodwin, C. J., and Holt, S. J., A critical review of the use of microculture tetrazolium assays to measure cell growth and function, Growth Regul., 5, 69–84, 1995. 28. Borenfreund, E. and Puerner, J. A., Toxicity determined in vitro by morphological alterations and neutral red absorption, Toxicol. Lett., 24, 119–124, 1985. 29. Reader, S. J., Blackwell, V., O’Hara, R., Clothier, R. H., Griffin, G., and Balls, M., A vital dye release method for assessing the short term cytotoxic effects of chemicals and formulations, ATLA, 17, 28–37, 1989. 30. Harbell, J. W., Koontz, S. W., Lewis, R. W., Lovel, D., and Acosta, D., IRAG working Group 4: cell cytotoxicity assays, Food Chem. Toxicol., 35, 79–126, 1997. 31. Harbell, J. W., Southee, J. A., and Curren, R. C., The path to regulatory acceptance of in vitro methods is paved with the strictest standards, in Animal Alternatives, Welfare and Ethics, van Zutphen, L. F. M. and Balls, M., Eds., Elsevier Science, Amsterdam, 1997, 1177–1181. 32. Centers for Disease Control, Biosafety in Microbiological and Biomedical Laboratories, 3rd ed., U. S. Government Printing Office, Washington, D. C., 1993. 33. Gautheron, P., Dukic, M., Alix, D., and Sina, J. F., Bovine corneal opacity and permeability test: an in vitro assay of ocular irritancy, Fundam. Appl. Toxicol., 18, 442–449, 1992.


34. Sina, J. F., Galer, D. M., Sussman, R. G., Gautherone, P. D., Sargent, E. V., Leong, B., Shah, P. V., Curren, R. D., and Miller, K., A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical intermediates, Fundam. Appl. Toxicol., 26, 20–31, 1995. 35. Bagley, D. M., Waters, D., and Kong, B. M., Development of a 10-day chorioallantoic membrane vascular assay as an alternative to the Draize rabbit eye irritation test, Food Chem. Toxicol., 33(12), 1155–1160, 1994. 36. Bagley, D. M., Cervin, D., and Harbell, J. W., Assessment of the chorioallantoic membrane vascular assay (CAMVA) in the COLIPA in vitro eye irritation validation study, Toxicol. In Vitro, 13, 285–293, 1999. 37. Chamberlain, M., Gad, S. C., Gautherone P., and Prinsen, M. K., Organotypic models for the assessment/prediction of ocular irritation, Food Chem. Toxicol., 35, 23–37, 1997. 38. Tchao, R., Trans-epithelial permeability of fluorescein in vitro as an assay to determine eye irritants, in Alternative Methods in Toxicology, Vol. 6, Goldberg, A. M., Ed., Mary Ann Liebert, New York, 1988, 271–283. 39. Shaw, A. J., Clothier, R. H., and Balls, M., Loss of trans-epithelial impermeability of a confluent monolayer of Madin-Darby canine kidney (MDCK) cells as a determinant of ocular irritancy potential, ATLA, 18, 145–151, 1990. 40. Kruszewski, F. H., Walker, T. L., Ward, S. L., and Dipasquale, L. C., Progress in the use of human ocular tissues for in vitro alternative methods, Comments Toxicol., 5, 203–224, 1995. 41. Luepke, N. P. and Kemper F. H., HET-CAM: an alternative to the Draize eye test, Food Chem. Toxicol., 24, 495–496, 1986. 42. Balls, M., Botham, P. A., Bruner, L. H., and Spielmann, H., The EC/HO international validation study on alternatives to the Draize eye irritation test, Toxicol. In Vitro, 9, 871–929, 1995. 43. Kristen, U. and Kappler, R., The pollen tube growth test, in In Vitro Testing Protocols. Methods in Molecular Biology, Vol. 43, O’Hare, S. and Atterwill, C. K., Humana Press, Totowa, NJ, 1995, 189–198. 44. Pape, W. J. W., Pfannenbecker, U., and Hoppe, U., Validation of the red blood cell test system as an in vitro assay for the rapid screening of irritation potential of surfactants, Mol. Toxicol., 1, 525–536, 1987. 45. Osborne, R., Perkins, M. A., and Roberts, D. A., Development and intralaboratory evaluation of an in vitro human cell-based test to aid ocular irritancy assessments, Fundam. Appl. Toxicol., 28, 139–153, 1995. 46. Stern, M., Klausner, M., Alvarado, R., Renskers, K., and Dickens, M., Evaluation of the EpiOcular™ tissue model as an alternative to the Draize eye irritation test, Toxicol. In Vitro, 12, 455–461, 1998. 47. Blazka, M. E., Harbell, J. W., Klauzner, M., Raabe, H., Kubilus, J., Hsia, F., Minerath, B., Kotler, M., and Bagley, D. M., Colgate-Palmolive’s program to validate the EpiOcular™ human tissue construct model, Toxicologist, 54(1), 188, 2000. 48. Brantom, P. G., Bruner, L. H., Chamberlain, M., De Silva, O., Dupuis, J., Earl, L. K., Lovell, D. P., Pape, W. J. W., Uttley, M., Bagley, D. M., Baker, F. W., Bracher, M., Courtellemont, P., Declercq, L., Freeman, S., Steiling, W., Walker, A. P., Carr, G. J., Dami, N., Thomas, G., Harbell, J., Jones, P. A., Pfannenbecker, U., Southee, J. A., Tcheng, M., Argembeaux, H., Castelli, D., Clothier, R., Esdaile, D. J., Itigaki, H., Jung, K., Kasai, Y., Kojima, H., Kristen, U., Larnicol, M., Lewis, R. W., Marenus, K., Moreno, O., Peterson, A., Rasmussen, E. S., Robles, C., and Stern, M., A summary report of the COLIPA international validation study on alternatives to the Draize Rabbit Eye Irritation Test, Toxicol. In Vitro, 11, 141–179, 1997. 49. Shopsis, C. and Eng, B., In vitro ocular irritancy prediction: assays in serum-free medium correlate better with in vivo data, in Alternative Methods in Toxicology, Vol. 6, Goldberg, A. M., Ed., Mary Ann Liebert, New York, 1988, 253. 50. Gettings, S. D., Lordo, R. A., Hintze, K. L., Bagley, D. M., Casterton, P. L., Chudkowski, M., Curren, R. D., Demetrulias, J. L., DiPasquale, L. C., Earl, L. K., Feder, P. I., Galli, C. L., Glaza, S. M., Gordon, V. C., Janus, J., Kuntz, P. J., Marenus, K. D., Moral, J., Pape, W. J. W., Renskers, K. J., Rheins, L. A., Roddy, M. T., Rozen, M. G., Tedeschi, J. P., and Zyracki, J., The CTFA evaluation of alternatives program: an evaluation of in vitro alternatives to the Draize primary eye irritation test (Phase III). Surfactant-based formulations, Food Chem. Toxicol., 34, 79–117, 1996. 51. Harbell, J. W. and Curren, R. D., The bovine corneal opacity and permeability assay: observations on assay performance, In Vitro Mol. Toxicol., 11, 337–341, 1998.


52. Harbell, J., Raabe, H., Dobson, T., Evans, M., and Curren, R., Histopathology associated with opacity and permeability changes in bovine corneas in vitro, ATLA, 27, 347, 1999. 53. Curren, R. D., Evans, M. G., Raabe, H. A., Ruppalt, R. R., and Harbell, J. W., An histological analysis of damage to bovine corneas in vitro by selected ocular toxicants, Toxicologist, 54(1), 188, 2000. 54. Wilmer, J. L., Burleson, F. G., Kayama, F., Kanno, J., and Luster, M. I., Cytokine induction in human epidermal keratinocytes exposed to contact irritants and its relation to chemical-induced inflammation in mouse skin, J. Invest. Dermatol., 102, 915–922, 1994. 55. Spielmann, H., Balls, M., Dupuis, J., Pape, W. J., Pechovitch, G., de Silva, O., Holzhütter, H. G., Clothier, R., Desolle, P., Gerberick, F., Liebsch, M., Potthast, J. M., Csato, M., Sladowski, D., Steiling, W., and Brantom, P., The international EU/COLIPA In Vitro Phototoxicity Validation Study; Results of Phase II (blind trial). Part 1: The 3T3 NRU Phototoxicity Test, Toxicol. In Vitro, 12, 305–327, 1998. 56. Holzhütter, H. G., A general measure of the in vitro phototoxicity derived from pairs of dose response curves and its use for predicting in vivo phototoxicity of chemicals, ATLA, 25, 445–462, 1997. 57. Spielmann, H., Balls, M., Dupuis, J., Pape, W. J., de Silva, O., Holzhütter, H. G., Gerberick, F., Liebsch, M., Lovell, W. W., and Pfannenbecker, U., A study on UV filter chemicals from Annex VII of European Union Directive 76/768/EEC, in the In Vitro 3T3 Phototoxicity Test, ATLA, 26, 679–708, 1998. 58. Zerler, B., Roemer, E., Raabe, H, Reeves, A., and Harbell, J. W., Evaluation of the phototoxic potential of chemically modified tetracyclines using the 3T3 Neutral Red Assay, ATLA, 27, 107, 1999. 59. Bronaugh, R. L. and Collier, S. W., In vitro methods for measuring skin permeation, in Skin Permeation Fundamentals and Applications, Zatz, J. L., Ed., Allured Publishing Corporation, Wheaton, IL, 1993, 93–111. 60. Heylings, J. R., Clowes, H. M., Trebilcock, K. L., and Hughes, L., Prediction of skin irritation potential using the skin integrity function test (SIFT), Kosmetica Speciale Dermatologia, May 2001. 61. van de Sandt, J., Roguet, R., Cohen, C., Esdaile, D., Ponec, M., Corsini, E., Barker, C., Fusenig, N., Liebsch, M., Benford, D., de Brugerolle de Fraissinette, A., and Fartasch, M., The use of human keratinocytes and human skin models for predicting skin irritation. The report and recommendations of ECVAM workshop 38, ATLA, 27, 723–743, 1999. 62. Koschier, F. J., Roth, R. N., Wallace, K. A., Curren, R. D., and Harbell, J. W., A comparison of three dimensional human skin models to evaluate the dermal irritation of selected petroleum products, In Vitro Toxicol., 10(4), 391–406, 1997. 63. Liebsch, M., Traue, D., Barrabas, C., Spielmann, H., Uphill, P., Wilkins, S., McPherson, J. P., Wiemann, C., Kaufmann, T., Remmele, M., and Holzhütter, H-G., The ECVAM prevalidation study on the use of EpiDerm for skin corrosivity testing, ATLA, 28, 371–401, 2000. 64. Roguet, R., Cohen, C., Robles, C., Courtellemont, P., Tolle, M., Guillot, J. P., and Pouradier Duteil, X., An interlaboratory study of the reproducibility and relevance of Episkin™, a reconstructed human epidermis, in the assessment of cosmetics irritancy, Toxicol. In Vitro, 12, 295–304, 1998. 65. Osborne, S., Mayer, F. K., Spake, A., Rosdy, M., De Wever, B., Ettlin, R. A., and Cordier, A., Predictivity of an in vitro model for acute and chronic skin irritation (SkinEthic) applied to the testing of topical vehicles, Cell Biol. Toxicol., 15, 121–135, 1999. 66. Medina, J., de Brugerolle de Fraissinette, A., Chibout, S. D., Kolopp, M., Kammermann, R., Burtin, P., Ebelin, M. E., and Cordier A., Use of human skin equivalent Apligraf for in vitro assessment of cumulative skin irritation potential of topical products, Toxicol. Appl. Pharmacol., 164, 38–45, 2000. 67. Fentem, J. H., Briggs, D., Chesné, C., Elliott, G. R., Harbell, J. W., Heylings, J. R., Portes, P., Roguet, R., Van De Sandt, J. J. M., and Botham, P. A., A prevalidation study on in vitro tests for acute skin irritation: results and evaluation by the management team, Toxicol. In Vitro, 15, 57–93, 2001. 68. Anonymous, Corrostitex: An In vitro Test Method for Assessing Dermal Corrosivity Potential of Chemicals, National Institute of Health Publication 99-4495, Interagency Coordinating Committee on the Validation of Alternative Methods, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 1999. 69. Fentem, J. H., Archer, G. E. B., Balls, M., Botham, P. A., Curren, R. D., Earl, L. K., Esdaile, D. J., Holzhütter, H. -G., and Liebsch, M., The ECVAM international validation study on in vitro tests for skin corrosivity. 2. Results and evaluation by the management team, Toxicol. In Vitro, 12, 483–524, 1998.


21

Ecotoxicology David J. Hoffman, Ph.D., Barnett A. Rattner, Ph.D., G. Allen Burton, Jr., Ph.D., and Daniel R. Lavoie, M.S.

CONTENTS Section 1. Introduction and Historical Overview Table 21.1 Historical Overview: First Observations of Ecotoxic Effects of Different Classes of Environmental Contaminants Figure 21.1 Ecological Risk Assessment Section 2. Aquatic Toxicity Testing Table 21.2 Summary of Published EPA, ASTM, and EC Methods for Conducting Aquatic Toxicity Tests Table 21.3 Aquatic Toxicity Tests Required by the EPA for the Development of Water Quality Criteria Table 21.4 Summary of the Aquatic Toxicity Test Requirements by Regulatory Guidelines Table 21.5 Summary of Published EPA, ASTM, and EC Methods for Conducting Sediment Toxicity Tests Table 21.6 Useful Internet Web Sites for Standardized Toxicity Test Methods Section 3. Wildlife Toxicity Testing Table 21.7 Acute Avian Toxicity Testing of Organophosphorus Pesticides of Widely Variable Mammalian Toxicity Table 21.8 Summary of Ecological Effects Test Guidelines for Terrestrial Wildlife and Other Terrestrial Organisms Figure 21.2 Protocols Used in Avian Toxicity Testing Table 21.9 Toxicity Testing of Herbicides by Application to Bird Eggs Table 21.10 “Neonatal” Toxicity Testing of Environmental Contaminants in Nestling Birds Section 4. Biomarkers Used in Aquatic and Terrestrial Monitoring Figure 21.3 The Use of Biomarkers to Assess Ecosytem Integrity Figure 21.4 Potential Effects of Agrichemicals and Other Environmental Contaminants on Survival and Reproduction of Wild Birds Table 21.11 Biomarkers for Environmental Monitoring Section 5. Contaminant Effects Table 21.12 Principal Contaminants for Which Critical Concentrations and Diagnostic Guidelines Have Been Established for Wild Terrestrial Vertebrates A. Heavy Metals Table 21.13 Some Criteria for Determining Lead Problem Areas for Waterfowl Table 21.14 Tissue Lead Concentration Thresholds of Lead Exposure and Poisoning in Waterfowl


B.

C.

D.

E.

F.

G.

Table 21.15 Interpretation of Tissue Lead Residues in Falconiformes, Columbiformes, and Galliformes Table 21.16 Total Mercury Concentrations in Tissues of Fish Exhibiting Symptoms of Methylmercury Toxicity Table 21.17 Lethality of Mercury to Birds Table 21.18 Kidney and Liver Cadmium Concentrations in Species of Small Mammals from Contaminated Habitats Selenium Table 21.19 Toxic Effects Thresholds for Selenium Concentrations in Water, Food Chain Organisms, and Fish Tissues Table 21.20 Laboratory Studies on Effects of Excess Selenium in Aquatic Birds Pesticides Table 21.21 Residues of DDE in Sample Eggs of Wild Birds Related to Eggshell Thinning and Reproductive Success Table 21.22 Physical Signs and Symptoms in Vertebrates Commonly Associated with Acute Exposure to Cholinesterase Inhibitors Table 21.23 Acute Response and Toxicity Ranking of Some Anticholinesterase Pesticides in Fish, Birds, and Laboratory Rats PCBs and Dioxins Table 21.24 Summary of PCB Concentrations in Aquatic Organisms for Adverse Effects Based on Laboratory Studies Table 21.25 Reproductive Effects of Aroclors and Other PCB Mixtures in Birds Table 21.26 Egg Injection Studies with Planar PCBs and Dioxin PAHs and Petroleum Figure 21.5 Sources of Petroleum and PAHs in the Environment Table 21.27 Effects of Petroleum and Individual PAHs on Living Organisms Radionuclides Figure 21.6 Ranges of Acute Lethal Doses of Radiation to Various Taxonomic Groups Table 21.28 Radiation Effects on Birds Summary of Studies of Persistent Environmental Contaminants in Terrestrial Vertebrates Table 21.29 Summary of Studies of Persistent Environmental Contaminants in Terrestrial Vertebrates

References Additional Related Information A. Glossary of Ecotoxicological Terms

SECTION 1. INTRODUCTION AND HISTORICAL OVERVIEW The term ecotoxicology was first designated by Truhaut in 1969 as a natural extension of toxicology to include the ecological effects of pollutants.1 Ecotoxicology employs ecological parameters to assess toxicity. In the broadest sense, ecotoxicology has been described as toxicity testing on one or more components of any ecosystem as described by Cairns.2 The definition of ecotoxicology


TABLE 21.1 Historical Overview: First Observations of Ecotoxic Effects of Different Classes of Environmental Contaminants Date

Contaminant(s)

1850s

Industrial revolution; soot from coal burning Industrial wastewater Spent lead shot Industrial wastewater Arsenic emissions from metal smelters Crude oil spill Lead and zinc mine runoff Hydrogen sulfide fumes in oil field DDT and organochlorines

1863 1874 1887 1907 1924 1927 1950s

1960s 1970s 1980s 1986

Anticholinesterase pesticides Mixtures of toxic wastes including dioxins at hazardous waste sites Agricultural drain water containing selenium and other contaminants Radioactive substances from Chernobyl nuclear power station

Effects Industrial melanism of moths Toxicity to aquatic organisms, first acute toxicity tests Ingestion resulted in death of waterfowl and pheasants Zones of pollution in rivers established by species tolerance Death of fallow deer and foxes Death of thousands of puffins Toxicity of metal ions to fish Large die-off of both wild birds and mammals Decline in American robins linked to DDT use for Dutch Elm disease; eggshell thinning in bald eagles, osprey, and brown pelicans linked to DDT; fish-eating mammals at risk Die-offs of wild birds, mammals, and other vertebrate species Human, aquatic, and wildlife health at risk Multiple malformations and impaired reproduction in aquatic birds in central California Worst nuclear incident in peacetime, affecting a wide variety of organisms and ecosystems

Derived from Hoffman et al.3

can be further expanded as the science of predicting effects of potentially toxic agents on natural ecosystems and nontarget species. Historically, as shown in Table 21.1, some of the earliest observations of anthropogenic ecotoxic effects such as industrial melanism of moths date back to the industrial revolution of the 1850s.3 In the field of aquatic toxicology, Forbes4 recognized the significance of the presence or absence of species and communities within an aquatic ecosystem, and reported approaches for classifying rivers into zones of pollution based on species tolerance. During the same era, some of the earliest acute aquatic toxicity tests were first performed by Penny and Adams (1863)5 and Weigelt, Saare, and Schwab (1885)6 who were concerned with toxic chemicals in industrial wastewater. It was recognized that the presence or absence of species (especially populations or communities) living in a given aquatic ecosystem provides a more sensitive and reliable indicator of the suitability of environmental conditions than do chemical and physical measurements alone. In the field of terrestrial toxicology, reports of anthropogenic contaminants affecting freeranging wildlife included cases of arsenic pollution and smoke stack emission toxicity. One early report described the death of fallow deer (Dama dama) due to arsenic emissions from a silver foundry in Germany in 1887. Another report described hydrogen sulfide fumes in the vicinity of a Texas oil field that resulted in a large die-off of many species of wild birds and mammals,7 thus affecting multiple species within an ecosystem. With the advent of modern pesticides, most notably the introduction of DDT in 1943, a marked decline in the population of American robins (Turdus migratorius) was linked to DDT spraying against Dutch Elm disease by the early 1950s. It soon became evident that ecosystems with bald eagles (Haliaeetus leucocephalus), osprey (Pandion haliaetus), brown pelicans (Pelecanus occidentalis), and populations of fish-eating mammals were at risk.8,9 More recently, mortality of multiple species of vertebrates has occurred as a consequence of exposure to anticholinesterase pesticides.10,11


Integrate Available Information PROBLEM FORMULATION Assessment Endpoints

Conceptual Model

Analysis Plan

Characterization of Exposure

Measures of Ecosystem and Receptor Characteristics

Measures of Exposure

A N A L Y S I S

Characterization of Ecological Effects

Exposure Analysis

Measures of Effect

Ecological Response Analysis

StressorResponse Profile

Exposure Profile

As Necessary: Acquire Data, Iterate Process, Monitor Results

Planning (Risk Assessor/ Risk Manager/ Interested Parties Dialogue)

Risk Estimation

RISK CHARACTERIZATION Risk Description

Communicating Results to the Risk Manager Risk Management and Communicating Results to Interested Parties

FIGURE 21.1 Ecological risk assessment examines the likelihood that adverse ecological effects are occurring as a result of exposure to one or more stressors. This process examines data, information, assumptions, and uncertainties in order to understand and predict the relationship between chemical, physical, or biological stressors and ecological effects. (Adapted from U.S. Environmental Protection Agency.12)


An important tool of modern ecotoxicology is ecological risk assessment (ERA), which examines the likelihood that adverse ecological effects are occurring as a result of exposure to one or more stressors12 as shown in Figure 21.1. This process examines data, information, assumptions, and uncertainties in order to understand and predict the relationship between chemical, physical, or biological stressors and ecological effects. The two major elements of this process include characterization of exposure and characterization of effects, and help focus problem formulation, analysis, and risk characterization. The overall effort expended in conducting an ERA should be consistent with the perceived magnitude of the problem. Results of an ERA, along with social, economic, political, and legal issues, can be used by professionals such as natural resource managers and risk managers as part of a cost–benefit analysis. In addition, results of an ERA can identify alternative chemicals or processes to mitigate risk. The body of this chapter is presented in four sections including aquatic toxicity testing, wildlife toxicity testing, bioindicators used in aquatic and terrestrial monitoring, and classes of major environmental contaminants and their effects.

SECTION 2. AQUATIC TOXICITY TESTING There have been a large number of aquatic toxicity test methods developed during the past two decades (Tables 21.2 through 21.4). During the past 10 years, methods for sediment toxicity testing have also increased dramatically (Table 21.5). Most of the standardized test methods have been developed by three North American entities: the U.S. Environmental Protection Agency (EPA), the American Society for Testing and Materials (ASTM), and Environment Canada (EC). In Europe, the Organisation of Economic Cooperation and Development (OECD), European Economic Community (EEC), and International Standards Organisation (ISO) have been the leading standards development organizations. In addition, some European countries, such as the Netherlands and Germany, have developed test methods that may be appropriate for use. Tables 21.2 through 21.5 list the available test methods that are currently available; however, the reader should verify whether new or revised methods have been published because the science of ecotoxicology is a rapidly developing field. Table 21.6 lists some useful Web sites to assist the reader in finding the most recent methods. It is of interest that many of the laboratory toxicity test methods are quite similar in their basic experimental design. Most are conducted for 2 to 14 days, in static or static-renewal systems, which assess survival, growth, or reproduction as measurement end points. The freshwater test methods utilize laboratory-cultured test organisms of known quality and health, whereas the marine test methods often must use field-collected organisms from presumably clean reference areas. Most of the test organisms are available from commercial suppliers. The primary differences among the test methods are that they are species specific for different geographic areas or regulatory programs, which may dictate the need for species-specific feeding, culturing, testing, and end-point measurement requirements. In addition, the required quality assurance/quality control requirements may vary between protocols and standards-setting organizations. In addition to laboratory toxicity test methods, there has been a significant increase in the use of in situ (field-based) toxicity testing. (See the recent review by Chappie and Burton.13) In situ toxicity testing here is defined as exposure of test organisms in confined chambers in the field, followed by measurement of typical toxicity or bioaccumulation test end points. Many of these methods, particularly for marine systems, have involved deployment of bivalves or fish for determining bioaccumulation and exposure by measuring tissue residues or biomarkers, respectively. The in situ approaches provide unique information on receiving water conditions, not possible in laboratory methods. In situ exposures are more realistic than laboratory tests, as water/sediment quality conditions and interacting factors such as suspended solids, temperature, and light will occur naturally. In addition, possible alterations in contaminant bioavailability are less likely to occur as a result of sampling, handling, and manipulation effects required for laboratory testing. Copyright © 2002 by Taylor & Francis

TABLE 21.2 Summary of Published EPA, ASTM, and EC Methods for Conducting Aquatic Toxicity Tests Agency EPA

ASTM

EC

Test Description

Reference ID

Methods for Acute Toxicity Tests with Fish, Macroinvertebrates, and Amphibians Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Waters to Marine and Estuarine Organisms Methods Guidance and Recommendations for Whole Effluent Toxicity (WET) Testing (40 CFR Part 136) Methods for Aquatic Toxicity Identification Evaluations: Phase I. Toxicity Characterization Procedures Methods for Aquatic Toxicity Identification Evaluations: Phase II. Toxicity Identification Procedures for Samples Exhibiting Acute and Chronic Toxicity Methods for Aquatic Toxicity Identification Evaluations: Phase III. Toxicity Confirmation Procedures for Samples Exhibiting Acute and Chronic Toxicity Toxicity Identification Evaluation: Characterization of Chronically Toxic Effluents, Phase I Practice for Algal Growth Potential Testing with Selenastrum capricornutum Practice for Conducting Static Acute Toxicity Tests on Wastewaters with Daphnia (discontinued 1990) Guide for Conducting Static Acute Toxicity Tests Starting with Embryos of Four Species of Saltwater Bivalve Mollusks Guide for Conducting Acute Toxicity Tests Starting with Fishes, Macroinvertebrates, and Amphibians Practice for Conducting Bioconcentration Tests with Fishes and Saltwater Bivalve Mollusks Guide for Assessing the Hazard of a Material to Aquatic Organisms and Their Uses Guide for Conducting Life Cycle Toxicity Tests with Saltwater Mysids Guide for Conducting Acute Toxicity Tests on Aqueous Effluents with Fishes, Macroinvertebrates, and Amphibians Guide for Conducting Renewal Life Cycle Toxicity Tests with Daphnia magna Practice for Using Brine Shrimp Nauplii as Food for Test Animals in Aquatic Toxicology Guide for Conducting Static 96-h Toxicity Tests with Microalgae Guide for Conducting Early Life-Stage Toxicity Tests with Fishes Practice for Using Octanol-Water Partition Coefficient to Estimate Median Lethal Concentration for Fishes Due to Narcosis Guide for Conducting Three-Brood, Renewal Toxicity Tests with Ceriodaphnia dubia Guide for Conducting Static Acute Aquatic Toxicity Screening Tests with Mosquito, Wyeomyia smithii (Coquillett) Practice for Standardized Aquatic Microcosm: Fresh Water Guide for Conducting Static Toxicity Tests with Lemna gibba Guide for Conducting the Frog Embryo Teratogenesis Assay — Xenopus (FETAX) Guide for Acute Toxicity Tests with the Rotifer Brachionus Guide for Conducting Static and Flow-through Acute Toxicity Tests with Mysids from the West Coast of the United States Acute Lethality Test Using Rainbow Trout Acute Lethality Test Using Threespine Stickleback Acute Lethality Test Using Daphnia spp. Test of Reproduction and Survival Using the Cladoceran Ceriodaphnia dubia

EPA-660/3-75-009 EPA/600/4-90/027F


EPA/600/4-91/002 EPA/600/R-95/136 EPA/821/B-00/004 EPA-600/6-91/003 EPA-600/R-92/060 EPA-600/R-92/061 EPA-600/6-91/005F ASTM D 3978-80 ASTM D 4229-84 ASTM E 724-89 ASTM E 729-88 ASTM E 1022-84 ASTM E 1023-84 ASTM E 1191-90 ASTM E 1192-88 ASTM ASTM ASTM ASTM ASTM

E 1193-87 E 1203-87 E 1218-90 E 1241-92 E 1242-88

ASTM E 1295-89 ASTM E 1365-90 ASTM ASTM ASTM ASTM ASTM EPS EPS EPS EPS

E 1366-91 E 1415-91 E 1439-91 E 1440-91 E 1463-92

1/RM/9 1/RM/10 1/RM/11 1/RM/21

TABLE 21.2 (Continued) Summary of Published EPA, ASTM, and EC Methods for Conducting Aquatic Toxicity Tests Agency

Test Description Test of Larval Growth and Survival Using Fathead Minnows Toxicity Test Using Luminescent Bacteria (Photobacterium phosphoreum) Growth Inhibition Test Using the Freshwater Alga (Selenastrum capricornutum) Fertilization Assay with Echinoids (Sea Urchin and Sand Dollars) Toxicity Testing Using Early Life Stages of Salmonid Fish (Rainbow Trout), second edition Test for Measuring the Inhibition of Growth Using the Freshwater Macrophyte Lemna minor Reference Method for Determining Acute Lethality of Effluents to Rainbow Trout

Reference ID EPS EPS EPS EPS EPS

1/RM/22 1/RM/24 1/RM/25 1/RM/27 1/RM/28

EPS 1/RM/37 EPS 1/RM/13

TABLE 21.3 Aquatic Toxicity Tests Required by the EPA for the Development of Water Quality Criteria Type of Testing Acute toxicity tests

Recommended Aquatic Tests Eight different families must be tested for both freshwater and marine species (16 acute tests): Freshwater: 1. A species in Family Salmonidae 2. A species in another family of Class Osteichthyes 3. A species in another family of Phylum Chordata 4. A plankton species in Class Crustacea 5. A benthic species in Class Crustacea 6. A species in Class Insecta 7. A species in a phylum other than Chordata or Arthropoda 8. A species in another order of Insecta or another phylum Marine: 1. Two families in Phylum Chordata 2. A family in a phylum other than Arthropoda or Chordata 3. Either Family Mysidae or Penaeidae 4. Three other families not in Phylum Chordata (may include Mysidae of Penaeidae, whichever was not used above) 5. Any other family

Chronic toxicity tests

Three chronic or partial life cycle studies are required: One invertebrate and one fish One freshwater and one marine species

Plant testing

At least one algal or vascular plant test must be performed with a freshwater and marine species

Bioconcentration testing

At least one bioconcentration study with an appropriate freshwater and saltwater species is required


TABLE 21.4 Summary of the Aquatic Toxicity Test Requirements by Regulatory Guidelines Regulatory Guidelines

Type of Testing Required

Clean Water Act (CWA) EPA NPDES Regulations

Aquatic Tests for the Protection of Surface Waters Effluent Biomonitoring Studies Toxicity Identification and Reduction Evaluations

Water Quality Standards Toxic Substances Control Act (TSCA) Premanufacture Notification, PMN Section Four Test Rule

Aquatic Test for the Development of Water Quality Criteria (WQC) Industrial and Specialty Chemicals: Aquatic Assessments Algae, daphnid and other fish species Data set requirements may include mulitple acutes with fish, algae, and invertebrates, freshwater and marine; followed by one to three chronic or partial life-cycle studies; a sediment study with midge and a bioconcentration study may be required if low Kow > 3.0

TSCA Aquatic Test Guidelines Number: 795.120 797.1050 797.1160 797.1300 797.1310 797.1330 797.1400 797.1520 797.1600 797.1800 797.1830 797.1930 797.1950 797.1970 Adams et al. (1985)57 Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) Subdivision E Wildlife and Aquatic Organisms (Aquatic Test Guideline Number): 72-1 72-2 72-3 72-4a 72-4b 72-5 72-6 72-7 Food and Drug Administration (FDA) Environmental Effects test number: 4.01 4.08 4.09 4.10 4.11 4.12


Hyalella azteca flow-through acute Algal toxicity test (Selenastrum capricornutum) Duckweed acute (Lemna sp.) Daphnia magna acute test Gammaraid acute test (Gammarus sp.) Daphnia magna chronic test Fish acute test (freshwater and marine) Fish bioconcentration test (bluegill, fathead minnow, rainbow trout) Fish early life stage test (fathead minnow, rainbow trout, sheephead minnow) Oyster shell deposition test Oyster bioconcentration study Mysid shrimp acute test Mysid shrimp chronic test Penaeid shrimp acute test Midge partial life cycle test with sediments

Acute test for freshwater fish Acute test for freshwater invertebrates Acute test for estuarine and marine organisms Fish early life stage study Aquatic invertebrate life cycle studies Life cycle test if fish Aquatic organism accumulation tests Simulated or actual field tests for aquatic organisms (mesocosms) New Drug Environmental Assessments Algal test Daphnia magna acute toxicity Daphnia magna chronic toxicity Hyalella azteca acute toxicity Freshwater fish acute toxicity Earthworm subacute toxicity

TABLE 21.4 (Continued) Summary of the Aquatic Toxicity Test Requirements by Regulatory Guidelines Regulatory Guidelines Organisation of Economic Cooperation and Development (OECD) and European Economic Community (EEC) Aquatic Effects Testing; 201 202 C2 203 204 210 211 212 215 305

Type of Testing Required European Community Aquatic Testing Requirements

Algal growth inhibition test Daphnia magna acute immobilization test and reproduction test Acute toxicity in Daphnia magna Fish, acute toxicity test Fish, prolonged toxicity test: 14-day study Fish, early life-stage toxicity test Daphia magna reproduction test Fish, short-term toxicity test on embryo and sac-fry stages Fish, juvenile growth test Bioconcentration: flow-through fish test

TABLE 21.5 Summary of Published EPA, ASTM, and EC Methods for Conducting Sediment Toxicity Tests Agency EPA ASTM

EC

Test Description

Reference ID

Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates Guide for Conducting 10-day Static Sediment Toxicity Tests with Marine and Estuarine Amphipods Guide for Conducting Sediment Toxicity Tests with Freshwater Invertebrates Guide for Collection, Storage, Characterization, and Manipulation of Sediments for Toxicological Testing Guide for Conducting Biological Tests with Sediment Standard Test Methods for Measuring the Toxicity of Sediment-Associated Contaminants with Freshwater Invertebrates Standard Guide for Conducting Sediment Toxicity Tests with Marine and Estaurine Ploychaetous Annelids Standard Guide for Determination of Bioaccumulation of Sediment-Associated Contaminants by Benthic Invertebrates Acute Test for Sediment Toxicity Using Marine and Estuarine Amphipods Test for Survival and Growth in Sediment Using Freshwater Midge Larvae Chironomus tentans or riparius Test for Survival and Growth in Sediment Using Freshwater Amphipod Hyalella azteca Test for Survival and Growth for Sediment Using a Marine Ploychaete Worm Reference Method for Determining Acute Lethality of Sediments to Estuarine or Marine Amphipods Reference Method for Determining Sediment Toxicity Using Luminescent Bacteria

EPA/600/R-99/064


ASTM E 1367-92 ASTM E 1383-93a ASTM E 1391-90 ASTM E 1525-93 ASTM E 1706-95b ASTM E 1611 ASTM E 1688-97a EPS 1/RM/26 EPS 1/RM/32 EPS 1/RM/33 EPS 1/RM/** EPS 1/RM/35 EPS 1/RM/**

TABLE 21.6 Useful Internet Web Sites for Standardized Toxicity Test Methods Organisation of Economic Cooperation and Development (OECD) Main Web site: www.oecd.org/ Guidelines for the Testing of Chemicals: www.oecd.ehs/test/testlist.htm U.S. Environmental Protection Agency (U.S. EPA) Main Web site: www.epa.gov/ Index to EPA Test Methods: www.epa.gov/epahome/index/ American Society for Testing and Materials (ASTM) Main Web site: www.astm.org/ Search for test methods: www.astm.org/cgi-bin/SoftCart.exe/STORE/store.htm?E+mystore U.S. Geologic Survey (USGC) Columbia Environmental Research Center (CERC): related Toxicity Test Method information Main Web site: www.cerc.usgs.gov/ Sediment methods: www.cerc.usgs.gov/pubs/sedtox/index.htm International Organisation for Standardization (ISO) Main Web site: www.iso.ch/ International Standards: www.iso.ch/cate/cat.html

SECTION 3. WILDLIFE TOXICITY TESTING Avian toxicity testing protocols were first utilized by the U.S. Fish and Wildlife Service as a consequence of wildlife losses in the 1950s because of the increased use of DDT and other pesticides. The first testing protocols focused on single-dose acute oral toxicities with lethality as the major end point; Table 21.7 shows acute avian sensitivities to certain organophosphorus pesticides of widely variable mammalian toxicity.14 Further protocol development resulted in subacute 5-day dietary tests, which, along with the single acute oral dose tests, are currently required by the EPA for regulatory purposes under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) in support of pesticide registration and also under the Toxic Substances Control Act (TSCA) (Table 21.8). Figure 21.2 summarizes the protocols used in avian toxicity testing, including acute, subacute, subchronic, chronic, developmental, field, and behavioral tests of avian wildlife toxicity.15 The avian subchronic dietary toxicity test was developed as an extension of the subacute test as a precursor to full-scale reproductive studies, but is not routinely required for regulatory purposes. Subchronic testing has been applied to compare the sublethal effects of different chemical forms including hepatotoxicity, and to study delayed neurotoxicity of certain organophosphorus insecticides. Avian chronic toxicity tests are designed with reproduction as the primary end point and are required for both waterfowl and upland gamebirds during chemical registration (see Avian reproduction test in Table 21.8). Persistent chemicals such as chlorinated hydrocarbons require relatively long-term exposures (at least 10 weeks) in advance of breeding, whereas shorter-term exposures may be utilized for less-persistent chemicals such as organophosphorus insecticides. Avian terrestrial field studies are basically of two types: (1) screening studies to ascertain whether impacts are occurring, and (2) definitive studies to estimate the magnitude. Single-dose avian embryotoxicity and teratogenicity tests were developed, in part, to assess the potential contaminant hazard of external exposure of birds’ eggs. Results of these tests with multiple species and chemicals have revealed differential toxicities of a spectrum of chemicals and sensitivities among species.16 Table 21.9 summarizes the effects of herbicides applied by this exposure route. Developmental toxicity testing has also focused on the vulnerability of “neonatal” nestling altricial birds, as shown in kestrels and starlings, following oral ingestion of a variety of environmental contaminants (Table 21.10).15 Copyright © 2002 by Taylor & Francis

Additionally, behavioral testing in field and pen studies has documented aberrations in wildlife behavior such as changes in nest attentiveness, brood behavior, and increased vulnerability to predation. Response time to maternal call, avoidance of fright stimulus, tests of operant learning ability, as well as time–activity budgets, have been successfully applied to laboratory studies. Laboratory studies with environmental contaminants and mammalian wildlife have been limited compared with avian studies. Mammalian toxicity data of EPA FIFRA (Tier 1) has consisted largely of laboratory rat data for pesticide registration, whereas Tier 2 testing requires a dietary LC50 or acute oral LD50 study with a nonendangered representative species likely to be exposed, quite often a microtine rodent (see Table 21.8). Regulatory test guidelines for other terrestrial organisms including insects, plants, microbes, and earthworms are summarized in Table 21.8.

TABLE 21.7 Acute Avian Toxicity Testing of Organophosphorus Pesticides of Widely Variable Mammalian Toxicity Single-Dose Oral LD50 Pesticide Phorate Azinphos-methyl Ethion Dimethoate Fenitrothion Temephos

Rata

Pheasantb

Blackbirdc

2 13 65 215 740 8600

7 75 1297 20 26 35

1 8 45 7 25 42

a

Sherman strain male laboratory rats, 3 months old, n = 50 to 60 per test. Farm-reared male and female ring-necked pheasants, 3 to 4 months old, n = 8 to 29 per test. c Wild-captured pen-conditioned male and female red-winged blackbirds, adult, n = 8 to 28 per test. b

Source: Adapted from Hill.14

TABLE 21.8 Summary of Ecological Effects Test Guidelines for Terrestrial Wildlife and Other Terrestrial Organisms OPPTSa No.

Name of Test

OTSb

OPPc

OECDd

EPA Pub. No.

71-1 71-2 71-4

None 205 206

712-C-96-139 712-C-96-140 712-C-96-141

850.2400 850.2450 850.2500

Terrestrial Wildlife Tests 797.2175 797.2050 797.2130, .2150 Wild mammal acute toxicity None Terrestrial (soil-core) microcosm test 797.3775 Field testing for terrestrial wildlife None

71-3 None 71-5

None None None

712-C-96-142 712-C-96-143 712-C-96-144

850.3020 850.3030 850.3040

Beneficial Insects and Invertebrates Tests Honeybee acute contact toxicity None 141-1 Honeybee toxicity of residues on foliage None 141-2 Field testing for pollinators None 141-5

None None None

712-C-96-147 712-C-96-148 712-C-96-150

850.2100 850.2200 850.2300

Avian acute oral toxicity test Avian dietary toxicity test Avian reproduction test


TABLE 21.8 (Continued) Summary of Ecological Effects Test Guidelines for Terrestrial Wildlife and Other Terrestrial Organisms 850.4000 850.4025 850.4100 850.4150 850.4200 850.4225 850.4230 850.4250 850.4300 850.4600 850.4800

Nontarget Plants Tests (except aquatic) Background — nontarget plant testing None Target area phytotoxicity None Terrestrial plant toxicity, Tier I (seedling emergence) None Terrestrial plant toxicity, Tier I (vegetative vigor) None Seed germination/root elongation toxicity test 797.2750 Seedling emergence, Tier II 797.2750 Early seedling growth toxicity test 797.2800 Vegetative vigor, Tier II 797.2750 Terrestrial plants field study, Tier III None Rhizobium-legume toxicity 797.2900 Plant uptake and translocation test 797.2850

120-1 121-1 122-1 122-1 122-1 123-1 123-1 123-1 124-1 None None

None None None None None None None None None None None

712-C-96-151 712-C-96-152 712-C-96-153 712-C-96-163 712-C-96-154 712-C-96-363 712-C-96-347 712-C-96-364 712-C-96-155 712-C-96-158 712-C-96-159

850.5100

Toxicity to Microorganisms Tests Soil microbial community toxicity test 797.3700

None

None

712-C-96-161

None None

207 209

712-C-96-167 712-C-96-168

None

None

712-C-96-348

850.6200 850.6800

850.7100 a b c d

Chemical Specific Tests Earthworm subchronic toxicity test 795.150 Modified activated sludge, respiration inhibition test 795.170 for sparingly soluble chemicals Field Test Data Reporting Data reporting for environmental chemistry methods None

Office of Pollution Prevention and Toxics. Office of Toxic Substances (for TSCA). Office of Pesticide Programs (for FIFRA). Organisation for Economic Cooperation and Development.


ACUTE TESTS

1. Adult bobwhite, mallard 2. Mallard embryo

LD50 LD50, embryotoxicity teratogenicity

SUBACUTE TEST Five-day feeding, juvenile bobwhite, mallard

LC50

SUBCHRONIC TESTS

Behavioral tests

1. Subchronic feeding (extended subacute)

LC50, EC50

"Neonatal" tests

2. Delayed neurotoxicity

CHRONIC TESTS 1. Persistent chemical pen reproductive test 2. Nonpersistent chemical pen reproductive test

TERRESTRIAL FIELD STUDIES

1. Screening field studies 2. Definitive field studies FIGURE 21.2 Protocols used in avian toxicity testing, which include acute, subacute, subchronic, chronic, developmental, field, and behavioral tests. The avian subchronic dietary toxicity test was developed as an extension of the subacute test as a precursor to full-scale reproductive studies, but is not routinely required for regulatory purposes. Avian chronic toxicity tests are designed with reproduction as the primary end point and are required for both waterfowl and upland gamebirds during chemical registration. (Adapted from Hoffman, D.J., Wildlife toxicity testing, in Handbook of Ecotoxicology, Hoffman.15)


TABLE 21.9 Toxicity Testing of Herbicides by Application to Bird Eggsa Herbicide Formulation

Species

Effects

2,4-D (Esteron® 99) 2,4-D (butyl ester)

Mallard Mallard Mallard Chicken, ring-necked pheasant Chicken, Japanese quail, gray partridge Japanese quail Ring-necked pheasant, Japanese quail Chicken Chicken

2,4-D (dimethylamine salt) Dalapon (Dowpon® M) Dicamba (Banvel®) Fosamine ammonium (Krenite®)

Mallard Mallard Mallard Northern bobwhite, mallards

Glyphosate (Roundup®) Methyldiclofop (Hoelon® 3EC) Paraquat Paraquat (Gramoxone®) Paraquat (Ortho® paraquat CL) Pichloram (Tordon® 10K) Prometron (Pramitol® 25E) Propanil (Stampede® 3E) 2,4,5-T (unspecified formulation)

Mallard Mallard Chicken, Japanese quail Japanese quail Mallard Mallard Mallard Mallard Chicken

2,4,5-T (Tormona® 80)

Japanese quail, ring-necked pheasant Chicken Mallard Mallard Northern bobwhite

LC50 = 211 g/l; stunted growth LC50 > 479 g/l; not teratogenic LC50 = 9 g/l; eye defects; edema; stunted growth No apparent effects at 6 g/l Abnormal gonads; stunted growth; fused vertebrae at 3.75 ml/l No apparent effects at 4 l/ha (conc. was 500 g/l) Decreased hatching at 30× normal application, not teratogenic No apparent effects at 15 g/l LD50 = 5 mg/egg; motor dysfunction; altered brain biochemistry LC50 = 230 g/l; not teratogenic LC50 > 449 g/l; not teratogenic LC50 > 240 g/l; eye defects; stunted growth More reduced hatching in mallards than bobwhite at 65 g/l; not highly teratogenic LC50 = 213 g/l; not teratogenic LC50 = 12 g/l; eye defects 4 g/l caused mortality and defects of the lung 0.5 g/l decreased hatching success LC50 = 1.8 g/l; brain defects; edema; stunted growth LC50 = 120 g/l; stunted growth LC50 = 46 g/l; edema; stunted growth LC50 = 8 g/l; limb and neck defects 4 g/l caused embryonic mortality; growth inhibition; abnormal gonads Decreased hatching at 30× normal application; not teratogenic No effects apparent at 15 g/l LC50 = 127 g/l; not teratogenic LC50 = 2 g/l; bill defects, stunted growth Hatching success not affected but hatching weight lower at 38 g/l; decreased ratios of muscle and liver DNA to protein

®

Amitrole (Amitrol T ) Atrazine (Aatrex® 4L) Bromoyxnil + MCPA (Bronate®) 2,4-D (isoctyl ester) 2,4-D (unspecified formulation) 2,4-D (Hedonal®) 2,4-D (U-46-D-Fluid)

2,4,5-T (Esteron® 245) 2,4,5-T (isoctyl ester) Trifluralin (Treflan®) Trifluralin (Treflan®)

a

Herbicides were applied by immersion or spraying of eggs in these studies.

Source: Adapted from Hoffman.16

TABLE 21.10 “Neonatal” Toxicity Testing of Environmental Contaminants in Nestling Birds Species American kestrel

Exposure Method

Chemical

Observation Period

Daily oral

Lead, metallic

Days 1–10

Daily oral

Paraquat

Days 1–10


Effects 525 mg/kg, high mortality; 125 mg/kg, reduced growth; 25 mg/kg, altered physiology 60 mg/kg, high mortality; 10–25 mg/kg, reduced growth, altered physiology

TABLE 21.10 (Continued) “Neonatal” Toxicity Testing of Environmental Contaminants in Nestling Birds

European starling

Herring gull Black guillemot

Daily oral

Bifenox

Days 1–10

Daily oral

Nitrofen

Days 1–10

Daily oral Single oral (day 5 or 15)

Oxyfluorfen Dicrotophos

Days 1–10 24 h postdose

Single oral

Diazinon

24 h postdose

Single oral (3–4 weeks old) Single oral

Crude oil

For 9 days

Crude oil (weathered)

For 22 days

500 mg/kg, high mortality, 250 mg/kg, reduced growth, altered physiology 500 mg/kg, complete mortality; 250 mg/kg, reduced growth; 50 mg/kg, altered physiology 500 mg/kg, few effects Day 5 LD50 = 4.9 mg/kg; day 15 LD50 = 9.0 mg/kg; reduced growth, brain cholinesterase Day 1 LD50 =13 mg/kg; fledgling LD50 = 145 mg/kg 0.3 mg/kg reduced growth, altered physiology 0.1–0.2 ml reduced growth, altered physiology

Source: Adapted from Hoffman.15

SECTION 4. BIOMARKERS USED IN AQUATIC AND TERRESTRIAL MONITORING Measures of exposure and effect may be quantified at a variety of levels of biological organization (molecular, cellular, organ system, organismal, population, and even biotic community). There have been efforts to classify toxicological measures with terminology linking them to levels of biological organization. The term biomarker has been used most broadly to encompass “any biological response to an environmental chemical at or below the level of the individual demonstrating a departure from the normal status.” This definition encompasses biochemical, physiological, histological, morphological, and behavioral measurements. This is the most generally accepted definition, and will be used in this chapter. Ideally, all such response end points are contaminant dose– and exposure time–dependent phenomena. Population level effects are referred to as “bioindicator” responses, and changes at the community and ecosystem level are categorized as “ecological indicators.” Biomarkers may be used to assess chemical exposure and the cumulative, adverse effects of toxicants on biota in situ.17 Biomarkers in ecological risk assessment have been used to assess ecosystem integrity (Figure 21.3). Biomarkers also play an important role in the evaluation of the effectiveness of remedial action to alleviate pollution. When selecting appropriate biomarkers for such studies, there are many important factors that must be accounted for since they can affect the outcome, including species sensitivity, physiological condition, behavioral traits, and the time, rate, and frequency of chemical exposure to the ecosytem. As well as the ability to measure direct toxicological effects of chemical exposure, equally important are indirect effects (Figure 21.4). For example, for many avian species the reproductive season corresponds with peaks in invertebrate populations, which also occurs at the same time insects with insecticides are treated or unwanted vegetation may be sprayed with herbicides. All of these factors may impair the chance of successful reproduction and growth of young by decreasing the food supply, as well as decreasing important ground cover in nesting areas, thus increasing the likelihood of exposure to predators.11


Of the hundreds of potential biomarker end points that have been used in laboratory studies, few have gained widespread application in ecotoxicological “field” investigations. Table 21.11 summarizes ones used successfully in field studies. At the molecular level of organization, these include inhibition of esterases, porphyria, adduct formation (DNA and hemoglobin), cytochrome P-450 induction, inhibition of delta-aminolevulinic acid dehydratase, oxidative stress (shifts in ratios of oxidized:reduced glutathione concentration and associated enzymes), alterations in concentrations of hormones, metallothionein,, and increased activities of plasma/serum enzymes released as a consequence of cellular damage.18 At the cellular and tissue level, substantial literature exists describing contaminant-linked histopathology, cytogenetic and cytotoxic end points, as well as an emerging literature on immunological end points. A few remarkable organ-system and wholeanimal end points (e.g., gross anomalies such as bird deformity, teratogenesis, eggshell abnormalities, and behavior) have been linked to contaminant exposure. Many of these end points are sensitive, precise and specific “indicators of exposure” to various contaminants, although even fewer end points have well-documented association with population-level effects in wildlife. Select reviews on the use of biomarkers in ecotoxicology include those by Hugget et al.,19 Fossi and Leonzio,20 and Melancon.21

ECOSYSTEM INTEGRITY

THE USE OF BIOMARKERS TO ASSESS ECOSYSTEM INTEGRITY

Increasingly abnormal structure and function

Resistance (replacement of one specles by another, etc.)

Normal structure and function

Normal baseline biomarkers in all populations

Exposure biomarkers in one or more populations

Exposure and effects biomarkers showing damage to sensitive populations

Exposure and effects biomarkers signal damage in at least some individuals of all populations investigated

FIGURE 21.3 Biomarkers in ecological risk assessment have been used to help assess ecosystem integrity through association with such changes as replacement of sensitive species by more tolerant species, followed by eventual disruption of structure and function of the ecosystem. (From Depledge, M.H. and Fossi, M.C., Ecotoxicology 3, 161–172, 1994. With permission.)


Contaminant Deposition

Invertebrate Vertebrate Prey

Clutch Size

Embryo Mortality

Hatching Success

Nestling Mortality

Fledging Success

Recruitment

Vegetation

Adult Mortality

Behavior

Food Availability

Parental Care

Clutch Size

Breeding Density

Post-fledging Survival

Population Size

FIGURE 21.4 Potential effects of agrichemicals and other environmental contaminants on survival and reproduction of wild birds. Indirect effects, such as changes in invertebrate prey populations and vegetation, are as equally important as ones that measure direct toxicological effects of chemical exposure. All of these factors may impair the chance of successful reproduction and growth of young birds by decreasing the food supply and important ground cover vegetation in nesting areas. (Adapted from Grue et al.11)


TABLE 21.11 Biomarkers for Environmental Monitoring Biomarker

Biological Response

Esterases

Enzymer inhibition

Porphyrins Cytochrome P-450 Blood chemistry

Metabolic disorder Enzyme induction Various enzymes

Retinols Thyroid function delta-Aminolevulinic acid dehydratase Immunotoxicology

Retinol changes Thyroid function alteration Inhibition

Hemoglobin adducts Stress proteins DNA strand breakage Adducts Sister chromatid exchange

Adducts Protein induction Strand breaks Adducts Chromosome

a b c

Various

Pollutanta Organophosphates and carbamates Toxic metals, PHAHs PAHs, PHAHs Toxic metals, PHAHs, organophosphates PHAHs PHAHs Toxic metals Toxic metals, PAHs, PHAHs, organophosphates PAHs, PHAHs Toxic metals, PHAHs PAHs, PHAHs PAHs, PHAHs PAHs, PHAHs

Invasive Techniques

Nondestructive Techniques

Temporal Occurrenceb

Reliability Indexc

Brain

Blood

Early

S, D, P

Liver Liver

Blood, excreta, feathers Skin, mucosa, blood Blood

Middle Early Middle

S, D, P S, D S, D

Liver Thyroid

Blood Blood Blood

Early Middle Early

S S S, D, P

Lymphatic cells

Blood

Middle late

S

Several tissues Several tissues Several tissues

Blood Blood Blood, skin Blood, skin Blood

Early Early Early Early Middle Late

S, D S S S,D,P S,D,P

Pollutants: PAHs = polynuclear aromatic hydrocarbons; PHAHs = polyhalogenated aromatic hydrocarbons. Temporal occurrences: early — hours to days; middle — days to weeks/months; late — weeks/months to years. Reliability Index: S = signal of potential problem; D = definitive indicator of type or class of pollutant; P = predictive indicator of a long-term adverse effect.

Source: Adapted from Fossi et al.18


SECTION 5. CONTAMINANT EFFECTS Table 21.12 lists the principal contaminants for which critical concentrations and diagnostic guidelines have been established for wild terrestrial vertebrates.22

TABLE 21.12 Principal Contaminants for Which Critical Concentrations and Diagnostic Guidelines Have Been Established for Wild Terrestrial Vertebrates Organochlorine Pesticides Chlordecone p,p′ -DDE Dicofol Dieldrin Endrin Heptachlor epoxide Hexachlorobenzene Hexachlorocyclohexane Methoxychlor Mirex Oxychlordane Toxaphene Organophosphorus and Carbamate Pesticides

Polychlorinated biphenyls Total aroclors Coplanar congeners Dioxins Dibenzofurans Petroleum hydrocarbons Crude oils Oil fractions Metals, Metalloids, and Trace Elements Cadmium Fluoride Lead Mercury Selenium

Source: Adapted from Rattner et al.22

A. HEAVY METALS 1. Lead Lead exposure in birds results from direct consumption of spent lead shot, consumption of shot or bullet fragments in food items, ingestion of lead fishing sinkers, and lead-based paints, and environmental contamination of urban and industrial areas. Lead-poisoning mortality has occurred in waterfowl and at least 30 species of birds other than waterfowl. Despite restrictions on the use of lead shot for hunting waterfowl and coots in the United States, lead shot and other lead-containing ammunition continue to be used for hunting many wild species.23 Ingestion of merely one number 4 lead shot (about 230 mg lead) can be lethal to waterfowl under certain conditions. Lead exposure criteria include presence of lead shot in gizzards, lead residues in body tissues, and blood enzyme measurements as illustrated in Table 21.13. Tissue lead concentrations corresponding to background contamination, subclinical poisoning, clinical poisoning, and severe clinical poisoning24 are presented in Table 21.14. Considerable toxicity data exist for Falconiformes, Columbiformes, and Galliformes, allowing categorization of lead residues according to the increasing severity of effects: (1) subclinical, the range of residues associated with physiological effects only; (2) toxic, the approximate threshold concentration consistent with the development of clinical signs of lead toxicosis; and (3) compatible with death, the approximate threshold concentration associated with mortality in field or laboratory cases of lead poisoning (Table 21.15). 25


TABLE 21.13 Some Criteria for Determining Lead Problem Areas for Waterfowl Endpoint/Measurement

Samples Required

Examination Process

Mortality

Whole carcasses

Postmortem examination and laboratory assays, including microbiology and toxicology

Lead concentrations in body tissues

Liver, kidney, and blood

Lead shot in gizzard samples

Intact gizzards

Liver and kidney are removed from dead birds; chemical analysis is usually by atomic absorption spectrophotometry Examination of gizzard contents for presence or absence of lead shot

Measurement of deltaaminolevulinic acid dehydratase (delta-ALAD) in soft tissues Measurement of protoporphyrin IX concentration in blood

Blood, brain, or liver

Red blood cells examined most often; analysis is usually by spectrophotometry

Drop of blood

Fluorometric measurement of red blood cells

Substrate samples

Soil and bottom samples taken at predetermined depths Calculation of relative amounts of lead shot deposited

Amount of lead shot in environment Hunting pressure

Information on hunteruse days or birds shot per unit area

Evaluation of Data Diagnosis of lead poisoning as a cause of mortality depends on a combination of pathological findings supported by appropriate toxicological results; liver is most often used for chemical analysis; lead values of 8 μg/g (wet weight) and above are consistent with lead intoxication when supported by pathology Values indicate the amount of lead present; concentrations of 2 μg/g or greater for liver and 0.2 μg/g or greater for blood should be considered elevated; these data must be supported by pathological findings before confidence can be placed in a lead poisoning diagnosis Presence of lead shot reflects exposure just prior to sampling and constitutes an index for lead poisoning risk; shot ingestion may be independent of cause of death, particularly if dissolution and absorption have not occurred; one or more ingested shot in 5% or more of gizzards examined is the threshold criteria for potential problem areas Inhibition of delta-ALAD is a sensitive indicator of lead exposure; suppression below normal values can occur within 24 h of lead absorption; recovery of delta-ALAD requires a month or more; the degree of suppression is directly correlated with blood lead levels Concentration of protoporphyrin IX results from lead inhibition of heme synthetase, an enzyme responsible for incorporation of iron into protoporphyrin IX; values above 40 μg/dl indicate exposure to lead; recovery to normal levels occurs within a month following exposure; high values are correlated with body function impairment and reflect toxicity The amount of shot present represents the potential for lead shot ingestion, but variables influence its probability of occurrence Provides an index of potential lead shot availability to waterfowl; many variables impact the probability of lead shot ingestion and subsequent intoxication

Source: Adapted from Interpretation of Criteria Commonly Used to Determine Lead Poisoning Problem Areas, U.S. Department of the Interior, Fish and Wildlife Service, Fish and Wildlife Leaflet 2. Washington, D.C., 1985. 23


TABLE 21.14 Tissue Lead Concentration Thresholds of Lead Exposure and Poisoning in Waterfowl Tissue Lead Blood (μg/dl)

Liver (ppm of wet weight)

Bone (ppm of dry weight)

Threshold Concentration >20 (elevated) >40 (indicative of poisoning) >50 (acute toxicity) >2 (elevated) >6 (indicative of poisoning) >6–20 (acute exposure and absorption) >7 (poisoning) >8 (consistent with signs of poisoning) >12.5 (poisoning) >20 (excessive exposure and absorption)

Source: Adapted from Pain.24

TABLE 21.15 Interpretation of Tissue Lead Residues (ppm wet weight) in Falconiformes, Columbiformes, and Galliformesa Order

Blood

Liver

Kidney

Falconiformes Subclinical Toxic Compatible with death

0.2–1.5 >1 >5

2–4 >3 >5

2–5 >3 >5

Columbiformes Subclinical Toxic Compatible with Death

0.2–2.5 >2 >10

2–6 >6 >20

2–20 >15 >40

Galliformes Subclinical Toxic Compatible with Death

0.2–3 >5 >10

2–6 >6 >15

2–20 >15 >50

a Subclinical = physiological effects, e.g., ALAD depression, only, with no overt clinical signs; toxic = clinical signs, such as muscle wasting, green diarrhea, weakness, anemia, muscular incoordination; Compatible with death = residues associated with mortality in field reports of lead poisoning or in experimental dosing studies.

Source: Adapted from Franson.25


2. Methylmercury Recent reports of high mercury concentrations in fish, particularly in newly flooded reservoirs and in low-alkalinity lakes have renewed concerns about mercury in the environment.26 Neurotoxicity seems to be the most probable chronic response of wild adult fish to dietary methylmercury, even though other effects have been observed in laboratory studies. In the brain, concentrations of 7 μg/g wet weight or greater probably cause severe, potentially lethal effects. In mercury-sensitive species, such as the walleye, brain tissue concentrations of 3 μg/g wet weight or greater probably indicate significant toxic effects (Table 21.16). For axial muscle tissue, field studies indicate that residues of 6 to 20 μg/g wet weight are associated with toxicity. Sublethal and lethal effects on fish embryos are associated with mercury residues in eggs that are much lower than (perhaps 1 to 10% of) the residues associated with toxicity in adult fish. In birds and mammals, methylmercury is primarily neurotoxic, damaging the central nervous system. For some species of birds, such as mallards, adverse effects on reproduction have been associated with 3 mg of Hg/kg of diet (Table 21.17).27 Additional interpretation on mercury poisoning in wildlife is provided by Heinz.28

TABLE 21.16 Total Mercury Concentrations in Tissues of Fish Exhibiting Symptoms of Methylmercury Toxicity Hg Concentration (μg/g wet weight) Type of Study and Species of Fish

Location or Mode and Duration of Exposure

Brain

Liver

Muscle

Whole Body

Walleye

Diet (42–63 d)

Laboratory Studies 3–6 6–14 5–8

Rainbow trout

Diet (240–314 d) Diet (105 d) Diet (84 d)

15–40 — —

18–50 — —

15–45 12–23 —

— — 30–35

Diet (84 d)

—

—

—

10–30

Diet (270 d)

16–30

26–68

20–28

19

Water (4 μg/l)b (30–98 d)

7–32

32–114

9–52

—

Water (9 μg/l)b (12–33 d)

—

—

—

4–27

Water (2.9 μg/l)b (273 d)

42

58

24

24

Water (0.93 μg/l)b (273 d)

17

24

10

5–7

Brook trout


—

Toxic Effect(s)a

Onset of mortality Fish emaciated (–) locomotor activity (–) coordination (–) appetite (+) mortality (–) growth Darkened skin Lethargic behavior (–) appetite (–) growth (–) appetite (–) activity (–) visual acuity (–) growth Darkened skin Loss of equilibrium Death, preceded by (–) appetite and (–) activity Death, preceded by (–) appetite and (–) activity Mortality, preceded by loss of appetite, muscle spasms, and deformities (+) mortality (–) growth Sluggish behavior

TABLE 21.16 (Continued) Total Mercury Concentrations in Tissues of Fish Exhibiting Symptoms of Methylmercury Toxicity Hg Concentration (μg/g wet weight) Type of Study and Species of Fish

Nibea schlegeli Latcolabrax japonicus Sparus macrocephalus Scomberomorus niphonicus Striped mullet Mugil cephalus Cardinalfish Apogon sp. Northern pike

a b

Location or Mode and Duration of Exposure

Brain

Liver

Muscle

Whole Body

Water (0.27 μg/l)b (273 d)

5

8

5

3

Deformities None observed

Toxic Effect(s)a

Minamata Bay Minamata Bay

Field Studies — — — —

8–15 17

— —

Fish enfeebled Fish enfeebled

Minamata Bay

—

—

24

—

Fish enfeebled

Minamata Bay

—

15

9

—

Fish enfeebled

Minamata Bay

—

—

11

—

Fish enfeebled

Minamata Bay

—

—

19

—

Fish enfeebled

Clay Lake, Ontario

—

—

6–16

—

Fish emaciated, with biochemical symptoms of starvation and (–) immunity

An increase is indicated by (+); a decrease is indicated by (–). Concentration of mercury in water, administered as methylmercuric chloride.

Source: Adapted from Wiener and Spry.26

TABLE 21.17 Lethality of Mercury to Birds

Species Chukar (Alectoris chukar) Mallard (Anas platyrhynchos)

Northern bobwhite (Colinus virginianus) Coturnix (Coturnix japonica)

Rock dove (Columba livia) Fulvous whistling duck (Dendrocygna bicolor) Chicken (Gallus domesticus)

Form of Mercury

Exposure

Concentration (mg Hg/kg body weight)

Effects

Ethyl Methyl Ethyl Phenyl Methyl

Acute Acute Acute Acute Acute

oral oral oral oral oral

26.9 2.2–23.5 75.7 524.7 23.8

LD50 LD50 LD50 LD50 LD50

Methyl Inorganic Ethyl Inorganic Ethyl Methyl

Acute Acute Acute Acute Acute Acute

oral oral oral oral oral oral

11.0–33.7 26.0–54.0 21.4 31.1 22.8 37.8

LD50 LD50 LD50 LD50 LD50 LD50

Phenyl

Acute oral

60.0

LD50


TABLE 21.17 (Continued) Lethality of Mercury to Birds

Species House sparrow (Passer domesticus) Gray partridge (Perdix perdix) Ring-necked pheasant (Phasianus colchicus)

Prairie chicken (Tympanuchus cupido) Mallard, hens

Coturnix

Form of Mercury

Acute oral

12.6–37.8

LD50

Ethyl Ethyl

Acute oral Acute oral

17.6 11.5

LD50 LD50

Methyl Phenyl Ethyl

Acute oral Acute oral Acute oral

11.5–26.8 65.0–101.0 11.5

LD50 LD50 LD50

Methyl

Dietary (mg Hg/kg diet), for two reproductive seasons Dietary, hatch to 9 weeks Dietary, 5 d +7 d observation period Dietary, 28 d Dietary, 28 d

Inorganic

Inorganic in dry salt Inorganic in ethanol, methanol, or water Inorganic in caseinpremix Methyl Methyl Methyl

Ring-necked pheasant

Birds, 4 spp. Birds, 3 spp.

Effects

Methyl

Inorganic

Zebra finch (Poephila guttata)

Exposure

Concentration (mg Hg/kg body weight)

Methyl Methyl Ethyl Ethyl Ethyl Ethyl Methyl Methyl

Dietary, 28 d Dietary, hatch to 9 weeks Dietary, 5 d Dietary, 5 d +7 d observation period Dietary, 77 days Dietary, 77 days Dietary, 70 days Dietary, 70 days Dietary, 28 days Dietary, 15 days Dietary, 6–11 d Dietary, 35 d

3.0

Reduced duckling survival

32.0

LD0

2956–5086

LD50

500.0 500.0

LD86 LD55

500.0

LD33

4.0

LD0

8.0 31.0–47.0

Some deaths LD50

2.5 5.0 4.2 12.5 37.4 112.0 40.0 33.0

LD50 LD25 LD0 LD50 LD50 LD50 LD33 LD8 to LD90

Source: Adapted from Eisler.27

3. Cadmium Increased cadmium in the terrestrial habitats of small mammals is derived from a variety of anthropogenic sources including atmospheric deposition, the application of phosphatic fertilizers and sewage sludge to land, and disused mine waste. Field studies have shown that cadmium, whether derived from the atmosphere or soil, is commonly found in most biotic components within a terrestrial ecosystem. The concentrations of cadmium measured in wild small mammals caught in contaminated sites are elevated in many tissues and organs, but most of the body burden is in the kidney and liver (Table 21.18).29 Kidney concentrations ranged from 2 to 90 mg of cadmium/kg of dry weight in mice and voles and were two to eight times the corresponding liver value. In


shrews, liver values were usually higher, at 200 to 600 mg of cadmium/kg of dry weight as against kidney concentrations of 100 to 250 mg of cadmium/kg. Using response criteria similar to those for human cadmium exposure and using primarily laboratory experiments with rats and mice, it has been suggested that 100 mg of cadmium/kg of wet weight or 350 mg of cadmium/kg of dry weight could be considered as the critical kidney concentration on a whole-organ basis.

TABLE 21.18 Kidney and Liver Cadmium Concentrations in Species of Small Mammals from Contaminated Habitats Species Bank vole (Clethrionomys glareolus) Wood mouse (Apodemus sylvaticus)

Short-tailed field vole (Microtus agretis)

Meadow vole (Microtus pennsylvanicus) White-footed mouse (Peromyscus leucopus) Common shrew (Sorex araneus)

European mole (Talpa europea) a

Contaminated Site

Kidney Conc. (mg/kg dry wt)

Dulowa Forest, Poland Y Fan, Pb/Zn mine, Wales Smelter waste, Wales Minera, Pb/Zn mine, Wales Y Fan, Pb/Zn mine, Wales Cu/Cd refinery, England Fluorspar waste, England Y Fan, Pb/Zn mine, Wales Cu/Cd refinery, England Fluorspar waste, England Budel, The Netherlands Sludge-treated fields, USA Wastewater-irrigated site, USA Cu/Cd refinery, England Fluorspar waste, England (Oct.–Nov.) Budel, The Netherlands (Feb.–Mar.) Budel, The Netherlands Budel, The Netherlands

Liver Conc. (mg/kg dry wt)

Ratio Kidney/Liver

29.6 16.8 18.0 39.7 10.3 41.7 1.78 8.91 88.8 5.3 2.7 23a

12.8 5.1 5.5 9.8 2.49 18.2 0.71 1.06 22.7 1.8 0.57 7.9a

2.3 3.3 3.3 4.1 4.1 2.3 2.5 8.4 3.9 2.9 4.7 2.9

2.3a

0.5a

5.0

253 158 126

578 236 180

0.43 0.67 0.7

200

268

0.75

224

227

0.99

Values changed from wet weight (dry weight = wet weight × 3.5).

Source: Adapted from Cooke.29

B. SELENIUM Agricultural drain water, sewage sludge, fly ash from coal-fired power plants, and mining of phosphates and metal ores contribute to the selenium (Se) burden in the aquatic environment. Once in aquatic systems, selenium is readily taken up from solution by food-chain organisms and can quickly reach concentrations that are toxic to the fish and wildlife that consume them.30 Selenium transferred to the eggs of fish from parents can result in edema, hemorrhaging, spinal deformities, and death of embryos. Reproductive success is more sensitive to selenium toxicity than are the growth and survival of juvenile and adult fish. Waterborne selenium concentrations of 2 μg/l or greater (on a total recoverable basis in 0.45-μm filtered samples) should be considered hazardous to the health and long-term survival of fish and wildlife populations because of the high potential for food-chain bioaccumulation, dietary toxicity, and reproductive effects. The dietary toxicity threshold for fish is 3 μg/g. Thresholds for tissue concentrations that affect the health and repro-


ductive success of freshwater and anadromous fish are as follows: whole-body, 4 μg/g; skeletal muscle (skinless fillets), 8 μg/g; liver, 12 μg/g; and ovaries and eggs, 10 μg/g (Table 21.19). In birds, historically, plant-derived Se in excess of 4 ppm in the diet of chickens was found to impair reproductive success and to be teratogenic.31 In more recent years, adverse effects of Se in wild aquatic birds has been documented as a consequence of pollution of the aquatic environment by subsurface agricultural drain water and other sources.32 Selenomethionine is a form of Se that wild aquatic birds are most likely exposed to since the toxic thresholds in eggs for decreased hatching success and teratogenicity from laboratory studies with mallards are very close to those derived from field studies.33 Biological and physiological effects observed have included avian mortality, impaired reproduction and teratogenesis, and histopathological lesions with alterations in hepatic glutathione (Table 21.20).34-42

TABLE 21.19 Toxic Effects Thresholds for Selenium Concentrations in Water, Food Chain Organisms, and Fish Tissues Selenium Source

Selenium Concentrationa

Water Inorganic selenium

2 μg/l

Organic selenium

0.5–1 >0.5–1

>0.5 >0.5 >0.5

>25 mg/kg >25 mg/kg >25 mg/kg

a

>100 μg/l Low mg/kg

Fish >100 mg/kg >50 mg/kg >100 mg/kg >50 mg/kg >100 μg/l mg/kg range High μg/kg to low mg/kg High μg/kg to low mg/kg

Estimates for algae include those for phytoplankton, and zooplankton estimates were primarily based on Daphnia. A maximum concentration of >100 mg/kg of PCBs in tissues was used because higher concentrations may have limited environmental relevance. Some threshold concentrations are expressed on a waterborne exposure basis, where estimates of tissue concentrations were poorly defined. Source: Adapted from Niimi.48


TABLE 21.25 Reproductive Effects of Aroclors and Other PCB Mixtures in Birds Conc. in Tissue (ppm wet wt)

Species, Age Chickens, laying hens Chickens, white leghorn hens Chickens, fertile white leghorn eggs Chickens, laying hens Chickens, fertile white leghorn eggs Chickens, fertile eggs Ringed turtledoves

5 in eggs 13.2 in eggs Above 4 in eggs Less than 1 in eggs 10 in eggs 23 in eggs 5 in eggs 0.05 to 0.1 in eggs 16 in eggs, 5.5 in adult brain 2.8 in brain

Mallard hens

Screech owls Atlantic puffins

23 in eggs, 30 in 3-week-old ducklings and 55 in hens 105 in eggs 4 to 18 in eggs 10 to 81 in eggs, 6 in adults

Source: Adapted from Hoffman et al.49


Effect

Experimental Treatment

Hatching reduced Did not affect hatching Embryo mortality and teratogenic Decreased hatching Embryonic mortality of 64% Decreased hatching Hatching reduced to 17% Decreased gluconeogenic enzyme activity Embryonic mortality; Decreased parental attentiveness depletion of brain dopamine and norepinephrine No effects

Aroclor 1254 in diet, up to 50 ppm Aroclor 1254 in diet, 20 ppm Aroclor 1254 in drinking water at 50 ppm Aroclor 1242 diet, up to 80 ppm Aroclor 1242 injected into air cell of eggs Aroclor 1248 in diet at 10 ppm Aroclor 1248 injected into yolk sac Aroclor 1254 injected into air cell Aroclor 1254 in diet

Eggshell thickness decreased; hatching success not affected No effects No effects detected

Aroclor 1242 in diet

Aroclor 1254 in diet Aroclor 1254 in diet at 25 ppm

Aroclor 1248 in diet at 3 ppm wet weight Aroclor 1254 dosed by implantation of 30–35 mg

TABLE 21.26 Egg Injection Studies with Planar PCBs and Dioxin Species, Age Chicken, white leghorn embryo

Pheasant, embryo

Bobwhite, embryo Common tern, embryo American kestrel, embryo Mallard, embryo; goldeneye, embryo

Compound, Conc. 2,3,7,8-TCDDa 10 ppt 10–20 ppt 40–50 ppt 63 ppt 147 ppt 115 ppt 180 ppt 240 ppt 302 ppt 1 ppb 3,3′ ,4,4′ 5-PeCB (PCB 126) 0.4 ppb 3.1 ppb 3,3′ 4,4′ -TeCB (PCB 77) 2.6 ppb 8.6 ppb 40 ppb 2,3,3′ 4,4′ -PeCB (PCB 105) 2,200 ppb 2,3,3′ 4,4′ 5-HxCB (PCB 157) 2000 ppb 1500 ppb 2,3,7,8-TCDD 1.4 ppb 2.2 ppb 3,3′ ,4,4′ 5-PeCB (PCB 126) 24 ppb 3,3′ ,4,4′ 5-PeCB (PCB 126) 104 ppb 3,3′ ,4,4′ 5-PeCB (PCB 126) 65 ppb 3,3′ 4,4′ -TeCB (PCB 77) 5,000 ppb

Effect

Two-fold AHH* induction Onset of embryotoxicity Mortality, edema, surface hemorrhaging ED50 for AHH induction LD50 (air cell injection) LD50 (yolk sac injection) LD50 (air cell injection) LD50 (air cell injection) ED50 for AHH induction 100% mortality LD50 (air cell injection), day 4 through hatch LD50 (air cell injection), day 7 through 10 LD50 (air cell injection) LD50 (air cell injection) LD50 (air cell injection) LD50 (air cell injection)

LD50 (air cell injection) LD50 (air cell injection) LD50 (albumin) LD50 (yolk) LD50 (air cell injection), through hatching LD50 (air cell injection), through hatching LD50 (air cell injection), through hatching No effects (air cell injection)

Source: Adapted from Hoffman et al.49 * Aryl hydrocarbon hydroxylase.

E. PAHS

AND

PETROLEUM

Natural sources of polyaromatic hydrocarbons (PAHs) in the environment include forest and grass fires, oil seeps, volcanoes, and plants. Figure 21.5 shows natural and anthropogenic sources of PAHs, including petroleum spills and discharges, electric power generation, refuse incineration, home heating, and internal combustion engines. The primary mechanism for atmospheric contamination by PAHs is incomplete combustion of organic matter. Aquatic contamination by PAHs is caused by petroleum spills, discharges, and seepages, industrial and municipal wastewater, urban


and suburban surface runoff, and atmospheric deposition. Sources of PAHs on land include natural fires, industrial activities, waste disposal and incineration, home heating, and automobile exhaust. Petroleum can cause environmental harm by toxic action, physical contact, chemical and physical changes within the soil or water medium, and habitat alteration (Table 21.27).52 Oil spills have caused major changes in local plant and invertebrate populations lasting from several weeks to many years. Effects of oil spills on populations of mobile vertebrate species, such as fish, birds, and mammals, have been difficult to determine beyond the immediate losses in local populations. The induction of lesions and neoplasms in laboratory animals by metabolites of PAHs and observations of lesions and neoplasms in fish from PAH-contaminated sites indicate potential health problems for animals with cytochrome P-450 capable of metabolizing PAHs. Although evidence linking environmental PAHs to the incidence of cancerous neoplasms in wild animals is limited and primarily circumstantial, the growing quantities of PAHs entering the environment are a cause for concern. Reptiles and amphibians can be killed by petroleum, but available information is inadequate to evaluate properly the sensitivity of these organisms to petroleum or individual PAHs. Birds are often killed by oil spills, primarily because of plumage oiling and oil ingestion. Birds that spend much of their time on the water surface are the most vulnerable to spilled oil. Ingested oil can cause many sublethal effects, and transmittal to nests and eggs is highly embryotoxic. Mammals that rely on fur for insulation (polar bear, otters, fur seals, muskrat) are the most likely to die from oiling.

2

1

4 10 5

3

1. Volcanoes 2. Natural fires 3. Industry, power generation 4. Internal combustion engines 5. Cities and towns, municipal discharges 6. Pipeline spills 7. Oil fields 8. Offshore oil platform 9. Natural oil seep 10. Shipping accidents, intentional oil discharges

8

6

7

9

FIGURE 21.5 Sources of petroleum and PAHs in the environment. The primary mechanism for atmospheric contamination by PAHs is incomplete combustion of organic matter. Aquatic contamination by PAHs is caused by petroleum spills, discharges, and seepages, industrial and municipal wastewater, urban and suburban surface runoff, and atmospheric deposition. Sources of PAHs on land include natural fires, industrial activities, waste disposal and incineration, home heating, and automobile exhaust. (Adapted from Albers.52)


TABLE 21.27 Effects of Petroleum and Individual PAHs on Living Organisms Effects

Death Impaired reproduction Reduced growth and development Impaired immune system Altered endocrine function Altered rate of photosynthesis Malformations Tumors and lesions Cancer Altered behavior Blood disorders Liver and kidney disorders Hypothermia Inflammation of epithelial tissue Altered respiration or heart rate Impaired salt gland function Gill hyperplasia Fin erosion

Local population changes Altered community structure Biomass change

Plant

Invertebrate

Fish

Individual Organisms ⻬⻬⻬⻬⻬⻬⻬⻬⻬

Reptile and Amphibian

Bird

Mammal

⻬⻬⻬

⻬⻬⻬

⻬

⻬⻬

⻬

⻬⻬⻬⻬

⻬

⻬⻬⻬⻬⻬⻬

⻬

⻬⻬⻬⻬⻬

⻬⻬

⻬⻬⻬⻬⻬

⻬⻬⻬⻬⻬⻬⻬

⻬

⻬⻬ Groups of Organismsa ⻬⻬⻬⻬⻬⻬

⻬⻬

a

Populations of chlorophyllous and nonchlorophyllous plants (bacteria, filamentous fungi, yeast, microalgae) can increase or decrease in the presence of petroleum, whereas animal populations decrease. Source: Adapted from Albers.52

F. RADIONUCLIDES On a global basis, radiation from natural sources is a much greater contributor to radiation dose to living organisms than is radiation from anthropogenic sources. However, ionizing radiation can harm biological systems, causing a range of syndromes from prompt lethality to reduced vigor, shortened life span, diminished reproductive rate, and genetic transmission of radiation-altered genes that are most commonly recessive and usually disadvantageous. In general, more-primitive organisms are the most-radioresistant taxonomic groups and the more-advanced, complex organisms, such as mammals, are the most radiosensitive (Figure 21.6).53 The early effects of exposure to ionizing radiation result primarily from cell death where dividing cells, frequently undergoing mitosis, are the most sensitive, and cells that do not divide are the most radioresistant. Thus, embryos and fetuses are particularly susceptible to ionizing radiation, and very young animals are consistently more radiosensitive than adults. Among birds, as in most other tested species, there is a direct relation between dose and mortality at single high doses of ionizing radiations (Table 21.28).54 For any given total dose, the survival of a bird is higher if the dose is delivered at a lower rate or over a longer period of time and suggests that biological repair processes compensate for radiation-induced cellular and tissue damage over a prolonged period or at a comparatively low dose rate.55


VIRUSES MOLLUSKS PROTOZOANS BACTERIA

TAXONOMIC GROUP

PRIMITIVE PLANTS (mosses, lichens, algae) INSECTS CRUSTACEANS REPTILES AMPHIBIANS FISHES HIGHER PLANTS BIRDS MAMMALS 100

101

102

103

104

105

DOSE (GRAY)

FIGURE 21.6 Ranges of acute lethal doses of radiation to various taxonomic groups. Dose of radiation is expressed in grays (Gy, where 1 Gy = 100 rad). In general, more primitive organisms are the most radioresistant taxonomic groups and more-advanced complex organisms, such as mammals, are the most radiosensitive. (Adapted from Talmage and Meyers-Schone.53)

TABLE 21.28 Radiation Effects on Birds Species Green-winged teal (Anas carolinensis) Northern shoveler (Anas clypeata) Blue-winged teal (Anas discors) Passerine species eggs Passerine species nestlings Common quail (Coturnix coturnix) eggs

Chicken (Gallus domesticus) eggs

Chicks, age 15 days

Dose/Exposure 4.8 Gy, single acute exposure 8.9 Gy, single acute exposure 7.2 Gy, single acute exposure 5–10 Gy, single acute exposure 1 Gy daily Exposed first 9 days of incubation, single acute exposure 5 Gy 7 GY 9 GY Exposed before incubation, single acute exposure, 0.05–2.1 GY

2.1 GY 6.6 GY


Effects LD50, 30 days postexposure LD50, 30 days postexposure LD50, 30 days postexposure LD100 Growth retardation

Negligible effect on survival Mortality > 50% All dead before hatch No adverse effects on embryonic development at 1.6 Gy and lower; at 2.1 Gy, adverse effects on development, survival, and body weight of hatched chicks Reversible changes in blood chemistry within 60 days; no deaths Irreversible and permanent damage to red blood cells, hemoglobin, and hematocrit; all dead within 7 days

TABLE 21.28 (Continued) Radiation Effects on Birds Species

Dose/Exposure

Effects

Laying hens

Fed diet containing 400 Bq of 137Cs/kg ration for 4 weeks

Broiler chickens

Fed diet containing 400 Bq of 137Cs/kg ration for 40 days; some diets contained up to 5% bentonite 9.6 GY over 20 days

Of total 137Cs ingested, 3% was distributed in egg contents (29–33 Bq/kg egg; 2 Bq egg); 9% in muscle (171 Bq/kg FW*); and 81% in excreta Feeding with bentonite reduced 137Cs concentration in muscle by 32%, from 155 to 105 Bq/kg FW LD50

Single acute exposure > 8 Gy

All dead by fledging

Black-headed gull (Larus ridibundus ridibundus) eggs Great crested flycatcher (Myiarchus crinitus) nestlings Eastern bluebird (Sialia sialis) nestlings, age 2 days

Single acute exposure 3 GY 3–5 GY 4–12 Gy

Eastern bluebird eggs European starling (Sturnus vulgaris) Tree swallow (Tachycineta bicolor)

Tree swallow nestlings

House wren (Troglodytes aedon) fledglings

5-6 Gy 25 Gy 30 Gy Single acute exposure 6 Gy Single exposure > 2 GY 0.006 mGy/h during breeding season equivalent to annual dose of about 50 mSv 0.9–4.5 Gy, single acute exposure 1.0 Gy daily, chronic 0.9 Gy, single acute exposure

Reduced growth after 16 days Reduced growth and shorter primary feathers at fledging Developed normally and fledged successfully LD50, nestling to fledgling LD50, 16 days postexposure All dead 4 days postexposure All dead before hatch Fatal No adverse effects on breeding performance of adults, or growth performance of nestlings Adverse effects on growth, survival, or both Reduced hatch, depressed growth Growth reduction

Source: Adapted from Eisler. 54 * FW = Fresh weight.

G. SUMMARY OF STUDIES OF PERSISTENT ENVIRONMENTAL CONTAMINANTS TERRESTRIAL VERTEBRATES

IN

Table 21.29 summarizes field studies of persistent environmental contaminants by species studied, contaminants detected, and locations.56

TABLE 21.29 Summary of Studies of Persistent Environmental Contaminants in Terrestrial Vertebrates Species Snapping turtle (Chelydra serpentina)


Contaminantsa HM OC, PCB Hg Pb

Area New Jersey, Maryland New York, New Jersey, Great Lakes, Canada Canada, Tennessee Missouri

TABLE 21.29 (Continued) Summary of Studies of Persistent Environmental Contaminants in Terrestrial Vertebrates Species Diamondback terrapin (Malaclemys terrapin) Brown pelican (Pelecanus occidentalis)

Double-crested cormorant (Phalacrocorax auritus)

Anhinga (Anhinga anhinga) Great blue heron (Ardea herodias) Snowy egret (Egretta thula)

Tricolored heron (Egretta tricolor)

Cattle egret (Bubulcus ibis) Black-crowned night heron (Nycticorax nycticorax) White-faced ibis (Plegadis chihi) Roseate spoonbill (Ajaia ajaja) Woodstork (Mycteria americana) Black vulture (Coragyps atratus) California condor (Gymnogyps californianus) Greater snow goose (Chen caerulescens atlantica) Mute swan (Cygnus olor)

Wood duck (Aix sponsa) Black duck (Anas rubripes) Canvasback duck (Aythya valisineria) Greater scaup (Aythya marila)

Mergansers (Mergus spp.)


Contaminantsa OC, PCB DDE OC, PCB PCB OC, PCB, HM DDE, PCB, Hg, Se OC, PCB, Hg OC, PCB TEQ HM OC, PCB, Hg DDE, Hg OC, PCB, Hg OC, PCB, TEQ, HM OC, PCB, TEQ DDE, PCB PCB OC, PCB, HM HM Hg OC Hg DDE, PCB, HM OC, PCB OC, PCB OC, PCB, TEQ, HM OC, PCB OC DDE, Se, Hg OC, PCB OC OC DDE, Pb OC, PCB, Hg DDE HM Pb DDE, PCB, HM OC OC, PCB, HM OC, PCB, Hg DDE, PCB HM PCB OC, HM OC, PCB Hg OC, PCB, Hg

Area Georgia U.S., Mexico, Puerto Rico, Virgin Islands Florida, South Carolina, California, Louisiana Georgia, Puerto Rico, Virgin Islands Texas, Southeastern U.S. Florida South Dakota Texas, Canada, Mexico, Maine Canada, Great Lakes Washington, Canada Great Lakes California Eastern U.S. U.S. Canada Eastern U.S., Idaho Nevada San Francisco Bay, Texas South Carolina, Florida New York Florida Florida Texas Eastern U.S. Baja, California U.S. Italy Texas Nevada Texas Eastern U.S. Mexico California Canada Scotland Sweden, Chesapeake Bay UK Denmark Mississippi Canada, Eastern U.S. North America Scotland, Finland, Northeastern U.S. British Columbia, Chesapeake Bay, Northeastern U.S. Netherlands, Poland San Francisco Bay, Poland New York, Michigan, Great Lakes Utah, Finland U.S.

TABLE 21.29 (Continued) Summary of Studies of Persistent Environmental Contaminants in Terrestrial Vertebrates Species Ruddy duck (Oxyura jamaicansis) Osprey (Pandion haliaetus) Mississippi kite (Ictinia mississippiensis) Bald eagle (Haliaeetus leucocephalus) Fish eagle (Haliaeetus vocifer) Spanish eagle (Aquila adalberti) Red-tailed hawk (Buteo jamaicensis) White-tailed eagle (Haliaeetus albicilla) Peregrine falcon (Falco peregrinus) Prairie falcon (Falco mexicanus) Clapper rail (Rallus longirostris) American oystercatcher (Haematopus palliatus) Willet (Catoptrophorus semipalmatus) Long-billed curlew (Numenius americanus) Laughing gull (Larus atricilla)

Herring gull (Larus argentatus)

Gull-billed tern (Sterna nilotica) Caspian tern (Sterna caspia) Common tern (Sterna hirundo)

Forster’s tern (Sterna forsteri) Black skimmer (Rynchops niger) Barn owl (Tyto alba) Great horned owl (Bubo virginianus) Loggerhead shrike (Lanius ludovicianus) Tree swallow (Tachycineta bicolor)

Contaminantsa

Area

HM Se OC, PCB, HM OC OC, PCB, Hg, Pb TEQ DDE OC, PCB, HM OC OC, PCB, Hg OC, PCB, DDE DDE OC, PCB OC, PCB, HM DDE, PCB OC, PCB, HM DDE, PCB, HM OC, PCB OC, PCB, HM DDE, PCB HM OC OC, PCB Hg HM OC, PCB, TEQ, HM DDE, PCB

Delaware River, Chesapeake Bay California U.S. U.S. U.S., Canada British Columbia Zimbabwe Spain Ohio Sweden Worldwide Colorado New Jersey, South Carolina, Virginia Georgia, California South Carolina Delaware Bay Texas Oregon Texas South Carolina New York Florida Germany, Newfoundland Denmark, Ontario UK, Germany, New York, New Jersey Great Lakes, Canada Maine, Scotland, Portugal, Gibraltar, Finland, Canada, Virginia, Denmark, Crete, Cyprus Norway South Carolina Southern California Rhode Island Great Lakes, Eastern U.S., Canada Netherlands, Massachusetts Germany Wisconsin Florida, South Carolina, Texas, Mexico Florida, New York, New Jersey, Texas Chesapeake Bay Ohio Illinois Colorado, Oregon, Wyoming, Montana, Idaho, Utah, South Dakota, New Mexico Wisconsin, Michigan, New York Pennsylvania, Indiana, Maryland New York, New Jersey, Canada Idaho Canada, Wisconsin Great Lakes

OC, PCB, Flouride OC, PCB OC, PCB DDE, PCB, HM OC, PCB, HM TEQ Hg PCB OC, PCB HM OC, PCB OC DDE OC, PCB PCB, TEQ PCB HM Pb DDE, PCB OC, PCB, Hg


TABLE 21.29 (Continued) Summary of Studies of Persistent Environmental Contaminants in Terrestrial Vertebrates Species Mink (Mustela vison)

Contaminantsa Hg

OC, PCB

OC HM Muskrat (Ondatra zibethicus)

River otter (Lontra canadensis)

Se HM Hg OC, PCB, HM DDE, PCB, Hg HM OC, Cs, Hg

Area Georgia, Wisconsin River, Wisconsin, Connecticut, Massachusetts, Ontario, Manitoba, Southern Atlantic Coast Canada, Maryland, Oregon, Great Lakes, New York, Connecticut, Massachusetts, Southern Atlantic Coast, Minnesota Iowa Virginia, Washington, Idaho, New York, Illinois, Minnesota, Canada New York Pennsylvania, Missouri, Manitoba Tennessee Virginia New York Virginia Georgia

a

OC = organochlorine insecticides; PCB = polychlorinated biphenyls; HM = heavy metals; PCS = polychlorinated styrenes; TEQ = toxic equivalents. Source: Adapted from Keith.56

REFERENCES 1. Moriarity, F., Ecotoxicology, the Study of Pollutants in Ecosystems, 2nd ed., Academic Press, San Diego, CA, 1988. 2. Cairns, J., Jr., Will the real ecotoxicologist please stand up? Environ. Toxicol. Chem., 8, 843, 1989. 3. Hoffman, D. J., Rattner, B. A., Burton, G. A., Jr., and Cairns, J., Jr., Introduction, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, G. A. Jr., and Cairns, J. Jr., Eds., CRC Press, Boca Raton, FL, 1995, 1–10. 4. Forbes, S. A., The lake as a microcosm, Bulletin of the Peoria Scientific Association, 1887; reprinted in Bull. Ill. State Natl. Hist. Sur., 15, 537–550, 1925. 5. Penny, C. and Adams, C., Fourth Report Royal Commission on Pollution in Scotland, London, 2, 377, 1863. 6. Weigelt, C., Saare, O., and Schwab, L., Die Schädigung van fischerei und fischzueht durch industrie und haus abwasser, Arch. Hyg., 3, 39, 1885. 7. Newman, J. R., Effects of industrial air pollution on wildlife, Biol. Conserv., 15, 181, 1979. 8. Carson, R., Silent Spring, Houghton Mifflin, Boston, 1962, 103. 9. Blus, L. J., Gish, C. D., Belisle, A. A., and Prouty, R. M., Logarithmic relationship of DDE residues to eggshell thinning, Nature, 235, 376, 1972. 10. Hill, E. F. and Fleming, W. J., Anticholinesterase poisoning of birds; field monitoring and diagnosis of acute poisoning, Environ. Toxicol. Chem., 1, 27, 1982. 11. Grue, C. E., Fleming, W. J., Busby, D. G., and Hill, E. F., Assessing hazards of organophosphate pesticide to wildlife, Trans. North Am. Wildl. Nat. Res. Conf., 48, 200, 1983. 12. U.S. Environmental Protection Agency, Guidelines for Ecological Risk Assessment, Washington, D.C., Fed. Reg., 63(93), 26846–26924, 1998. 13. Chappie, D. J. and Burton, G. A., Jr., Applications of aquatic and sediment toxicity testing in situ, Soil Sediment Contam., 9, 219–245, 2000. 14. Hill, E. F., Acute and subacute toxicology in evaluation of pesticide hazard to avian wildlife, in Wildlife Toxicology and Population Modeling: Integrated Studies of Agroecosystems, Kendall, R. J. and Lacher, T. E., Jr., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1994, 2077–226.


15. Hoffman, D. J., Wildlife toxicity testing, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, G. A., Jr., and Cairns, J., Jr., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1995, 47–69. 16. Hoffman, D. J., Measurements of toxicity and critical stages of development, in Wildlife Toxicology and Population Modeling: Integrated Studies of Agroecosystems, Kendall, R. J. and Lacher, T. E., Jr., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1994, 47–67. 17. Depledge, M. H. and Fossi, M. C., The role of biomarkers in environmental assessment (2). Invertebrates, Ecotoxicology, 3, 161–172, 1994. 18. Fossi, M. C., Leonzio, C., and Peakall, D. B., The use of nondestructive biomarkers in the hazard assessments of vertebrate populations, in Nondestructive Biomarkers in Vertebrates, Fossi, M.C. and Leonzio, C., Eds., Lewis Publishers, Boca Raton, FL, 1994, 3–34. 19. Huggett, R. J., Kimerle, R. A., Mehrle, P. M., Jr., and Bergman, H. L., Biomarkers: biochemical, physiological, and histological markers of anthropogenic stress, SETAC Special Publication Series, Lewis Publishers, Boca Raton, FL, 1992, 347 pp. 20. Fossi, M. C. and Leonzio, C., Eds., Nondestructive Biomarkers in Vertebrates, Lewis Publishers, Boca Raton, FL, 1994, 345 pp. 21. Melancon, M. J., Bioindicators used in aquatic and terrestrial monitoring, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, G. A., Jr., and Cairns, J., Jr., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1995, 220–240. 22. Rattner, B. A., Cohen, J. B., and Golden, N. H., Contaminant effect endpoints in terrestrial vertebrates at and above the level of the “individual,” in Environmental Contaminants and Terrestrial Vertebrates: Effects on Populations, Communities and Ecosystems, Albers, P. H., Ed., SETAC Press, Pensacola, FL, 2000, 61–94. 23. Interpretation of Criteria Commonly Used to Determine Lead Poisoning Problem Areas, U.S. Department of the Interior, Fish and Wildlife Service, Fish and Wildlife Leaflet 2, Washington, D.C., 1985. 24. Pain, D. J., Lead in waterfowl, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W. N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 251–264. 25. Franson, J. C., Interpretation of tissue lead residues in birds other than waterfowl, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W.N., Heinz, G.H., and RedmonNorwood, A. W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 265–280. 26. Wiener, J. G. and Spry, D. J., Toxilogical significance of mercury in freshwater fish, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W. N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 297–340. 27. Eisler, R., Handbook of Chemical Risk Assessment: Health Hazards to Humans, Plants, and Animals, Vol. 1, Lewis Publishers/CRC Press, Boca Raton, FL, 2000, 313–411. 28. Heinz, G. H., Mercury poisoning in wildlife, in Noninfectious Diseases of Wildlife, Fairbrother, A., Locke, L. N., and Hoff, G. L., Eds., Iowa State University Press, Ames, 1996, 118–122. 29. Cooke, J. A. and Johnson, M. S., Cadmium in small mammals, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W.N., Heinz, G.H., and Redmon-Norwood, A.W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 377–388. 30. Lemly, A .D., Selenium in aquatic organisms, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W. N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 427–446. 31. Poley, W. E. and Moxon, A. L., Tolerance levels of seleniferous grains in laying rations, Poult. Sci., 17, 72–76, 1938. 32. Ohlendorf, H. M., Bioaccumulation and effects of selenium in wildlife, in Selenium in Agriculture and the Environment, Jacobs, L. W., Ed., SSSA Special Publication 23, Soil Science Society of America, Madison, WI, 1989, 133-177. 33. Heinz, G. H., Selenium in birds, in Environmental Contaminants in Wildlife, Beyer, W. N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., CRC Press, Boca Raton, FL, 1996, 447–458. 34. Hoffman, D. J. and Heinz, G. H.,Embryotoxic and teratogenic effects of selenium in the diet of mallards, J. Toxicol. Environ. Health, 24, 477–490, 1988. 35. Smith, G. J., Heinz, G. H., Hoffman, D. J., Spann, J. W., and Krynitsky, A. J., Reproduction in blackcrowned night-herons fed selenium, Lake Reservoir Manage., 4, 175–180, 1988. 36. Heinz, G. H., Hoffman, D. J., and Gold, L. G., Toxicity of organic and inorganic selenium to mallard ducklings, Environ. Toxicol. Chem., 17, 561–568, 1988.


37. Hoffman, D. J., Heinz, G. H., and Krynitsky, A. J., Hepatic glutathione metabolism and lipid peroxidation in response to excess dietary selenomethionine and selenite in mallard ducklings, J. Toxicol. Environ. Health, 27, 263–271, 1989. 38. Hoffman, D. J., Sanderson, C. J., LeCaptain, L. J., Cromartie, E., and Pendleton, G. S., Interactive effects of arsenic, selenium, and dietary protein on survival, growth, and physiology in mallard ducklings, Arch. Environ. Contam. Toxicol., 20, 288–294, 1992. 39. Hoffman, D. J., Sanderson, C. J., LeCaptain, L. J., Cromartie, E., and Pendleton, G. S., Interactive effects of boron, selenium and dietary protein on survival, growth, and physiology in mallard ducklings, Arch. Environ. Contam. Toxicol., 20, 288–294, 1991. 40. Hoffman, D. J., Sanderson, C. J., LeCaptain, L. J., Cromartie, E., and Pendleton, G. S., Interactive effects of selenium, methionine and dietary protein on survival, growth, and physiology in mallard ducklings, Arch. Environ. Contam. Toxicol., 23, 163–186, 1992. 41. Fairbrother, A. and Fowles, J., Subchronic effects of sodium selenite and selenomethionine on several immune functions in mallards, Arch. Environ. Contam. Toxicol., 19, 836–844, 1990. 42. Hoffman, D. J., Heinz, G. H., LeCaptain, L. J., Bunck, C. M., and Green, D. E., Subchronic hepatoxicity of selenomethionine in mallard ducks, J. Toxicol. Environ. Health., 32, 449–464, 1991. 43. Blus, L. J., DDT, DDD, and DDE in birds, in Environmental Contaminants in Wildlife, Beyer, W.N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., Lewis Publishers, Boca Raton, FL, 1996. 44. Grue, C. E., Hart, A. D. M., and Mineau, P., Biological consequences of depressed brain cholinesterase activity in wildlife, in Cholinesterase-Inhibiting Insecticides: Their Impact on Wildlife and the Environment, Mineau, P., Ed., Elsevier, New York, 1991, 157–158. 45. Hardy, A. R., Fletcher, M. R., and Stanley, P. I., Pesticides and wildlife: twenty years of vertebrate wildlife incident investigations by MAFF, State Vet. J., 40, 182–192, 1986. 46. Mineau, P., Fletcher, M. R., Glasser, L. C., Thomas, N. J., Brassard, C., Wilson, L. K., Elliott, J. E., Lyon, L. A., Henny, C. J., Bollinger, T., and Porter, S. L., Poisoning of raptors with organophosphorus and carbamate pesticides with emphasis on Canada, U.S. and U.K., J. Raptor Res., 33, 1–37, 1999. 47. Hill, E. F., Organophosphorus and carbamate pesticides, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, A. G., Jr., and Cairns, J., Jr., Eds., Lewis Publishers, Boca Raton, FL, 1995, 243–273. 48. Niimi, A. J., PCBs in aquatic organisms, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W. N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 117–152. 49. Hoffman, D. J., Rice, C. P., and Kubiak, T. J., PCBs and dioxins in birds, in Environmental Contaminants in Wildlife: Interpreting Tissue Concentrations, Beyer, W. N., Heinz, G. H., and RedmonNorwood, A. W., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1996, 165–208. 50. Rice, C. P. and O’Keefe, P., Sources, pathways, and effects of PCBs, dioxins, and dibenzofurans, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, G. A., Jr., and Cairns, J., Jr., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1995, 424–468. 51. Giesy, J. P. and Kannan, K., Dioxin-like and non-dioxin-like toxic effects of polychlorinated biphenyls (PCBs): implications for risk assessment, Crit. Rev. Toxicol., 28(6), 511–569, 1998. 52. Albers, P. H., Petroleum and individual polycyclic aromatic hydrocarbons, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, G. A., Jr., and Cairns, J., Jr., Eds., Lewis Publishers,/CRC Press, Boca Raton, FL, 1995, 330–355. 53. Talmage, S. S. and Meyers-Schone, L., Nuclear and thermal, in Handbook of Ecotoxicology, Hoffman, D. J., Rattner, B. A., Burton, G. A., Jr., and Cairns, J., Jr., Eds., Lewis Publishers/CRC Press, Boca Raton, FL, 1995, 469–491. 54. Eisler, R., Handbook of Chemical Risk Assessment: Health Hazards to Humans, Plants, and Animals, Vol. 3, Lewis Publishers/CRC Press, Boca Raton, FL, 2000, 1707–1828. 55. Brisbin, I. L., Jr., Avian radioecology, in Current Ornithology, Vol. 8, Power, D. M., Ed., Plenum Press, New York, 1991, 69–140. 56. Keith, J. O., Residue analyses: how they were used to assess the hazards of contaminants to wildlife, in Environmental Contaminants in Wildlife, Beyer, W. N., Heinz, G. H., and Redmon-Norwood, A. W., Eds., Lewis Publishers, Boca Raton, FL, 1996. 57. Adams, W. J., Kimerle, R. A., and Mosher, R. G., An approach for assessing the environmental safety of chemicals sorbed to sediments, in Aquatic Toxicology and Hazard Evaluation: Seventh Symposium, Purdy, R. and Bahner, R. C., Eds., American Society for Testing and Materials, Philadelphia, 1985, 429–453.


ADDITIONAL RELATED INFORMATION* A. GLOSSARY

OF

ECOTOXICOLOGICAL TERMS

Bioaccumulation General term describing a process by which chemicals are taken up by aquatic organisms directly from water as well as from exposure through other routes, such as consumption of food and sediment containing chemicals. Bioaccumulation Factor (BAF) The ratio of tissue chemical residue to chemical concentration in an external environmental phase (i.e., water, sediment, or food). BAF is measured as steady state in situations where organisms are exposed from multiple sources (i.e., water, sediment, and food), unless noted otherwise. Biochemical oxygen demand (BOD) Sometimes called biological oxygen demand, a measure of the rate at which molecular oxygen is consumed by microorganisms during oxidation of organic matter. The standard test is the 5-day BOD test, in which the amount of dissolved oxygen required for oxidation over a 5-day period is measured. The results are measured in mg of oxygen/l (mg/l), or parts per million (ppm). Bioconcentration A process by which there is a net accumulation of a chemical directly from water into aquatic organisms resulting from simultaneous uptake (e.g., by gill or epithelial tissue) and elimination. Bioconcentration factor (BCF) A term describing the degree to which a chemical can be concentrated in the tissues of an organism in the aquatic environment as a result of exposure to waterborne chemical. At steady state during the uptake phase of a bioconcentration test, the BCF is a value that is equal to the concentration of a chemical in one or more tissues of the exposed aquatic organisms divided by the average exposure water concentration of the chemical in the test. Biodegradation The transformation of a material resulting from the complex enzymatic action of microorganisms (e.g., bacteria, fungi). It usually leads to disappearance of the parent chemical structure and to the formation of smaller chemical species, some of which are used for cell anabolism. Although typically used with reference to microbial activity, it may also refer to general metabolic breakdown of a substance by any living organism. Chemical oxygen demand (COD) COD is measured instead of BOD when organic materials are not easily degraded by microorganisms. Strong oxidizing agents (e.g., potassium permanganate) are used to enhance oxidation. COD values will be larger than BOD values. EC50 (median effective concentration) The concentration of chemical in water to which test organisms are exposed that is estimated to be effective in producing some sublethal response in 50% of the test organisms. The EC50 is usually expressed as a time-dependent value (e.g., 24-h or 96-h EC50). The sublethal response elicited from the test organisms as a result of exposure to the chemical must be clearly defined (e.g., test organisms may be immobilized, lose equilibrium, or undergo physiological or behavioral changes). Fate Disposition of a material in various environmental compartments (e.g., soil or sediment, water, air, biota) as a result of transport, transformation, and degradation. Flow-through system An exposure system for aquatic toxicity tests in which the test material solutions and control water flow into and out of test chambers on a once-through basis either intermittently or continuously. LC50 (median lethal concentration) The concentration of chemical in water to which test organisms are exposed that is estimated to be lethal to 50% of the test organisms. The LC50 is often expressed as a time-dependent value (e.g., 24-h or 96 h LC50).

* Source: Rand, G.M., Ed., Fundamentals of Aquatic Toxicology, 2nd ed., Taylor & Francis, Washington, D.C., 1995. With permission.


Life cycle study A chronic study in which all the significant life stages of an organism are exposed to a test material. Generally, a life cycle test involves an entire reproductive cycle of the organism. Lowest observed effect concentration (LOEC) The lowest concentration of a chemical used in a toxicity test that has a statistically significant adverse effect on the exposed population of test organisms compared with the controls. Also called the lowest observed adverse effect level (LOAEL). Maximum acceptable toxicant concentration (MATC) The hypothetical toxic threshold concentration lying in a range bounded at the lower end by the highest tested concentration having no observed effect (NOEC) and at the higher end by the lowest concentration having a statistically significant toxic effect (LOEC) in a life cycle (full chronic) or a partial life cycle (partial chronic) test. This can be represented by NOEC < MATC < LOEC. No observed adverse effect level (NOAEL) See NOEC. No observed effect concentration (NOEC) The highest concentration of chemical in a toxicity test at which no statistically significant adverse effect was observed on the exposed population when compared with the controls. Octanol-water partition coefficient (Kow) The ratio of the solubility of a chemical in n-ocatanol and water at steady state; also expressed as P. The logarithm of P or Kow (i.e., log P or log Kow) is used as an indication of the propensity of a chemical for bioconcentration by aquatic organisms. Static system An exposure system for aquatic toxicity tests in which the test chambers contain still solutions of the test material or control water. TLm or TL50 (median tolerance limit) The concentration of material in water at which 50% of the test organisms survive after a specified time of exposure. The TLm (or TL50) is usually expressed as a time-dependent value (e.g., 24-h or 96 h TL50).


22

Metal Toxicology Akira Yasutake, Ph.D. and Kimiko Hirayama, Ph.D.

CONTENTS Abbreviations Section 1. Introduction Section 2. Arsenic Table 22.1 Table 22.2 Section 3. Beryllium Table 22.3

Toxic Effects of Arsenate and Arsenite Toxic Effects of GaAs and As2O3 Pulmonary Toxicity of Beryllium Aerosol Inhalation or Intratracheal Injection

Section 4. Cadmium A. Acute Toxicity Table 22.4 Acute Effects of Cadmium Toxicity Table 22.5 Effects of Cadmium on Pregnant Animals B. Subacute to Chronic Toxicities Table 22.6 Subacute to Chronic Effects of Cadmium Table 22.7 Pulmonary Toxicity of Aerosol Inhalation or Intratracheal Injection of Cadmium Table 22.8 Renal Toxicities by Cadmium-Metallothionein or Cadmium Plus Thiol Coinjection Section 5. Chromium Table 22.9 Toxic Effects of Hexavalent Chromium in Rats Section 6. Cisplatin Table 22.10 Toxic Effects of CDDP Section 7. Cobalt Table 22.11 Toxic Effects of Cobalt in Experimental Animals Section 8. Copper Section 9. Iron Table 22.12 Toxic Effects of Iron Overload in Experimental Animals Section 10. Lead A. Inorganic Lead Toxicity Table 22.13 The LD50 of Lead Salts for Rats and Mice after Intraperitoneal Dose Table 22.14 Neurobehavioral Effects of Lead Table 22.15 Hematopoietic Effects of Lead B. Organic Lead Toxicity Section 11. Manganese Table 22.16 Toxic Effects of Manganese Chloride Table 22.17 Toxic Effects of Methylcyclopentadienyl Manganese Tricarbonyl (MMT) Section 12. Mercury Table 22.18 Acute Effects of Mercuric Chloride


Table Table Table Table

Section 13. Section 14. Section 15. Section 16. Section 17. References

22.19 22.20 22.21 22.22

Subchronic to Chronic Effects of Mercuric Chloride Toxic Effects by Single Methylmercury Injection Toxic Effects of Multiple Methylmercury Injection Effects of Methylmercury Exposure from Food or Drinking Water Table 22.23 Effects of Prenatal Exposure to Methylmercury Nickel Table 22.24 Toxic Effects of Nickel Selenium Table 22.25 Toxic Effects of Sodium Selenite Thallium Table 22.26 Toxic Effects of Thallium on Rats Tin Table 22.27 Toxic Effects of Trialkyltin Compounds Zinc

ABBREVIATIONS ALA δ-aminolevulinic acid ALAD δ-aminolevulinic acid dehydratase ALT alanine aminotransferase AST aspartate aminotransferase BUN blood urea nitrogen Cat catalase CNS central nervous system D6PD glucose-6-phosphate dehydrogenase DA dopamine γ-GCS γ-glutamylcysteine synthetase GFR glomerular filtration rate GPx glutathione peroxidase GSH reduced glutathione GSR glutathione reductase GSSG oxidized glutathione γ-GTP γ-glutamyltranspeptidase IgM immunoglobulin M LDH lactate dehydrogenase LPO lipid peroxide MAO monoamineoxidase MT metallothionein NE norepinephrine PAH p-aminohippurate RBC erythrocytes ZPP zinc protoporphyrin

SECTION 1. INTRODUCTION The various toxic effects caused by a number of metals have been documented in experimental animals. The specific organ toxicity closely related to the tissue distribution has been documented for each metal. Since susceptibility to metal toxicity sometimes varies significantly between sex


or strain, this information has also been cited. Here, the focus is on recent rodent data. Since the amount of data cited here may not be sufficient for some colleagues, previous publications1,2 are recommended for additional information.

SECTION 2. ARSENIC Arsenic (As) occurs in the environment in its inorganic and organic forms; the toxicity of the latter is very low. In a variety of inorganic arsenic compounds, arsenite (trivalent), arsenate (pentavalent), arsenic oxide, gallium arsenide, and arsine are mentioned in the literature. Toxic effects of arsenite and arsenate are shown in Table 22.1. The LD50 value for sodium arsenite (NaAsO2) was shown as 6.7 mg As/kg in mice after subcutaneous (sc) injection.6 Hyperglycemia and glucose intolerance were documented as acute effects of sodium arsenite in rats.3 Oral exposure to arsenite using drinking water (100 ppm) for 4 to 11 days caused decreased acetylcholine esterase and sorbitol dehydrogenase activities in axons and increased leucine amino peptidase activity in glial cells.4 Giving 0.5 to 10 ppm As-containing water for 3 weeks caused immunosuppressive effects in mice.7 Fowler and Woods8 observed mitochondria swelling and decreased monoamine oxidase activity in the liver of mice given water containing 40 or 85 ppm As as sodium arsenate for 6 weeks. Chronic exposure of rats to arsenite or arsenate in food up to 400 ppm caused reduced body weight and survival period and enlargement of common bile duct.5 When NaAsO2 was given to pregnant mice at dose levels of 40 (for oral, po) or 12 (for intraperitoneal, ip) mg As/kg, fetal death or resorption resulted.9,10 Injection of arsenate (45 mg As/kg, ip) also caused fetal resorption.11 Gallium arsenide (GaAs) is an excellent semiconductor material used in microcircuits. Intratracheal injection of GaAs up to 200 mg/kg or oral administration of up to 2000 mg/kg caused pathological changes in lung and kidney,14 inhibition of tissue δ-aminolevulinic acid dehydratase (ALAD) activities,12,14 and increased urinary elimination of porphyrin and δ-aminolevulinic acid (ALA) (Table 22.2).12-14 Acute fibrogenic responses of the lung were also documented in rats intratracheally injected with GaAs (100 mg/kg).15 Sikorski et al.16 found that female mice intratracheally treated by 50 to 200 mg GaAs/kg showed reduced IgM antibody response to sheep erythrocytes. The LD50 value for arsenic oxide (As2O3) was shown to be 11.3 mg/kg after sc injection in mice.17 Webb et al.15 reported that As2O3 caused effects similar to GaAs in rat lung. Serial oral administration of As2O3 (3 mg/kg) for 10 days resulted in increased motor activity, whereas higher dose level (10 mg/kg × 10 days) brought about the reverse result.18 Inhalation of As2O3 aerosol (270 to 940 μg As/m3) for 3 h caused decreased pulmonary bacterial activity and increased infectious mortality in mice.19 Arsine gas was documented to cause various hematological alterations in mice, such as decreased hematocrit, erythropoiesis, and hemolytic anemia.20,21

TABLE 22.1 Toxic Effects of Arsenate and Arsenite Animal Strain, Sex, (Weight)a

Chemical

Rats CD, M

NaAsO2

5 or 10

NaAsO2

100 ppm/water × 4–11 d

Wistar, M

Dose (mg As/kg or ppm As)


Route

ip

Effects

Ref.

Hyperglycemia, glucose intolerance (1.5–3 h) Axons: acetylcholine esterase, sorbitol dehydrogenase ↓ Glial cells: leucine amino peptidase ↑

3 4

TABLE 22.1 (Continued) Toxic Effects of Arsenate and Arsenite Animal Strain, Sex, (Weight)a Osborne, M + F Mice CD-1, M Swiss cross, M C57BL/6, ?, (10–15 g) CD-1, pregnant CD-1, pregnant Swiss, pregnant

Chemical

Dose (mg As/kg or ppm As)

NaAsO2 Na2HAsO4

125–400 ppm/food × 2 y 250–400 ppm/food × 2 y

NaAsO2 NaAsO2 Na2HAsO4

6.7 0.5–10 ppm/water × 3 wk 40, 85 ppm/water × 6 wk

sc

NaAsO2 NaAsO2

40 or 45 12 (G.D. 9 or 13)

po ip

Na2HAsO4

45

ip

Route

Effects

Ref.

Body weight, survival ↓; enlargement of common bile duct

5

50% mortality Immunosuppressive effect Liver: mitochondrial swelling, MAO ↑ Fetal death (max: G.D. 13 injection) Fetal death, resorption (G.D. 13, 44–51%; G.D. 9, 73–95%) Fetal abnormality, resorption (G.D. 8, injection)

6 7 8 9 10 11

a Data from adult animals, unless otherwise specified. Note: G.D. = gestation day; MAO = monoamine oxidase.

TABLE 22.2 Toxic Effects of GaAs and As2O3 Animal Strain, Sex, (Age)a

Chemical

Dose (mg GaAs or As2O3/kg)

Route

Rats ?, M

GaAs

500–2000

po

F344, M

GaAs

CD, M

GaAs

10, 30, 100 1000 100, 200

it po it

F344, M

GaAs

100

it

As2O3

17

it

GaAs

50, 100, 200

it

NMRI, M ddY, ?, (4 wk)

As2O3 As2O3

11.3 3 × 10 d 10 × 10 d

sc po po

CD-1, F, (4–5 wk)

As2O3 (aerosol)

270–940 μg As per m3 × 3 h

Mice B6C3F1, F

a

Data from adult animals, unless otherwise specified. Note: it = intratracheal.


Effects Blood: ALAD, Zn-protoporphyrin ↑ Brain, liver: ALAD ↑ Lung weight, urinary porphyrin ↑ Urinary porphyrin ↑ Blood, liver, kidney: ALAD ↓ Urine: ALA ↑ Lung, kidney: pathological change Lung: tissue weight, protein, DNA, 4-HyPro ↑; acute fibrogenic response Lung: tissue weight protein, DNA, 4-HyPro ↑; acute fibrogenic response Splenic accessory cell function ↓ IgM antibody response to sheep RBC ↓ 50% mortality (30 d) Motor activity ↑ Motor activity ↓ Brain: alteration in monoamine metabolite Infectious mortality ↑ Bactericidal activity ↓

Ref.

12 13 14

15

16

17 18

19

SECTION 3. BERYLLIUM Toxic effects of beryllium (Be) have been well investigated by the inhalation route, because most cases in humans occur via this route. The lung is the major target in beryllium inhalation (Table 22.3). Consecutive exposure for 14 days to 2.59 mg Be/m3 × 2 h as BeSO4 caused 80% mortality in male rats.22 Single (1-h) exposure to the same salt in a relatively higher dose (3.3 to 13 mg Be/m3) resulted in pathological changes23 or increase in lavage LDH and alkaline phosphatase activities.24 Inhalation of beryllium oxide (0.45 mg Be/m3 for 1 h) also caused similar effects.25 Chronic exposure to a much lower dose caused lung tumor.26 Intratracheal injection of beryllium was also effective for examination of pulmonary toxicity. Groth et al.27 showed that beryllium (metal or passivated) and its alloy with high beryllium content could induce lung neoplasms in rats 16 to 19 months after intratracheal injection (>0.3 mg Be), but alloys containing a low amount (50% mortality (7 d) Testis: hemorrhage (12 h) Decreased motor activity (8–11 d) Brain lesions, hyperactivity (4–18 d) 30% mortality (48 h) 4% mortality (48 h) Liver: lesions (10 h) No damage 10% mortality hGC-induced serum testosterone ↓ Brain: hypomyelination (5 d)

Ref.

31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

TABLE 22.4 (Continued) Acute Effects of Cadmium Toxicity Doseb (mg Cd/kg)

Animal Strain, Sex, (Age)a Wistar (d 19) Wistar, M Wistar, F SD, M SD, M

Mice Swiss-Webster, M Swiss-Webster, M CFLP, M ICR, F ICR, M C57BL/6, M, (50 d) C57BL/6, M, (7 d) C3H, F 5 strains, M CBA, M a b

Route

Effects

Ref.

11.2 μg/fetus 2.6 64 75 3.35 3.55 9.3 225

ip ip po po iv ip sc po

Hydrocephalus, brain necrosis Heart: GSH, LPO, MT ↑ 22% mortality (96 h) Liver: MT induction; GSH (4 h) ↓ LD50 (14 d) LD50 (14 d) LD50 (14 d) LD50 (14 d)

46 47 48 49 50

4 0.2 1 1.2 5 112 4.08 1.65 2.8 3.36 53

iv ip ip ip ip po ip ip sc sc po

60% mortality (14 d) Liver: MT induction Testis: pathological change (3 d) Blood: ALAD (1 d) ↑ 40% mortality (10 d) 60% mortality (10 d) LD50 (7 d) LD50 (7 d) Sheep RBC-induced immune response ↓ Liver, testis: alterations in essential metal levels 11% mortality (10 d)

51 52 53 54 55 56 57 58 59

Data from adult animals, unless otherwise specified. CdCl2 was used, unless otherwise specified.

TABLE 22.5 Effects of Cadmium on Pregnant Animals Animal Strain Rats SD Wistar

LE SD

Wistar Mice CD-1 a

Dose (mg Cd/kg or ppm Cd)

Injection Time (G.D.)a

Route

2.1 × 4 4.5

8, 10, 12, 14 18

ip sc

1.25 1.25 0.49 × 20 4.9 × 4 50, 100 ppm water × 15 d

12 12 1–20 12–15 6–20

iv ip or iv sc sc

60 ppm/ water × 20 d

1–20

5.6

10

G.D. = gestation day.


sc

Effects

Ref.

Offspring: 75% mortality (12 d) Placental necrosis (24 h) Fetal death Fetus: DNA and protein synthesis ↓ Fetus: skeletal malformation Neonatal: thymus weight and liver Zn ↓ Fetal: lung DNA and protein ↓ Fetus: body weight and liver Zn ↓ Mother: body weight, serum ALAD, and alkaline phosphatase ↓ Offspring: impaired movement

62 63 64 65 66 67 68

Embryo: malformation

70

69

B. SUBACUTE

TO

CHRONIC TOXICITIES

Continuous exposure to Cd was carried out by repeated injection or by giving Cd-contaminated food or drinking water. Long-term treatment at low levels increases the damage to other organs, including the kidney (Table 22.6). Chronic and subchronic renal damage induced by cadmium salt was recognized by the pathological changes71,72,77 or the appearance of abnormal urinary components.74,78,82 Urinary B2-microglobulin was reported to be an earlier marker than albumin.82 Abnormalities of bone tissue were also documented as chronic effects of cadmium.84,88 It should be noted that, particularly in a repeated injection experiment, Cd injection of even a sublethal dose induced metallothionein synthesis in the target organs, which lead to increased resistance to Cd (and other metal) toxicities.52,90,91 Nishimura et al.92 suggested urinary trehalase activity was a sensitive indicator for cadmium-induced chronic renal failure in rabbits. When animals were exposed to aerosol of CdO or CdCl2 or when the metal was instilled intratracheally, the lung primarily was damaged (Table 22.7). Increase in tissue weight93,96,99 and various cytosolic enzyme activities93,97 and decrease in mitochondrial enzymes94,98 have been documented. Long-term treatment caused an increase in the connective-tissue components.96 Damage to the kidney in the acute phase of cadmium salt injection is very rare. However, when cadmium was injected as Cd-metallothionein or coinjected with an SH compound such as cysteine even via an alternative route, the kidney became a primary target organ (Table 22.8). The renal damage was detected, for example, in the case of chronic Cd toxicity. Suppression of PAH uptake by the renal slice prepared from the intoxicated rats was suggested to be a sensitive marker for Cd plus cysteine-induced nephrotoxicity.109 Goering et al.106 found induction of renal stress proteins at the initial phase of renal damage in rats treated by Cd-cysteine.

TABLE 22.6 Subacute to Chronic Effects of Cadmium Animal Strain, Sex, (Weight)a Rats SD, M

Wistar, M BN, F Lewis, F ?, M, (40–50 g) SD, M Wistar, F Wistar, M SD, F Wistar, M

LE, F SD, M Mice C57BL/6, M CF1, F QS, F a

Dose (mg/kg or ppm Cd) 1.8 × 14 d 0.6 × 2–6 wk (5 d/wk) 0.025 × 6 wk (5 d/wk) 1.5 × 26 d 0.49 × 3 wk (5 d/wk) 0.49 × 3 wk (5 d/wk) 0.4 × 30 d 0.5 × 26 wk (6 d/wk) 0.4 × 13 wk (5 d/wk) 6.12 × 1–3 months 1.49 × 11 wk (5 d/wk) 3.4 × 1 100 ppm/water × 7 months 50 ppm/water × 10 months 50–100 ppm/food × 6–8 wk 50 ppm/food × 52 wk 1 ppm/water × 18 months

Route iv sc po sc sc sc ip sc sc po sc

Effects

Ref. 71 72 73 74 75 75 76 77 78 79 80 81 82 83 84

50 ppm/water × 30 d

Liver, kidney: pathological change Kidney: membrane degeneration Liver: cytochrome c-oxidase ↓ Urinary protein, AST, amino acids ↑ S-phase thymocytes ↓ G2-phase thymocytes ↑ Brain: SOD ↓; LPO ↑ Liver damage (4 wk); renal damage (8 wk) Urinary protein, HyPro, HyLys ↑ Duodenum: Ca2+ transport ↓ Intestinal mucosa: alkaline phosphatase ↓ Chronic nephropathy, testicular tumors (90 wk) Urine: transferrin, IgG, β2-M, albumin ↑ Alteration in skeletal muscle ultrastructure Bone: pathological change; lysil oxidase ↓ Bone: collagen cross-linking ↓ Hypertension (2 months) Heart, liver, kidney: ATP ↓; ADP ↑ Small intestine: hemoxygenase activity ↑

50–200 ppm/water × 3 wk 50 ppm/food × 252 d 10, 100 ppm/water × 22 wk

Proliferative response of spleen cell ↑ Femur Ca levels ↓ Brain: degenerative damage in choroid plexus

87 88 89

Data from adult animals, unless otherwise specified.


85 86

TABLE 22.7 Pulmonary Toxicity of Aerosol Inhalation or Intratracheal Injection of Cadmium Animal Strain, Sex Rats SD, M

Exposure Time (h × days)

Cd Level (mg/m3)

Chemicals

× × × × × ×

Wistar, M F344, F Lewis, M

CdO, CdCl2 CdO CdO CdCl2 CdO

0.45–4.5 4.5 5 1 1.6 8.4

CD-1, M Wistar, M

CdCl2 CdO, CdCl2

2×1

F344, F

CdCl2

0.85 0.5–10 μg Cd/rat (it) 0.1, 0.4 mg Cd/kg (it) 4.9 4.9

1×1 1×1

Mice BALB/c, F

CdCl2 CdCl2

2 0.5 3 6 3 3

1 1 1 62 20 1

Effects on Lung

Ref.

Tissue weight, GSR, GST, and G6PD ↑ Monooxygenase and cytochrome P-450 ↓ Nonprotein SH ↑ Tissue weight, elastin, collagen ↑ G6PD, GSR, Cat, and GPx ↑ Alkaline and acid phosphatase, LDH, and protein ↑ Mitochondrial enzymes ↓ Tissue weight, lavage fluid cell number ↑

93 94 95 96 97

HyPro ↑

100

Pathological change Cell proliferation ↑

101 102

98 99

TABLE 22.8 Renal Toxicities by Cadmium-Metallothionein or Cadmium Plus Thiol Co-Injection Animal Strain, Sex Rats SD, M Wistar, M

SD, M

Chemicals

Dose (mg Cd/kg) Routes

Effects

Ref.

Cd-MT Cd-MT

0.3 0.4

ip sc

Cd + Cys Cd + Cys-peptide Cd-MT Cd + Cys CD + β-ME

1.3–1.7 0.51–0.64 0.16–0.23 2 1.68

iv

Pathological changes; PHA uptake by kidney slice ↓ 103 Ca2+ uptake by luminal and batholateral membrane 104 vesicles ↓ Urinary protein, glucose, amino acids ↑ 105

iv ip

Stress protein synthesis ↑ Urinary protein, amino acids ↑

106 107

0.4–1.6 1.5

sc iv

Urinary glucose ↑ Urinary glucose, protein ↑ PHA uptake by kidney slice ↓

108 109

Mice 4 strains, M Cd-MT ICR, M Cd + Cys

SECTION 5. CHROMIUM Trivalent chromium (Cr) is essential in animals including humans, whereas hexavalent chromium, which is easily absorbed from the gastrointestinal tract, is very toxic. Hepatic and renal damages have been reported in the acute phase of chromium parenteral injection at dose levels 7.9 to 15.8 mg Cr/kg in rats (Table 22.9).110-113 Tsapakos et al.109 detected DNA-protein cross-links in liver and kidney of Cr-injected rats, suggesting relationships to carcinogenicity and toxicity of Cr(VI). In the subacute to chronic phase, reproductive tissues were also affected. Serial ip injection of 1 to 4 mg


Cr/kg as dichromate for 5 to 90 days caused pathological changes in testis cells or altered enzyme activities in rats.114-116 Vyskocil et al.117 showed that female rats manifested renal dysfunction after 6-month exposure to 25 ppm Cr (as chromate) in drinking water, but males did not. Increased DNAprotein cross-link was detected in lymphocytes and liver of rats given drinking water contaminated by 100 to 200 ppm Cr as chromate.118 Chronic inhalation experiments of Cr aerosol were reported to cause adverse effects in the lung. Glaser and co-workers119 found that although exposure to low Cr level caused activation of alveolar macrophages, high-level exposure inactivated it. They also showed that Cr5O12 aerosol was much more toxic to lung and blood cells than Na2Cr2O7 aerosol.120 If pregnant mice were exposed to K2Cr2O7 from drinking water containing 250 to 1000 ppm Cr throughout the gestation period, embryonic death and malformation of offspring occurred.121

TABLE 22.9 Toxic Effects of Hexavalent Chromium in Rats Animal Strain, Sex (Age)a Rats SD, M

a

Chemical

Dose (mg Cr/kg or ppm Cr)

Route

Wistar, M

Na2Cr2O7 Na2Cr2O7 Na2CrO4

7.9, 19.8 10.5 4.7 × 3

ip ip ip

SD, M Wistar, M

Na2Cr2O7 Na2CrO4

7.9, 15.8 1, 2, or 4 × 5

sc ip

ITRC, M, (weanling) Druckrey, M Wistar, M + F

K2Cr2O7

1–3 × 90

ip

K2Cr2O7 Na2CrO4

ip

F344, M

K2CrO4

2 × 15 25 ppm/water × 6 months 100–200 ppm/water × 3–6 wk

Effects

Ref.

Liver, kidney: DNA-protein cross-links Liver: GSH ↑ Liver microsome: Cr(VI) reductase, cytochrome P-450, cytochrome b5 ↓ Serum: BUN, lactate, glucose ↑; insulin ↓ Testis: tissue weight, epididymal sperm number ↓; pathological change Testis: pathological change; γ-GTP, LDH ↑; sorbitol dehydrogenase, G6PD ↓ Testis: pathological change in epithelial cells Female: urinary albumin, β2-M ↑ Male: no change Liver: DNA-protein cross-link

110 111 112 113 114 115 116 117 118


SECTION 6. CISPLATIN Cisplatin (CDDP; cis-diaminodichloroplatinum) is an effective anticancer drug widely used in cancer chemotherapy. However, it also acts as a nephrotoxin. The nephrotoxic actions of cisplatin have been well documented in laboratory animals (Table 22.10); 50% mortality was documented, for example, in rats (7.7 mg/kg, ip)123 and mice (9.5 to 13.4 mg/kg, sc, iv).142,143 Cisplatin-induced renal damage was detected in the form of increased BUN and serum creatinine levels, pathological change or decreased enzyme activities in the kidney, and alteration in urine constituents. Urinalysis was suggested to be a more sensitive method.124 De Witt et al.126 demonstrated that increase in the Ca2+ pump activity of the renal endoplasmic reticulum (ER) was the earlier marker, followed by renal failure in the cisplatin-treated rat. Litterst125 showed that a high salt concentration of the vehicle markedly lowered the toxic effects of cisplatin in rat. Cisplatin affects neural tissue, not only through its nephrotoxic action but also through glucose metabolism, testes, blood cells, and embryo. Neurotoxic action could be detected by pathological change,132,133 electrophysiological methods134 and abnormal behavior.147 Goldstein and coworkers135,136 demonstrated the increased plasma glucagon half-life and impaired glucose tolerance,


which possibly lead to renal failure. Effects on rat testes were observed as decreased plasma testosterone levels137 and functional and morphological alterations of Sertoli cells.138 Reduction in reticulocytes was documented in cisplatin-treated mice.145,146 Aggarwal and co-worker140,141 found that embryonic resorption or death occurred in cisplatin-treated rats and mice. They suggested that the cisplatin-induced decreases of sex hormone levels are responsible for the embryonic toxicity.

TABLE 22.10 Toxic Effects of CDDP Animal Strain, Sex, (Age)a Rats F344, M

Dose (mg CDDP/kg)

Route

6, 25 0.5–12 2.5–15 9 5, 7.5

ip ip iv ip ip

SD, ? Wistar, M

7 5 5 5

iv ip iv ip

BN, M

3 × 3 (21-d interval)

ip

Wistar, (10 d) Wistar, F

5 2 × 9 (1 or 2/wk)

sc ip

Wistar, M SD, M

1 × 15 or 34 (2/wk) 15 5 5 9 2×5

ip ip iv iv iv ip

F344, M

5

iv

SD, M

F344, M

Wistar, pregnant

Mice BDF1, ? Swiss, M + F Swiss, M B6D2F1, M B6C3F1, M + F B6D2F1, M CD1, M Swiss, pregnant

ip 4, 7 (on G.D. 6)

ip

9.5

sc iv ip iv

18, 20 5 × 2 months (1 d/wk) 15.5 6.5 10 5.24


iv iv ip ip

Effects GFR ↓; jejunal crypt cell survival (3–5 d) ↓ LD50: 7.7 mg/kg; renal damage: BUN ↑; histology Renal damage: urine analysis as sensitive method Decreased toxicity (lethality) by high-salt vehicle Renal endoplasmic reticulum: Ca2+ pump activity, Ca2+ content (4–24 h) ↑ Renal cytochrome P-450, b5, γ-GCS, γ-GTP (7 d) ↓ Defect in papillary hypertonicity; damage at S3 segment Creatinine clearance ↓ Kidney, liver: LPO ↑ GPx, GST, Cat ↓; kidney SOD ↓ Kidney: cytochrome P-450, GSR, GST, GPx, GSH ↓; N-glucuronyl transferase, GSSG, LPO ↑ Brain: abnormal shape of the dendritic tree (24 h) Pathological change in spinal ganglia neuron and sciatic and peroneal nerve Sensory nerve condition velocities (48 h) ↓ Cisplatin-DNA binding in DRG satellite cells (6 h) Plasma glucagon half-life ↑; renal failure (96 h) Impaired glucose tolerance (48 h) Plasma testosterone, testis cytochrome P-450 (72 h) ↓ Leakage of the Sertoli cell tight junction (24 h); abnormal Sertoli cell secretory function Intestinal epithelium (ileum): pathological change (24 h) Embryonic LD 50: G.D. 6, 2.88 mg/kg; G.D. 8, 1.28 mg/kg; G.D. 11, 1.0 mg/kg Serum: LH, progesterone, 20 α-hydroxysteroid dehydrogenase ↓; embryonal resorption Plasma: BUN, CRT ↑; 50% mortality (30 d) LD50: M, 13.4 mg/kg; F, 12.32 mg/kg Decreased nephrotoxicity in high NaCl vehicle Lesion in renal cortical tubules and bone marrow; circadian rhythm affected Reticulocytes ↓ Immature WBC, PMN ↑; immature RBC ↓ Tail flick temperature and distal sensory latency ↑ Embryonic LD50 (day 8)

Ref.

122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141

142 143 125 144 145 146 147 140

TABLE 22.10 Toxic Effects of CDDP Animal Strain, Sex, (Age)a

Dose (mg CDDP/kg)

Route

Effects

Ref.

a

Data from adult animals, unless otherwise specified. Note: G.D. = gestation day.

SECTION 7. COBALT Cobalt (Co) is an essential component of vitamin B12. Its toxicity is summarized in Table 22.11. The increase of lysozyme level in lavage fluid was documented in rabbits exposed to CoCl2 aerosol (0.5 mg Co/m3 × 6 h/day) for a month.156 Increased tissue weight and inflammatory response of the rat lung were documented in rat after intratracheal injection (10 mg Co/kg) of metallic Co dust.148 Chronic exposure experiments using Co-contaminated food or water revealed testicular atrophy and reduced behavioral activity in rats150 and abnormal testis morphology in mice.154 Di Giulio et al.149 indicated that consecutive ip injection of CoCl2 (10 mg Co/kg) for 42 days caused increased hematocrit and hypertrophy of glomus cells in the rat carotid body. CoCl2 treatment (10 to 20 mg Co/mg × 2, sc) caused a significant reduction in the hepatic P-450 levels of rats in the acute phase,151 an effect much more prominent in Co-protoporphyrin-treated rats153 and hamsters.155 Since serial injection of 100 mg Co/kg as CoCl2 in pregnant rats for 10 days caused no damage to the fetus,152 embryo toxicity of CoCl2 was considered to be very low.

TABLE 22.11 Toxic Effects of Cobalt in Experimental Animals Animal Strain, Sexa Rats SD, F

Dose (mg/kg or ppm Co)

Compound

Route

Co (metal dust)

10

it

SD, pregnant

CoCl2 CoCl2 CoCl2 CoCl2

ip Food sc po

SD, M

Co-protoporphyrin

10 × 42 20 × 69 10–20 × 2 100 × 10 (G.D. 6–15) 0.88–3.5

CD-1, M

CoCl2

400 ppm/water × 13 wk

Co-protoporphyrin

5.3

SD, M

Hamsters Syrian, M a

Data from adult animals, unless otherwise specified. Note: G.D. = gestation day.


sc

sc

Effects

Ref.

Lung: moderate inflammatory response, weight ↑ Glomus cell hypertrophy; hematocrit ↑ Testicular atrophy; slower lever press Liver: cytochrome P-450 (24 h) ↓ No fetotoxicity

148 149 150 151 152

Liver: cytochrome P-450, cytochrome b5, NADPH-P-450 reductase ↓ Pathological change in testis cells

153

Liver: cytochrome P-450 ↓

155

154

SECTION 8. COPPER Although copper (Cu) is an essential metal, its excessive intake has been shown to have a variety of toxic effects. Metabolism and pulmonary toxicity of intratracheally instilled CuSO4 or CuO have been reported in rat at dose levels of 2.5 to 50 μg Cu/rat.157, 158 The biochemical and elemental inflammatory indices in bronchoalveolar lavage fluid reached maximum values at 12 to 72 h after instillation of 5 μg Cu/rat. Toxic effects on liver or kidney have been reported in rats fed a diet containing 1500 ppm Cu as CuSO4 for 15 or 16 weeks, respectively.159,160 Hemolytic anemia has also been documented in rats after consecutive ip injection of Cu-nitrilotriacetate at a dose level of 4 to 7 mg Cu/kg.161 Mascular mutant mouse, which has been proposed as an animal model of Menke’s Kinky-hair disease, was reported to be sensitive to the acute hepatotoxic effects of Cu as compared with normal mice.162 Clastogenic effects of Cu on the bone marrow chromosomes were shown in mice injected ip with CuSO4 (1.1 to 6.6 mg/kg).163

SECTION 9. IRON Despite its abundance and necessity in almost all living organisms, excess iron (Fe) causes various toxic effects if accumulated in human and animal tissues. The effects of iron overload in experimental animals vary among species, strains, and sex (Table 22.12). Since the liver is the major storage organ of the excess iron, hepatotoxicity is the most common finding in animals undergoing iron overload experiments. Hepatic fibrosis was induced in rats by feeding them an iron carbonylcontaminated diet for as long as 8 months.168,169 On the other hand, single dosing of iron dextran caused hepatic fibrosis in gerbils,175 which were suggested to be a sensitive model for induction of hepatic fibrosis. Cardiotoxicity is the other major effect of iron overload observed in humans, but no experimental model has been reported in mice or rats. Recently, Garthew et al.174 demonstrated that repeated sc injection of gerbils with iron dextran resulted in hemochromatosis with a heart pathology similar to human cases. Since iron has a catalytic action in reactive oxygen generation in vivo, a considerable part of its toxicity may be the oxidative damage caused. Several studies demonstrated elevated lipid peroxidation in the tissue of iron-overloaded animals.164,165,170,171 The catalytic action of iron was also suggested to be involved in the carcinogenic action of polyhalogenated aromatic hydrocarbons.176

SECTION 10. LEAD Lead (Pb) may cause various adverse effects in experimental animals in both acute and chronic phases. Toxic effects of inorganic lead as related to hematopoietic, nervous, gastrointestinal, and renal systems, whereas those of organic lead are largely related to the nervous system. Although rats are the most frequently used animals in studies of metal toxicity, adult rats are relatively insensitive to lead toxicity,177 whereas perinatal animals are very sensitive. Accordingly, numerous experimental studies have been carried out using young animals.

A. INORGANIC LEAD TOXICITY There are marked differences in LD50 values for lead toxicity among species, sex, and age (Table 22.13).178-181 Acute lead encephalopathy occurs easily in young animals but only rarely in adults.184,188 Kumar and Desiraju184 reported that about 20% of rat pups orally administered lead acetate (0.4 g Pb/pup) for 10 to 11 days developed hind limb paralysis and died within 24 h. The characteristic disturbances in the central nervous system (CNS) functions during chronic lead exposure (lead encephalopathy) have stimulated numerous behavioral, pathological, and neurochemical investigations (Table 22.14). CNS disturbance seems more apt to occur during devel-


TABLE 22.12 Toxic Effects of Iron Overload in Experimental Animals Dose (mg/kg or ppm Fe)

Animal Strain, Sex (Age)a

Fe-compound

Rats Wistar, M

Dextran

500 mg/kg

ip

Dextran

116 mg Fe per kg × 3 d

ip

Wistar, F

Carbonyl

25,000 ppm/food × 10 wk

Wistar, F, (4 wk) SD, M

Saccharate

5 mg Fe per kg × 12 wk

Carbonyl

20,000 ppm/food × 4–15 months 25,000 ppm/food × 12 months 2 mg Fe per kg × 14 wk 25,000 ppm Fe per food × 28–44 d 305 ppm Fe per food × 10 wk

Carbonyl NTA Carbonyl Sulfate

Route

ip

ip

Mice SWR, M A/J, M + F

Dextran NTA

600 mg Fe per kg 1.8–2.7 mg Fe per kg × 12 wk (6 d/wk)

sc ip

Gerbil Mongolian

Dextran

1000 mg Fe per kg × 7 wk (1 d/wk)

sc

a

Effects

Ref.

Liver: chemiluminescence, LPO ↑; cytochrome P-450, SOD, Cat ↓ Liver: dimethylhydrazine demethylase, UDP Proliferative activity after a mitogenic stimulus ↓ Mesothelioma

164 165 166 167

Hemochromatosis

168

Hemochromatosis, hepatic fibrosis Liver mitochondria: LPO ↑ Liver mitochondria and microsome: LPO ↑ Liver: LPO ↑; non-Se-GPx ↑

169 170

Porphyria (25 wk) Nephrotoxicity, renal carcinoma

172 173

Liver, heart: hemosiderosis, hemochromatosis (12 wk)

171

174


opment,186,189 possibly due to high lead absorbability193,194 and high susceptibility.195 In the last decade, an increasing amount of evidence has accumulated to show that lead exposure, particularly in the perinatal period, disrupts the development of opioid peptide systems in the rat brain.196 Disturbance of the peripheral nerve has been documented by histopathological183 and electrophysiological methods.197 In the chronic phase, disturbances in hematological, nervous, and renal systems have been documented as lead toxicity in animals. Anemia is a common chronic systemic effect of lead, which is considered to be caused by a combined effect of the inhibition of hemoglobin (Hb) synthesis and shortened life span of circulating erythrocytes (RBC). Lead has been shown to interfere with heme biosynthesis even at a low level of exposure.209 Inhibition of the heme biosynthetic enzyme δ-aminolevulinic acid dehydratase (ALAD) and elevation of free RBC and zinc protoporphyrin (ZPP) are the earliest effects, followed by increase in urinary δ-aminolevulinic acid (ALA) and coprotoporphyrin, and fall in Hb and hematocrit (Ht) level (Table 22.15). ALAD activity in RBC is shown to be the most sensitive indicator of lead exposure. Maes and Gerber210 reported that when


rats were severely intoxicated, marked shortening of RBC survival led to increased ALAD activity in circulating erythrocytes. Kidney is also a target organ of chronic lead toxicity. A number of transient effects on renal function in experimental animals are consistent with pathological findings of reversible lesions.211-214 Irreversible lesions such as interstitial fibrosis have also been documented in animals following long-term lead exposure.214,215 Studies during the last decade have shown that chronic and low-level lead exposure might induce subtle alterations in the immune systems of experimental animals. Enhanced host susceptibility to bacteria and viral infections216-218 and increased growth and metastasis of implanted tumors219,220 have also been reported. Other studies demonstrated the ability of lead to reduce the number of antibody-forming cells,221 to suppress antibody synthesis,222,223 and to diminish the phagocytic function of the reticuloendothelial systems.223 Lead has also been shown to induce perivascular edema,183 teratogenicity,224 and testicular toxicity225 in experimental animals.

TABLE 22.13 The LD50 of Lead Salts for Rats and Mice after Intraperitoneal Dose Animal Strain Rats ?

SD Mice DBA/2 C57BL/6 Swiss-Webster ICR

Sex

M F M F M F

Age or Weight

3 3 18 18 20 20

M M M M F

wk wk wk wk wk wk

Salt

LD50 (mg/kg)

Days

Ref.

Acetate

225 231 170 258 172 280

8 8 8 8 8 8

178

74 102 148 278 280

10 10 10 8 8

180

Acetate

60–70 d

Nitrate

26 g 23 g

Acetate

179

181

B. ORGANIC LEAD TOXICITY Triethyllead (TEL) and other organic lead compounds have been shown to affect preferentially the nervous system.226 Degenerative changes have been observed in the cerebral cortex, cerebellum, and hippocampus of rabbits receiving lethal or near-lethal doses of tetraethyl lead.227,228 Rats subjected to both acute and short-term repeated TEL exposure experienced alterations in reactivity, locomotor activity, and avoidance learning.229,230 Moreover, organic lead has been shown to have several biochemical effects, including alteration of enkephalin levels,231 dopaminergic processes,232 and enhanced lipid peroxidation233 in brain.

SECTION 11. MANGANESE Manganese (Mn) is an essential metal, considered to have low toxicity. The chronic and subchronic effects on the neural tissues have been widely investigated (Table 22.16). Serial ip injection of 4 mg Mn/kg for 30 days caused reduced LPO236 or increased norepinephrine (NE) levels237 in rats. Exposure via drinking water containing 1000 ppm Mn for 14 days effected increases in brain dopamine (DA) and NE levels and activated behavior.243 A 30-day exposure to the same level increased turnover rates of DA and NE.242 Bonilla and Prasad244 have shown significant decreases of the biogenic amines in several regions of the brain of rats given 100 or 1000 ppm Mn-containing Copyright © 2002 by Taylor & Francis

TABLE 22.14 Neurobehavioral Effects of Lead Animal Strain, Sex Rabbits NZ, M + F Rats Wistar, M

Age or Weight

Salt

Dose

Route

Effects

Ref.

Newborn

Nitrate

4.5–18 mg/pup × 30 d

po

Pathological changes in CNS

182

400 g

Carbonate

1000 mg/kg × 600 d

po

183

Wistar, M + F

Newborn

Acetate

400 mg Pb per kg × 60 d

po

Wistar

Newborn

Acetate

po

Wistar, M SD, M

Perinatal Newborn

Acetate Nitrate

45–180 mg Pb per kg × 19 d 750 ppm/food × 17 d 10 mg/kg × 15 d

Histopathological changes in CNS Hind limb paralysis, brain edema and hemorrhages, biogenic amines and GABA/glutamate system changes Behavioral changes

Maternal ip

186 187

LE, M + F

Newborn

Acetate

600 mg/kg × 10 or 30 d

po

ITRC, M

Newborn 220–240 g

Acetate Acetate

10–90 mg/kg × 19 d 5–12 mg/kg × 14 d

po ip

Behavioral changes Histopathological changes in CNS Alteration in cerebellum development Behavioral changes Behavioral changes and alteration in biogenic amine levels

60 d, 1 year

Acetate

Behavioral changes

191

Perinatal

Acetate

5000 ppm/water × 7 wk 2500 ppm/water

Behavioral changes

192

Mice HET, M BK/W, M + F

Maternal

184

185

188 189 190

water for 8 months. Komura and Sakamoto250 have reported that effects of several Mn compounds (chloride, acetate, carbonate, and dioxide) on the brain biogenic amine levels in mice were different, within the highest toxicity shown by MnO2. In addition to its neurotoxic effects, MnCl2 was also documented to cause a pancreatitis-like reaction,245 hepatic damage,247 and reduced antibody production249 in mice or rats. Rogers et al.248 observed increases in the splenic natural killer cell activity and plasma interferon levels in three strains of mice. Methylcyclopentadienyl manganese tricarbonyl (MMT) is used as an octane enhancer in unleaded gasoline. LD50 values of MMT after po and ip injections were determined in rats to be 50 mg/kg254 and 12.1 to 23 mg/kg,251,254 respectively (Table 22.17). Fishman et al.256 showed the lethal effect of MMT was more potent in propyrene glycol vehicle than in corn oil vehicle. MMT affected lung tissue of experimental animals was detected as pathological change253,254 and increased lavage protein levels.252,255

SECTION 12. MERCURY In the terms of its toxicological properties, mercury (Hg) can be classified into metallic mercury, inorganic mercuric salt, and organic mercury. Among various inorganic and organic Hg compounds, the focus here is on the toxic actions of mercuric chloride (HgCl2) and methylmercury (MeHg) for the latter two mercurial species.


TABLE 22.15 Hematopoietic Effects of Lead Animal Strain, Sex Rabbits NZ, F

M+F F M+F M

?, ?

?, M Mice ddy, M + F NMRI, M

DBA/2, M C57BL/6, M Swiss-Webster, M

Salt

Dose

Route

Acetate

0.2 mg/kg × 48 d (3 d/wk)

90 d Newborn

Subacetate Nitrate

0.8–1.2 mg/kg × 97–181 d (3 d/wk) 5000 ppm/food × 90–120 d 4.5 mg/pup × 20 d

Perinatal

Acetate

2500 ppm Pb/water × 7 wk

Maternal

Newborn Pregnant Prenatal 180–250 g 180–200 g 160–180 g

Acetate Acetate

20,000 ppm/food × 20–22 d 500 ppm Pb/water × 3 wk

Maternal

Acetate Acetate Acetate

Newborn

Acetate

500 ppm Pb/water × 30 d 8 mg PB per kg × 7 d 550 ppm Pb/water × 1–4 months 10,000 ppm/water × 40–60 d

Maternal

40–60 d

Acetate

2 mg Pb per kg × 3

ip

150–170 d

Acetate

10 mg Pb per kg × 4 wk

po

30–40 g 20–25 g

Acetate Acetate

Acetate

500 ppm Pb/water × 30 d 500–5000 ppm/water × 30 d or 500–2500 ppm/water × 90 d 0.1 mg/kg

Carbonate

4000 ppm/food × 12 d

2.5–2.8 kg

NZ, M NZ, M + F Rats SD, M + F Wistar, Wistar, Wistar, Wistar, CF, M ?, M

Age or Weight

60–70 d

sc

Maternal ip

Effects RBC: ALAD ↓; urinary ZPP ↑ RBC: ALAD ↓; urinary ZPP and ALA ↑ Hb ↓; nephropathy Ht ↓

198

199

RBC, kidney: ALAD ↓; Ht ↓; 200 urinary ALA ↑ Brain, liver: ALAD ↓ 201 RBC: ALAD ↓ 202 RBC: ALAD ↓; Hb ↓; Ht ↓ Urinary ALA and CP ↑ 203 RBC: ALAD ↓ 204 RBC: ALAD ↓; ZPP ↑; 205 urinary ALA ↑ ALAD: RBC, liver, kidney 206 spleen ↓ ALAS: spleen ↑; liver ↓ ALA: brain, spleen, kidney, urine ↑ ALAD: RBC, liver, kidney, spleen ↓ ALAS: liver, spleen ↑; kidney ↓ ALA: spleen, kidney, urine ↑ RBC: ALAD ↓; ZPP ↑; 207 urinary ALA ↑ Urinary ALA ↑ RBC, liver, brain, bone marrow: ALAD ↓

iv

Ref.

RBC, liver, brain, bone marrow: ALAD ↓ Liver: ALAD ↓

203 208

180

Liver: ALAD ↓; Ht ↓

Exposure to metallic mercury can take place by inhalation of Hg vapor. Since metallic mercury easily penetrates the blood–brain barrier, exposure to low-dose levels ( C57BL/6 Antibody production ↓ Alteration in brain biogenic amines Toxicity: MnO 2 > MnCl2

249 250


TABLE 22.17 Toxic Effects of Methylcyclopentadienyl Manganese Tricarbonyl (MMT) Animals Strain, Sex Rats SD, M S/A, F SD, M

Dose (mg MMT/kg)

6–37.4 4 5

0.5–2.5 Mice CD-1, M BALB/c, F Hamster Syrian, F

120 180


Route

Effects

Ref.

ip sc ip po ip sc

LD50: 12.1 mg/kg (24 h) Lung: lavage protein (24 h) ↑ Lung cell damage LD50 (14 d): 50 mg/kg; pulmonary hemorrhagic edema LD50 (14 d): 23 mg/kg Bronchoalveolar lavage protein ↑

251 252 253 254

ip

LD50 (2 h): 152 mg/kg (propyrene glycol vehicle), 999 mg/kg (corn oil vehicle) Lung cell damage Lung cell damage

256

ip ip

255

253 253

22.18). The toxic effects could be detected by pathological change, enzyme activities and lipid peroxidation in the kidney, and alterations in urinary components. However, because of its poor absorbability by the gastrointestinal tract, the effective dose of this Hg species by oral administration is higher than by parenteral injection. Mortality within 7 days was documented in rats and mice with dose levels of 3.6 mg/kg or above via the sc or iv route.261,274 It should be noticed that young rats hardly show nephrotoxic symptoms even after injection of dose levels toxic for adult rats.261 Interestingly, HgCl2, particularly at low dose, increases the renal GSH level.275 Bernard et al.272 documented an increase in urinary albumin and beta-2-microglobulin in newborn rats by daily injection of dams during pregnancy with HgCl2 (0.74 mg Hg/kg). Holt and Webb271 found that variation of the LD50 value for a pregnant rat during the pregnancy was rather small (1.00 to 1.18 mg/kg) despite a drastic increase of body weight. In animals (sub)chronically treated by HgCl2, the toxic effects were observed in other tissues (Table 22.19). Abnormalities of the epididymis,277 heart,279 and, in the case of young animals, the brain278 have been documented in rats. In mice, disturbance of the immune system was reported.281,282 Among various organic Hg, toxicity of MeHg has been well investigated because of its natural occurrence and the history of Minamata disease. Acute and subacute toxic effects of MeHg after single and multiple injections were summarized in Tables 22.20 and 22.21. Although the LD50 values were reported to be around 10 mg Hg/kg (ip) in rats283 and mice291 after single injection, its variation should be considered between sexes and strains. Female mice showed higher resistance to MeHg acute toxicity than did males.276,290 However, after serial injection of a sublethal dose, male C57BL/6 strain mice survived much longer than females.306 Acute and subacute effects were observed not only in brain but also in liver and kidney. In the liver induction of protein synthesis,284 mild glycogen accumulation,304 and morphological changes295 were observed. Nephrotoxic actions of MeHg in the acute phase were also documented,276,288,301 although histochemical abnormality was very slight.276 A significant decrease in serum albumin in mice was documented 24 h after 16 mg Hg/kg dosing.307 It should be noted that, like HgCl2, MeHg could also induce GSH synthesis in the kidney of rats and mice in the acute and subacute phases.292,311 MeHg exposure using contaminated food or water is a useful method particularly for a longterm exposure experiment. Table 22.22 summarized the toxic effects of MeHg from food and drinking water. Similar effects in the form of abnormalities in neural tissue and kidney were often observed as in repeated injection studies. Woods et al.310 suggested urinary porphyrin might be a biomarker for renal damage induced by MeHg. From life-long exposure to MeHg, males manifested neurotoxic symptoms earlier than females in mice and rats.312,316 MeHg induced congenital abnormalities through prenatal exposure (Table 22.23). Effects could be documented as fetal death,317,323 renal failure319 and neural disorders detected by behavioral320 and pathological methods.318,321,322 It should be noted that the susceptibility of the fetus to MeHg toxicity varied with the time of exposure during the gestation period. For example, the induction rate of hydrocephalus in offspring of B10D2 strain mice was highest by injection on gestation day 15.322 Similar variations were seen with other metals. Inouye and Kajiwara324 observed abnormal morphology in the fetal brain of guinea pig, whose gestation period was much longer than in rats or mice, after a single injection of MeHg (7.5 mg Hg/animal) during pregnancy.

TABLE 22.18 Acute Effects of Mercuric Chloride Animal Strain, Sex (Age)a Rats SD, M + F, (1, 29 d)

Dose (mg Hg/kg)

3.7


Route

sc

Effects

29 d: 20% mortality; kidney damage 1 d: no effect

Ref.

261

TABLE 22.18 (Continued) Acute Effects of Mercuric Chloride Animal Strain, Sex (Age)a SD, M SD, F SD, M Wistar, M

Wistar, pregnant SD, pregnant Mice ?, M ICR, M NMRI, F C57BL/6, M + F

Dose (mg Hg/kg)

Route

0.37 3 11 1.1 1×2 1.5, 3 3 0.5, 1 4.4 0.79 (G.D. 8 or 16)

ip sc sc ip ip sc sc ip sc iv

1

sc

0.5 3.6 5–40

ip iv po

2, 4

iv

Effects

Ref.

Urine: glucose, maltase (24 h) ↑ Kidney damage: pathological (30 min), functional (6 h) Renal cortex amino acids ↓ Kidney mitochondria: GSH ↓; H2O2 formation, LPO ↑ Urine: Ca2+, Mg2+, MT ↑ Urine: alkaline phosphatase, LDH ↑ Kidney: LPO ↑; vitamin C, E ↓ Urine: alkaline phosphatase ↑; pathological change 100% mortality (48 h) Placental transport activity ↓ LD50 (G.D. 8–19): 1.00–1.18 mg/kg Dam and newborn: urinary β2-M, albumin ↑

262 263 264 265 266 267 268 269 270 271

Kidney: ribosome disaggregation Urine: NAG, LDH ↑; 100% mortality (7 d) Renal GSH, GPx, protein: dose-dependent alteration; necrosis (>20 mg/kg) Urinary PSP excretion ↓; pathological change

273 274 275

272

276

a Data from adult animals, unless otherwise specified. Note: G.D. = gestation day.

SECTION 13. NICKEL Nickel (Ni) is considered to be an essential element in several animal species. Inhalation of nickel compounds causes lesions in the lung. Dunnick et al.340 demonstrated that the lung toxicity and lethality in rats and mice of aerosols containing nickel sulfate and subsulfide depended on the solubilities of the salts. Parenteral injection of nickel salts affected various tissues in experimental animals (Table 22.24). Intraperitoneal injection of 6.75 mg Ni/kg as acetate to adult rats caused 57% mortality during the 14 days after injection.329 Hogan338 found LD50 values were higher in weanling mice than in adults. The acute effects of nickel toxicity proved to be increased tissue hemoxygenase activities,325 hyperglycemia, 329,333 hepatic dysfunction,331,335 and decreased natural killer cell activity.328,337 The incidence of sarcoma was documented in nickel subsulfide-treated rats.327 Nickel caused adverse effects also in embryos. LD50 values in rat embryos varied with the time of injection during the gestation period.326 Smith et al.334 reported that the lowest observed adverse effect level for pups was 10 ppm Ni in drinking water, which caused a significant number of embryo deaths in the second of two successive gestations.


TABLE 22.19 Subchronic to Chronic Effects of Mercuric Chloride Animal Strain, Sex (Age)a Rat Charles Foster, M Wistar, (2 d) SD, M Mice Swiss, F STL/N, F B6C3F1, M

a

Dose (mg/kg or ppm Hg)

Route

0.037, 0.074 × 30

ip

3 × 59 50 ppm/water × 320 d

po

6 × 10 1.8 or 3.7 ppm/water × 10 wk 3, 15, or 75 ppm/water × 7 wk

po

Effects

Ref.

Morphological change at epididymal epithelium; sperm count ↓ Brain: NE, DA ↑; acetylcholine esterase ↓ Alteration in cardiovascular response to epinephrine and NE

277 278 279

Renal UDP-glucuronyltransferase ↑ Autoimmunity, immune-complex disease

280 281

Bone marrow, thymus, and spleen: sugar metabolizing enzymes ↓

282


TABLE 22.20 Toxic Effects by Single Methylmercury Injection Animal Strain, Sex (Age)a Rat SD, M Wistar, M SD, ?, (10–20 d) CD, M Mice C57BL/6, M Swiss OF1, M BALB/c, M + F, (2 d) RF, M + F ICR, M + F Swiss Webster, M C57BL/6, F C57BL/6, M

a

Dose (mg Hg/kg)

Route

Effects

Ref.

9.5 8–40 8 8 4.65

ip sc ip ip ip

LD50 (24 h) Protein synthesis, RNA polymerase: liver ↑; brain ↓ Cerebellum: granule cell swelling (1–3 d) Brain: t-RNA amino acylation ↓ Cerebellum: reactive oxygen species ↑

283 284 285 286 287

0.93 74 4 30

ip po po po

287 288 289 290

10.8 32 40 16 8

ip po po po po

Cerebellum: reactive oxygen species ↑ Damage at renal proximal tubules Cerebellum: mitotic arrest; cell number ↓ Mortality (10 d): M > 70%; F 60% Mortality (10 d): M > 70%; F 3% LD50 (7 d) Kidney: γ-GCS activity, GSH ↑ 33% mortality (7 d) 67% mortality (7 d) Serum albumin (24 h) ↓

291 292 276 307


SECTION 14. SELENIUM Selenium (Se) is one of the essential trace elements, and its deficiency has widely been documented in human and experimental animals. However, excess selenium also causes various adverse effects (Table 22.25). Mortality from selenium overload has been documented in mice and rats. Jacobs and Forst343 showed that giving water containing 16 ppm Se (as Na2SeO3) to rats for 35 days caused a lethal effect of up to 80% mortality. They also found that susceptibility varied with sex and age. Copyright © 2002 by Taylor & Francis

TABLE 22.21 Toxic Effects of Multiple Methylmercury Injection Animal Strain, Sex (Age)a Rats Wistar, M SD, M Wistar, M + F LE, M SD, M Wistar, F SD, F SD, M Wistar, M SD, ?, (5 d) SD, M Mice ICR, F C57BL/6, M + F BALB/c, M + F a

Dose (mg Hg/kg)

8 8 8 8

× × × ×

Route

3 3 4 5

sc sc sc po

6.4 × 5 6.4 × 6 8×7 9.3 × 7 0.8 × 20 8 × 20 5 × 10–27 0.8 × 8

sc po sc po ip sc sc sc

10 × 5 4 × 49

sc po

Effects

Ref.

MT induction in liver and kidney Inflammation in kidney; urinary NE, DA (90 d) ↓ Ultrastructure change in liver Damage at cerebellar glandular layer and dorsal root ganglion (M < F, 10–12 d) Impaired auditory function (6 –7 wk) Brain: general blood flow (at silent phase) ↓ Sciatic nerve: phosphorylation of specific proteins (15 d) ↓ 14Leu incorporation into cerebellar slice ↓ Kidney: lysosome and mitochondria dysfunction Kidney, serum: LPO (2 d) ↑ Neurological symptoms (23 d) Liver: glycogen accumulation, SER proliferation (1–2 wk); pathological change (11 wk)

293 294 295 296

Brain: protein kinase C ↓ 50% mortality: C57BL/6 M, 45 d; F, 21 d 50% mortality: BALB/c M, 15 d; F, 17 d

297 298 299 300 301 302 303 304

305 306


TABLE 22.22 Effects of Methylmercury Exposure From Food or Drinking Water Animal Strain, Sex (Age)a Rats SD (offspring) Wistar, M F344, M SD, M + F

Mice CBA, M BALB/c, F

Swiss cross, M B6C3F1, M + F a

Hg Level × Duration (ppm Hg) 3.9/food before mating, → gestation, → lactation, → 50 d postpartum 16/water × 95 d (2-d intervals) 4.3/water × 2 wk, or 8.6/water × 1 wk 4.3, 8.6/water × 4 wk 8/food × 130 wk 1.6, 8/food × 130 wk

8–32/water × 2 wk 10/water × 60–71 d 20/water × 20–75 d 40/water × 7 d 0.5–10/water × 3 wk 8/food × 104 wk



Effect

Ref.

Cerebellar NE (50 d) ↑

308

Ataxia, Hg staining at CNS Urinary porphyrin (biomarker for renal damage) ↑ Kidney: GSH, γ-GCS ↑ Ataxia (M, F); renal failure (M) Pathological changes in spinal ganglion, spinal dorsal root, proximal tubules

309 310 311 312 313

LPO in liver, kidney, brain ↑ Ataxia

314 315

Immunosuppressive effects Neurotoxic signs, chronic nephropathy (M > F)

7 316

TABLE 22.23 Effect of Prenatal Exposure to Methylmercury Dose (mg/kg or ppm Hg)

Animal Strain Rats Wistar SD SD

Injection Time

Route

8 6.4 2.4 or 4.8 × 3

18 15 8, 10, 12

ip po ip

0.01 or 0.05 × 4

6–9

po

3.2 × 3

14–16

ip

B10D2, C57BL/10, DBA/2

8

15

po

IVCS

3.2 or 6.4 ppm/food

–30–18

7.5/animal

21, 28, 35, 42, or 49

Wistar Mice C57BL/6

Guinea pigs Hartley

po

Effect

Ref.

Fetal death (4 h) DA receptor density (14 d) ↑ Urine: γ-GTP, alkaline phosphatase, NAG (3 or 6 d) ↑ Behavioral performance deficits (4 months)

317 318 319

Abnormal neuronal migration at cortical layers II and III in newborn Hydrocephalus in newborn: B10D2, 88%; C57BL/10, 54%; DBA/2, 0% Litter size ↓; resorption, dead embryo ↑ Abnormal morphology in fetal brain

320

321

322

323

324

TABLE 22.24 Toxic Effects of Nickel Animal Strain, Sex (Age)a Rats F344, M

Dose (mg/kg or ppm Ni)

Compound

Route

NiCl2

15

sc

F344, pregnant

NiCl2

16

im

F344, M F344, M + F F344, M

Ni3S2 NiCl2 Ni(OAc)2 NiCl2

0.88 10–20 6.75 5.6 3.6–29

im im ip ip sc

Ni(OAc)2

6.28

ip

Ni(His)2

1.2

iv


Effects

Kidney, liver, lung, brain: hemeoxygenase (17 h) ↑ Fetal death: 18.5–20% (G.D. 8 and 18) LD50 (dam, 14 d): 22 mg Ni/kg (G.D. 8); 16 mg Ni/kg (G.D. 18) Sarcoma in 77% of rats (2 years) NK-cell activity (24 h) ↓ 57% mortality (14 d) Hyperglycemia, renal cytochrome P-450 ↓ Alveolar macrophage: cAMP ↑; 5′nucleochidase (1–4 h) ↓; LPO (72 h) ↑ Liver, kidney: LPO ↑; Cat, GPx, GSH, GSR (3 h) ↓ Serum: ALT, AST ↑ Oxidative damage at DNA

Ref.

325 326

327 328 329 330 331 332

TABLE 22.24 (Continued) Toxic Effects of Nickel Animal Strain, Sex (Age)a

Compound

Route

Ref.

Plasma: glucagon ↑; insulin ↓; hyperglycemia (1–4 h) Fetal death

333

NiCl2

4, 6

LE, pregnant

NiCl2

10–250 ppm/water

NiCl2 Ni (OAc)2

15 10

sc ip

Kidney: hemeoxygenase (17 h) ↑ Liver: Cat, GSH, SOD, GST ↓; GSR, LPO ↑

325 335

Ni3S2 NiCl2

2.85 8.3

it im

50% mortality (14 d) NK-cell activity ↓

336 337

ip

LD50 (5 d): 3 wk M, 29.6; F, 32.2 (mg Ni/kg) 9 wk M, 16.6; F, 17.9 (mg Ni/kg) 14 wk M, 13.0; F, 15.9 (mg Ni/kg) Bone marrow cellularity ↓

338

C3H, M BALB/c, M B6C3F1, M C57BL/6, M CBA, M ICR, M + F, (3, 9, 14 wk)

B6C3F1, F

ip

Effects

Wistar, F

Mice C57BL/6, M

a

Dose (mg/kg or ppm Ni)

Ni (OAc)2

NiSO4

380–3800 ppm water × 180 d

334

339


Note: G.D. = gestation day.

Mortalities in mice were documented from an experiment in which they were given water containing 64 ppm Se for 46 days.346 Since selenium functions as a potent SH oxidizing reagent in vivo, decreased GSH and increased GSSG were documented in tissues of Na2SeO3-treated rats. David and Shearer341 found that decreased lens GSH level was accompanied by cataract formation and increased insoluble protein levels in selenite-treated rats. LeBoeuf and Hoekstra342 observed increased GSSG, GSR, and γ-GTP in the liver of Na2SeO3-treated rats. Watanabe and Suzuki345 reported that selenite injection caused a transient hypothermia and cold-seeking behavior in mice. Inhalation of dimethylselenide gas up to 8000 ppm for 1 h caused alterations of DNA, RNA and protein levels in lung, liver, and spleen of rats, but no pathological change was observed.347

SECTION 15. THALLIUM Soluble thallium (Tl) salts are easily absorbed from the gastrointestinal tract and are widely distributed among various tissues to cause adverse effects (Table 22.26). The LD50 value (4 days) was documented as 24.8 mg Tl/kg in TlOAc-injected (ip) male rats.348 Around this dose level, increases in serotonin turnover rate and monoamine oxidase activity in the brain were observed within 24 h.351 Peele et al.349 showed that oral administration of Tl2SO4 caused flavor aversion effect in rats, whereas ip injection at the same dose level was much less effective. Decreased brain glutathione levels350 or the increased spontaneous discharge rate of Purkinje neurons352 were documented after serial injections of lower doses. Woods and Fowler353 observed dose-related ultrastructural changes in the liver with concomitant increases of mitochondrial membranous enzyme activities and decreases of microsomal enzyme


TABLE 22.25 Toxic Effects of Sodium Selenite Animal Strain, Sex (Age)a Rats SD, M

SD, M + F (5, 12 wk) Mice ICR, F ICR, M Swiss, M + F (7, 18 wk) a

Dose (mg/kg or ppm Se)

Route

1.6

sc

1.2 or 6 ppm/food × 6 wk 16 ppm/water × 35 d

ip

0.9 × 1–3 (2-d interval) 1.6–4.7 64 ppm/water × 46 d

ip sc

Effects Lens: GSH, insoluble protein ↑; cataract formation (4 d) Liver: GSSG, GSR, γ-GTP ↑ Mortality: 5 wk M, 60%; F, 80%; 12 wk M, 0%; F, 20%; liver damage WBC number (16 d) ↓ Transient hypothermia, cold-seeking behavior (1 h) Mortality: 20% (7 wk, M, F); 80% (18 wk, M); 40% (18 wk, F); liver necrosis

Ref. 341 342 343

344 345 346


activities after ip injection of TlCl3. Effects on the kidney were reduced GFR, proteinuria, and pathological change in the loop of Henle in TlSO4-treated female rats.354 Effects on testes were reported in rats given drinking water containing 10 ppm Tl for 60 days.355

TABLE 22.26 Toxic Effects of Thallium on Rats

Strain, Sex

Chemicals

Dose (mg/kg or ppm Tl)

Route

Effect

Ref.

TlOAc Tl2SO4 TlOAc

16–54 1.7–13.6 5×6

ip po ip

LD50 (4 d): 24.8 mg/kg Flavor aversion effect; ip less effective Brain: GSH, R-SH ↓

348 349 350

TlOAc TlOAc TlCl3

23 or 39 3.1 × 7 50–200

ip ip ip

351 352 353

Wistar, F

Tl2SO4

3.4–13.6

ip

Wistar, M

Tl2SO4

10 ppm/water × 60 d

Brain: serotonin turnover, MAO activity (24 h) ↑ Spontaneous discharge rate of Purkinje neurons ↑ Liver: change in ultrastructure (16 h) Mitochondrial membrane enzyme ↑; microsome enzyme ↓ Kidney: GFR ↓; protein level ↑; pathological change in loop of Henle Pathological change in Sertoli cells; spermatozoa motility, testicular β-glucuronidase ↓

Wistar, M LE, M Charles Foster, M Wistar, M SD, M

354 355

SECTION 16. TIN Oral toxicities of inorganic tin (Sn), including metallic Sn, are thought to be rather low because of poor gastrointestinal absorption. High dose exposure to inorganic Sn caused a decrease in weight gain and food intake as a result of damage of the gastrointestinal tract. de Groot et al.356 reported hemoglobin levels proved to be the most sensitive parameter in rats fed SnCl2-contaminated food above 1000 ppm. Macroscopic and microscopic examinations were reported in SnCl2-fed rats.357,358 Zareba and Chmielnicka359 showed decreased ALAD activity after seven sc or ip injections of 2 mg Sn/kg as SnCl2 in rats. Copyright © 2002 by Taylor & Francis

Toxicities of organic Sn compounds are much higher than those of inorganic compounds. Particularly, trialkylated Sn compounds are well known to penetrate and damage the brain tissue owing to high lipophilicity. Sublethal effects reported include abnormal behavior, such as hyperactivity, tremor, increase in hot plate or tail flick latency, disrupted learning, and flavor aversion (Table 22.27). Pathologically, lesions in the hippocampus were documented in trimethyltin-treated animals.361,371 Triethyltin showed the highest toxicity, followed by trimethyl-, tripropyl-, and tributyltin.371 Chang et al.361 demonstrated mice had higher susceptibility to trimethyltin toxicity than had rats. In addition to neural tissue, inhibitions of protein phosphorylation and Ca2+-ATPase activity in the heart367 and natural killer cell activity374 were documented. In contrast to trialkylated tin compounds, triphenyltin (TPT) was shown to damage hepatic functions in adult rats after three consecutive ip injections of 0.34 mg Sn/kg.376 Lehotzky et al.377 found that when pregnant rats were treated by TPT (1.74 mg/kg/day) during gestation days 7 to 15, the surviving offspring showed hyperactivity. Bis(tributyltin)oxide (0.5 ml/kg, intramuscular injection) brought about liver damage378 and corneal edema379 in rats. Feeding on dioctyltin chloride (DOTC)-contaminated food (50 to 150 ppm) for several weeks caused thymus atrophy in rats.380,381 Oral administration of DOTC (500 mg/kg) once a week for 8 weeks resulted in a suppressed antiself RBC antibody response in mice.382 Seinen et al.381 compared the effectiveness of various dialkylated tin compounds, and found that dioctyl- and dibutyl-compounds were most effective, whereas dimethyltin had no effect on the thymus.

TABLE 22.27 Toxic Effects of Trialkyltin Compounds Animal Strain, Sex (Age)a Rats LE, M SD and LE, M LE, M

F344, M

SD, M

LE, M F344, M LE, (3 d) LE, (newborn)

Compoundb

Dose (mg/kg or ppm Sn)

Route

TMT TMT

2.6 × 4 4.5

po po

TMT TMT TMT TMT TET TMT TET

3.6–5.1 3.6–5.1 2.9–5.1 2.2 (EC50) 1.0 (EC50) 2.5 1.12 1.68 1.8 × 6 0.87 × 6 1.2 × 6 0.62 × 6 0.21 × 14 0.22 × 27 0.17 × 27 2 1.3 4.2 10 0.66 × 24

po po po ip ip sc sc sc po po po ip sc po po po po po po po

TMT TET TBT TET TET TMT TET TMT TET TPT TBT TMT

Mice


Effects Hippocampus: Synapsin I ↓ Lesion in hippocampus, susceptibility: LE > SD Length of pyramidal cell line (30 d) ↓ Disrupted learning and memory (21 d) Visual system dysfunction Flavor aversion Hot plate latency (21–28 d) ↑ Hot plate latency ↑ 80% mortality Heart: 45Ca uptake by sarcoplasmic reticulum, Ca2+-ATPase ↓; phosphorylation of specific protein ↓ Visual evoked potential ↓; CNS depression Latency in tail flick and hot plate ↑ Hyperactivity; learning memory function (180–200 d) ↓ 100% mortality (5–6 d) 100% mortality (6–10 d) 100% mortality (15–20 d) 100% mortality (5–8 d) 67% mortality

Ref.

360 361 362 363 364 365 366

367

368 369 370 371

TABLE 22.27 (Continued) Toxic Effects of Trialkyltin Compounds Animal Strain, Sex (Age)a

a b

Compoundb

Dose (mg/kg or ppm Sn)

Route

C57BL/6, M

TMT

2.2

ip

BALB/c, M ICR, M

TMT TMT TET

1.8 1.8 0.58 or 2.8

po ip po

C3H, M

TBT

3.6 or 36 ppm/water × 1 wk

Effects Hippocampus, front cortex; O2-reactive species (48 h) ↑ Hippocampus: lesions (48 h) SMA (24 h) ↓ Anticonvulsant effect; interaction with adrenergic and GABAergic transmitter systems NK-cell activity ↓

Ref. 372 361 373 374

375

Data from adult animals, unless otherwise specified. TMT = trimethyltin; TET = triethyltin; TPT = tripropyltin; TBT = tributyltin.

SECTION 17. ZINC Zinc (Zn) is an essential metal and functions as a cofactor of various enzymes and insulin. Although zinc deficiency is well documented in humans and in animals, its toxic effects have also been reported in experimental animals. Young mice showed twofold higher LD50 values (115.2 mg Zn/kg) after ip injection of zinc acetate than did adults (44.4 to 50.4 mg Zn/kg).383 In the case of subchronic oral toxicity, the most severe histological lesions were observed in the kidney.384 Inhalation exposure (5 mg Zn/m3 × 3 h) or intratracheal instillation (20 μg/rat) of ZnO has been demonstrated to cause functional, morphological, or biochemical changes in lungs of rats385,386 and guinea pigs.387

REFERENCES 1. B. Venugopal and T.D. Luckey, Eds., Metal Toxicity in Mammals, Vol. 2. Chemical Toxicity of Metals and Metalloids, Plenum Press, New York, 1978. 2. L. Friberg, G.F. Nordberg, and V.B. Vouk, Eds., Handbook of the Toxicology of Metals, Vol. II, Elsevier Science Publishers, Amsterdam, 1986. 3. T. Ghafghazi, J.W. Ridlington, and B.A. Fowler, The effects of acute and subacute sodium arsenite administration on carbohydrate metabolism, Toxicol. Appl. Pharmacol., 55, 126–130, 1980. 4. S. Valkonen, H. Savolainen, and J. Jarvisalo, Arsenic distribution and neurochemical effects in peroral sodium arsenite exposure of rats, Bull. Environ. Contam. Toxicol., 30, 303–308, 1983. 5. W.R. Byron, G.W. Bierbower, J.B. Brouwer, and W.H. Hansen, Pathologic changes in rats and dogs from two-year feeding of sodium arsenite or sodium arsenate, Toxicol. Appl. Pharmacol., 10, 132–147, 1967. 6. H.V. Aposhian, C.H. Tadlock, and T.E. Moon, Protection of mice against the lethal effects of sodium arsenite: a quantitative comparison of a number of chelating agents, Toxicol. Appl. Pharmacol., 61, 385–392, 1981. 7. B.R. Blakley, C.S. Sisodia, and T.K. Mukkur, The effects of methylmercury, tetraethyl lead, and sodium arsenite on the humoral immune response in mice, Toxicol. Appl. Pharmacol., 52, 245–254, 1980. 8. B.A. Fowler and J.S. Woods, The effects of prolonged oral arsenate exposure on liver mitochondria of mice: morphometric and biochemical studies, Toxicol. Appl. Pharmacol., 50, 177–187, 1979.


9. M.N. Baxley, R.D. Hood, G.C. Vedel, W.P. Harrison, and G.M. Szczech, Prenatal toxicity of orally administered sodium arsenite in mice, Bull. Environ. Contam. Toxicol., 26, 749–756, 1981. 10. R.D. Hood and G.C. Vedel-Macrander, Evaluation of the effect of BAL (2,3-dimercaptopropanol) on arsenite-induced teratogenesis in mice, Toxicol. Appl. Pharmacol., 73, 1–7, 1984. 11. M.A. Bosque, J.L. Domingo, J.M. Llobet, and J. Corbella, Effects of meso-2,3-dimercaptosuccicic acid (DMSA) on the teratogenicity of sodium arsenate in mice, Bull. Environ. Contam. Toxicol., 47, 682–688, 1991. 12. S.J.S. Flora and S.D. Gupta, Effect of single gallium arsenide exposure on some biochemical variables in porphyrin metabolism in rats, J. Appl. Toxicol., 12, 333–334, 1992. 13. D.R. Webb, I.G. Sipes, and D.E. Carter, In vitro solubility and in vivo toxicity of gallium arsenide, Toxicol. Appl. Pharmacol., 76, 96–104, 1984. 14. P.L. Goering, R.R. Maronpot, and B.A. Fowler, Effect of intratracheal gallium arsenide administration on δ-aminolevulinic acid dehydratase in rats: relationship to urinary excretion of aminolevulinic acid, Toxicol. Appl. Pharmacol., 92, 179–193, 1988. 15. D.R. Webb, S.E. Wilson, and D.E. Carter, Comparative pulmonary toxicity of gallium arsenide, gallium (III) oxide, or arsenic (III) oxide intratracheally instilled into rats, Toxicol. Appl. Pharmacol., 82, 405–416, 1986. 16. E.E. Sikorski, L.A. Burns, K.L. McCoy, M. Stern, and A.E. Munson, Suppression of splenic accessory cell function in mice exposed to gallium arsenide, Toxicol. Appl. Pharmacol., 110, 143–156, 1991. 17. H. Kreppel, F.-X. Reichl, L. Szinicz, B. Fichtl, and W. Forth, Efficacy of various dithiol compounds in acute As2O3 poisoning in mice, Arch. Toxicol., 64, 387–392, 1990. 18. T. Itoh, Y.F. Zhang, S. Murai, H. Saito, H. Nagahama, H. Miyate, Y. Saito, and E. Abe, The effect of arsenic trioxide on brain monoamine metabolism and locomotor activity of mice, Toxicol. Lett., 54, 345–353, 1990. 19. C. Aranyi, J.N. Bradof, W.J. O’Shea, J.A. Graham, and F.J. Miller, Effects of arsenic trioxide inhalation exposure on pulmonary antibacterial defenses in mice, J. Toxicol. Environ. Health, 15, 163–172, 1985. 20. H.L. Hong, B.A. Fowler, and G.A. Boorman, Hematopoietic effects in mice exposed to arsine gas, Toxicol. Appl. Pharmacol., 97, 173–182, 1989. 21. P.C. Blair, M.B. Thompson, M. Bechtold, R.E. Wilson, M.P. Moorman, and B.A. Fowler, Evidence for oxidative damage to red blood cells in mice induced by arsine gas, Toxicology, 63, 25–34, 1990. 22. L.E. Sendelbach and H.P. Witschi, Protection by parenteral iron administration against the inhalation toxicity of beryllium sulfate, Toxicol. Lett., 35, 321–325, 1987. 23. L.E. Sendelbach, H.P. Witschi, and A.F. Tryka, Acute pulmonary toxicity of beryllium sulfate inhalation in rats and mice: cell kinetics and histopathology, Toxicol. Appl. Pharmacol., 85, 248–256, 1986. 24. L.E. Sendelbach and H.P. Witschi, Bronchoalveolar lavage in rats and mice following beryllium sulfate inhalation, Toxicol. Appl. Pharmacol., 90, 322–329, 1987. 25. B.A. Hart, A.G. Harmsen, R.B. Low, and R. Emerson, Biochemical, cytological, and histological alterations in rat lung following acute beryllium aerosol exposure, Toxicol. Appl. Pharmacol., 75, 454–465, 1984. 26. D.H. Groth, Carcinogenicity of beryllium: review of the literature, Environ. Res., 21, 56–62, 1980. 27. D.H. Groth, C. Kommineni, and G.R. Mackay, Carcinogenicity of beryllium hydroxide and alloys, Environ. Res., 21, 63–84, 1980. 28. H.P. Witschi and P. Marchand, Interference of beryllium with enzyme induction in rat liver, Toxicol. Appl. Pharmacol., 20, 565–572, 1971. 29. J. Vacher, R. Deraedt, and J. Benzoni, Compared effects of two beryllium salts (soluble and insoluble): toxicity and blockade of the reticuloendothelial system, Toxicol. Appl. Pharmacol., 24, 497–506, 1973. 30. R. Mathur, S. Sharma, S. Mathur, and A.O. Prakash, Effect of beryllium nitrate on early and late pregnancy in rats, Bull. Environ. Contam. Toxicol., 38, 73–77, 1987. 31. G.P. Samarawickrama and M. Webb, The acute toxicity and teratogenicity of cadmium in the pregnant rat, J. Appl. Toxicol., 1, 264–269, 1981. 32. R.E. Dudley, D.J. Svoboda, and C.D. Klaassen, Acute exposure to cadmium causes severe liver injury in rats, Toxicol. Appl. Pharmacol., 65, 302–313, 1982. 33. P.L. Goering, B.R. Fisher, and C.L. Kish, Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxicity, Toxicol. Appl. Pharmacol., 122, 139–148, 1993. 34. G. Lázár, D. Serra, and B. Tuchweber, Effect on cadmium toxicity of substances influencing reticuloendothelial activity, Toxicol. Appl. Pharmacol., 29, 367–376, 1974.


35. C.R. Caflisch and T.D. DuBose, Jr., Cadmium-induced changes in luminal fluid pH in testis and epididymis of the rat in vivo, J. Toxicol. Environ. Health, 32, 49–57, 1991. 36. N. Shiraishi, R.A. Barter, H. Uno, and M.P. Waalkes, Effect of progesterone pretreatment on cadmium toxicity in the male Fischer (F344/NCr) rat, Toxicol. Appl. Pharmacol., 118, 113–118, 1993. 37. A.-S. Chung and M.D. Maines, Differential effect of cadmium on GSH-peroxidase activity in the Leydig and the Sertori cells of rat testis: suppression by selenium and the possible relationship to heme concentration. Biochem. Pharmacol., 36, 1367–1372, 1987. 38. N. Konishi, J.M. Ward, and M.P. Waalkes, Pancreatic hepatocytes in Fischer and Wister rats induced by repeated injections of cadmium chloride, Toxicol. Appl. Pharmacol., 104, 149–156, 1990. 39. T. Koizumi and Z.G. Li, Role of oxidative stress in single-dose, cadmium-induced testicular cancer, J. Toxicol. Environ. Health, 37, 25–36, 1992. 40. P.H. Ruppert, K.F. Dean, and L.W. Reiter, Development of locomotor activity of rat pups exposed to heavy metals, Toxicol. Appl. Pharmacol., 78, 69–77, 1985. 41. K.-L. Wong and C.D. Klaassen, Neurotoxic effects of cadmium in young rats, Toxicol. Appl. Pharmacol., 63, 330–337, 1982. 42. J.U. Bell, Induction of hepatic metallothionein in the immature rat following administration of cadmium, Toxicol. Appl. Pharmacol., 54, 148–155, 1980. 43. P.L. Goering and C.D. Klaassen, Resistance to cadmium-induced hepatotoxicity in immature rats, Toxicol. Appl. Pharmacol., 74, 321–329, 1984. 44. J.W. Laskey, G.L. Rehnberg, S.C. Laws, and J.F. Hein, Reproductive effects of low acute doses of cadmium chloride in adult male rats, Toxicol. Appl. Pharmacol., 73, 250–255, 1984. 45. S. Gulati, K.D. Gill, and R. Nath, Effect of cadmium on lipid composition of the weanling rat brain, Acta Pharmacol. Toxicol., 59, 89–93, 1986. 46. T.E.K. White, R.B. Baggs, and R.K. Miller, Central nervous system lesions in the Wister rat fetus following direct fetal injections of cadmium, Teratology, 42, 7–13, 1990. 47. L. Yáñez, L. Carrizales, M.T. Zanatta, J.D.J. Mejía, L. Batres, and F. Díaz-Barriga, Arsenic-cadmium interaction in rats: toxic effects in the heart and tissue metal shifts, Toxicology, 67, 227–234, 1991. 48. L.G. Scharpf, Jr., F.J. Ramos, and I.D. Hill, Influence of nitrilotriacetate (NTA) on the toxicity, excretion and distribution of cadmium in female rats, Toxicol. Appl. Pharmacol., 22, 186–192, 1972. 49. M. Shimizu and S. Morita, Effects of fasting on cadmium toxicity, glutathione metabolism, and metallothionein synthesis in rats, Toxicol. Appl. Pharmacol., 103, 28–39, 1990. 50. F.N. Kotsonis and C.D. Klaassen, Toxicity and distribution of cadmium administered to rats at sublethal doses, Toxicol. Appl. Pharmacol., 41, 667–680, 1977. 51. L.R. Cantilena, Jr. and C.D. Klaassen, Comparison of the effectiveness of several chelators after single administration on the toxicity, excretion, and distribution of cadmium, Toxicol. Appl. Pharmacol., 58, 452–460, 1981. 52. G.S. Probst, W.F. Bousquet, and T.S. Miya, Correlation of hepatic metallothionein concentrations with acute cadmium toxicity in the mouse, Toxicol. Appl. Pharmacol., 39, 61–69, 1977. 53. A. Selypes, P. Serényi, I. Boldog, F. Bokros, and S. Takács, Acute and “long-term” genotoxic effects of CdCl2 on testes of mice, J. Toxicol. Environ. Health, 36, 401–409, 1992. 54. G.R. Hogan and S.L. Razniak, Split dose studies on the erythropoietic effects of cadmium, Bull. Environ. Contam. Toxicol., 48, 857–864, 1992. 55. M.M. Jones, M.A. Basinger, R.J. Topping, G.R. Gale, S.G. Jones, and M.A. Holscher, Meso-2,3dimercaptosuccinic acid and sodium N-benzyl-N-dithiocarboxy-D-glucamine as antagonists for cadmium intoxication, Arch. Toxicol., 62, 29–36, 1988. 56. D.J. Thomas, R.A. Winchurch, and P.C. Huang, Ontogenic variation in acute lethality of cadmium in C57BL/6J mice, Toxicology, 47, 317–323, 1987. 57. R.L. Shippee, D.H. Burgess, R.P. Ciavarra, R.A. DiCapua, and P.E. Stake, Cadmium-induced suppression of the primary immune response and acute toxicity in mice: differential interaction of zinc, Toxicol. Appl. Pharmacol., 71, 303–306, 1983. 58. T. Maitani and K.T. Suzuki, Effect of cadmium on essential metal concentrations in testis, liver and kidney of five inbred strains of mice, Toxicology, 42, 121–130, 1986. 59. O. Andersen, J.B. Nielsen, and P. Svendsen, Oral cadmium chloride intoxication in mice: effects of chelation, Toxicology, 52, 65–79, 1988.


60. D. Manca, A.C. Ricard, B. Trottier, and G. Chevalier, Studies on lipid peroxidation in rat tissues following administration of low and moderate doses of cadmium chloride, Toxicology, 67, 303–323, 1991. 61. J.W. Laskey, G.L. Rehnberg, S.C. Laws, and J.F. Hein, Age-related dose response of selected reproductive parameters to acute cadmium chloride exposure in the male Long-Evance rat, J. Toxicol. Environ. Health, 19, 393–401, 1986. 62. A.M. Saillenfait, J.P. Payan, M.T. Brondeau, D. Zissu, and J. de Ceaurriz, Changes on urinary proximal tubule parameters in neonatal rats exposed to cadmium chloride during pregnancy, J. Appl. Toxicol., 11, 23–27, 1991. 63. A.A. Levin and R.K. Miller, Fetal toxicity of cadmium in the rat: decreased utero-placental blood flow, Toxicol. Appl. Pharmacol., 58, 297–306, 1981. 64. D. Holt and M. Webb, Comparison of some biochemical effects of teratogenic doses of mercuric mercury and cadmium in the pregnant rat, Arch. Toxicol., 58, 249–254, 1986. 65. D. Holt and M. Webb, Teratogenicity of ionic cadmium in the Wister rat, Arch. Toxicol., 59, 443–447, 1987. 66. W.H. Roelfzema, A.M. Roelofsen, W. Leene, and J.H.J. Copius Peereboom-Stegeman, Effects of cadmium exposure during pregnancy on cadmium and zinc concentrations in neonatal liver and consequences for the offspring, Arch. Toxicol., 63, 38–42, 1989. 67. G.P. Daston, Toxic effects of cadmium on the developing rat lung. II. Glycogen and phospholipid metabolism, J. Toxicol. Environ. Health, 9, 51–61, 1982. 68. T.L. Sorell and J.H. Graziano, Effect of oral cadmium exposure during pregnancy on maternal and fetal zinc metabolism in the rat, Toxicol. Appl. Pharmacol., 102, 537–545, 1990. 69. B. Bara´nski, Effect of maternal cadmium exposure on postnatal development and tissue cadmium, copper and zinc concentrations in rats, Arch. Toxicol., 58, 255–260, 1986. 70. S.K. De, S.K. Dey, and G.K. Andrews, Cadmium teratogenicity and its relationship with metallothionein gene expression in midgestation mouse embryos, Toxicology, 64, 89–104, 1990. 71. O. Katsuta, H. Hiratsuka, J. Matsumoto, M. Tsuchitani, T. Umemura, and F. Marumo, Ovariectomy enhances cadmium-induced nephrotoxicity and hepatotoxicity in rats, Toxicol. Appl. Pharmacol., 119, 267–274, 1993. 72. R.A. Goyer, C.R. Miller, S.-Y. Zhu, and W. Victery, Non-metallothionein-bound cadmium in the pathogenesis of cadmium nephrotoxicity in the rat, Toxicol. Appl. Pharmacol., 101, 232–244, 1989. 73. L. Müller and N.H. Stacey, Subcellular toxicity of low level cadmium in rats: effect on cytochrome c oxidase, Toxicology, 51, 25–34, 1988. 74. S. Kojima, H. Ono, M. Kiyozumi, T. Honda, and A. Takadate, Effect of N-benzyl-D-glucamine dithiocarbamate on the renal toxicity produced by subacute exposure to cadmium in rats, Toxicol. Appl. Pharmacol., 98, 39–48, 1989. 75. A.F.W. Morsert, W. Leene, C. De Groot, J.B.A. Kipp, M. Evers, A.M. Roelofsen, and K.S. Bosch, Differences in immunological susceptibility to cadmium toxicity between two rat strains as demonstrated with cell biological methods. Effect of cadmium on DNA synthesis of thymus lymphocytes, Toxicology, 48, 127–139, 1988. 76. G.S. Shukla, T. Hussain, and S.V. Chandra, Possible role of regional superoxide dismutase activity and lipid peroxide levels in cadmium neurotoxicity: in vivo and in vitro studies in growing rats. Life Sci., 41, 2215–2221, 1987. 77. R.E. Dudley, L.M. Gammal, and C.D. Klaassen, Cadmium-induced hepatic and renal injury in chronically exposed rats: likely role of hepatic cadmium-metallothionein in nephrotoxicity, Toxicol. Appl. Pharmacol., 77, 414–426, 1985. 78. Y. Nagai, M. Sato, and M. Sasaki, Effect of cadmium administration upon urinary excretion of hydroxylysine and hydroxyproline in the rat, Toxicol. Appl. Pharmacol., 63, 188–193, 1982. 79. M. Ando and S. Matsui, Effect of cadmium on vitamin D-non-stimulated intestinal calcium absorption in rats, Toxicology, 45, 1–11, 1987. 80. I.G. O’Brien and L.J. King, The effect of chronic parenteral administration of cadmium on isoenzyme levels of alkaline phosphatase in intestinal mucosa, Toxicology, 56, 87–94, 1989. 81. M.P. Waalkes, R. Kovatch, and S. Rehm, Effect of chronic dietary zinc deficiency on cadmium toxicity and carcinogenesis in the male Wister [Hsd: (WI)BR] rat, Toxicol. Appl. Pharmacol., 108, 448–456, 1991.


82. A. Cárdenas, A. Bernard, and R. Lauwerys, Incorporation of [35S]sulfate into glomerular membranes of rats chronically exposed to cadmium and its relation with urinary glycosaminoglycans and proteinuria, Toxicology, 76, 219–231, 1992. 83. R. Toury, N. Stelly, E. Boissonneau, and Y. Dupuis, Degenerative processes in skeletal muscle of Cd2+-treated rats and Cd2+ inhibition of mitochondrial Ca2+ transport, Toxicol. Appl. Pharmacol., 77, 19–35, 1985. 84. H. Iguchi and S. Sano, Effect of cadmium on the bone collagen metabolism of rat, Toxicol. Appl. Pharmacol., 62, 126–136, 1982. 85. S.J. Kopp, H.M. Perry, Jr., E.F. Perry, and M. Erlanger, Cardiac physiologic and tissue metabolic changes following chronic low-level cadmium and cadmium plus lead ingestion in the rat, Toxicol. Appl. Pharmacol., 69, 149–160, 1983. 86. D.W. Rosenberg and A. Kappas, Induction of heme oxygenase in the small intestinal epithelium: a response to oral cadmium exposure, Toxicology, 67, 199–210, 1991. 87. B.A. Chowdhury, J.K. Friel, and R.K. Chandra, Cadmium-induced immunopathology is prevented by zinc administration in mice, J. Nutr., 117, 1788–1794, 1987. 88. M.H. Bhattacharyya, B.D. Whelton, D.P. Peterson, B.A. Carnes, E.S. Moretti, J.M. Toomey, and L.L. Williams, Skeletal changes in multiparous mice fed a nutrient-sufficient diet containing cadmium, Toxicology, 50, 193–204, 1988. 89. A.A. Valois and W.S. Webster, The choroid plexus as a target site for cadmium toxicity following chronic exposure in the adult mouse: an ultrastructural study, Toxicology, 55, 193–205, 1989. 90. P.L. Goering and C.D. Klaassen, Altered subcellular distribution of cadmium following cadmium pretreatment: possible mechanism of tolerance to cadmium-induced lethality, Toxicol. Appl. Pharmacol., 70, 195–203, 1983. 91. U. Wormser and I. Nir, Effect of age on cadmium-induced metallothionein synthesis in the rat, Arch. Toxicol., 62, 392–394, 1988. 92. N. Nishimura, H. Oshima, and M. Nakano, Urinary trehalase as an early indicator of cadmium-induced renal tubular damage in rabbit, Arch. Toxicol., 59, 255–260, 1986. 93. E.C. Grose, J.H. Richards, R.H. Jaskot, M.G. Ménache, J.A. Graham, and W.C. Dauterman, A comparative study of the effects of inhaled cadmium chloride and cadmium oxide: pulmonary response, J. Toxicol. Environ. Health, 21, 219–232, 1987. 94. M. Boisset and C. Boudene, Effect of a single exposure to cadmium oxide fumes on rat lung microsomal enzymes, Toxicol. Appl. Pharmacol., 57, 335–345, 1981. 95. B.J. Buckley and D.J.P. Bassett, Glutathione redox status of control and cadmium oxide-exposed rat lungs during oxidant stress, J. Toxicol. Environ. Health, 22, 287–299, 1987. 96. R.S. Kutzman, R.T. Drew, R.N. Shiotsuka, and B.Y. Cockrell, Pulmonary changes resulting from subchronic exposure to cadmium chloride aerosol, J. Toxicol. Environ. Health, 17, 175–189, 1986. 97. B.A. Hart, G.W. Voss, and C.L. Willean, Pulmonary tolerance to cadmium following cadmium aerosol pretreatment, Toxicol. Appl. Pharmacol., 101, 447–460, 1989. 98. P.V.V. Prasada Rao and D.E. Gardner, Effects of cadmium inhalation on mitochondrial enzymes in rat tissues, J. Toxicol. Environ. Health, 17, 191–199, 1986. 99. S. Hirano, N. Tsukamoto, S. Higo, and K.T. Suzuki, Toxicity of cadmium oxide instilled into the rat lung. II. Inflammatory responses in broncho-alveolar lavage fluid, Toxicology, 55, 25–35, 1989. 100. K.E. Driscoll, J.K. Maurer, J. Poynter, J. Higgins, T. Asquith, and N.S. Miller, Stimulation of rat alveolar macrophage fibronectin release in a cadmium chloride model of lung injury and fibrosis, Toxicol. Appl. Pharmacol., 116, 30–37, 1992. 101. P.J. Hakkinen, C.C. Morse, F.M. Martin, W.E. Dalbey, W.M. Haschek, and H.R. Witschi, Potentiating effects of oxygen in lungs damaged by methylcyclopentadienyl manganese tricarbonyl, cadmium chloride, oleic acid, and antitumor drugs, Toxicol. Appl. Pharmacol., 67, 55–69, 1983. 102. F.M. Martin and H.P. Witschi, Cadmium-induced lung injury: cell kinetics and long-term effects, Toxicol. Appl. Pharmacol., 80, 215–227, 1985. 103. C.A.M. Suzuki and M.G. Cherian, Renal glutathione depletion and nephrotoxicity of cadmiummetallothionein in rats, Toxicol. Appl. Pharmacol., 98, 544–552, 1989. 104. T. Jin, P. Leffler, and G.F. Nordberg, Cadmium-metallothionein nephrotoxicity in the rat: transient calcuria and proteinuria, Toxicology, 45, 307–317, 1987.


105. K.-S. Min, K. Kobayashi, S. Onosaka, N. Ohta, Y. Okada, and K. Tanaka, Tissue distribution of cadmium and nephropathy after administration of cadmium in several chemical forms, Toxicol. Appl. Pharmacol., 86, 262–270, 1986. 106. P.L. Goering, C.L. Kish, and B.R. Fisher, Stress protein synthesis induced by cadmium-cysteine in rat kidney, Toxicology, 85, 25–39, 1993. 107. J.Y. Zhao, E.C. Foulkes, and M. Jones, Delayed nephrotoxic effects of cadmium and their reversibility by chelation, Toxicology, 64, 235–243, 1990. 108. L.E. Sendelbach, W.C. Kershaw, F. Cuppage, and C.D. Klaassen, Cd-metallothionein nephrotoxicity in inbred strains of mice, J. Toxicol. Environ. Health, 35, 115–126, 1992. 109. T. Maitani, A. Watahiki, and K.T. Suzuki, Acute renal dysfunction by cadmium injected with cysteine in relation to renal critical concentration of cadmium, Arch. Toxicol., 58, 136–140, 1986. 110. M.J. Tsapakos, T.H. Hampton, and K.W, Jennette, The carcinogen chromate induces DNA cross-links in rat liver and kidney, J. Biol. Chem., 256, 3623–3626, 1981. 111. A.M. Standeven and K.E. Wetterhahn, Possible role of glutathione in chromium (VI) metabolism and toxicity in rats. Pharmacol. Toxicol., 69, 469–476, 1991. 112. A. Mikalsen, J. Alexander, R.A. Andersen, and M. Ingelman-Sundberg, Effect of in vivo chromate, acetone and combined treatment on rat liver in vitro microsomal chromium (VI) reductive activity and on cytochrome P450 expression, Pharmacol. Toxicol., 68, 456–463, 1991. 113. E. Kim and K.J. Na, Effect of sodium dichromate on carbohydrate metabolism, Toxicol. Appl. Pharmacol., 110, 251–258, 1991. 114. E. Ernst, Testicular toxicity following short-term exposure to tri- and hexavalent chromium: an experimental study in the rat, Toxicol. Lett., 51, 269–275, 1990. 115. D.K. Saxena, R.C. Murthy, B. Lal, R.S. Srivastava, and S.V. Chandra, Effect of hexavalent chromium on testicular maturation in the rat, Reprod. Toxicol., 4, 223–228, 1990. 116. R.C. Murthy, D.K. Saxena, S.K. Gupta, and S.V. Chandra, Ultrastructural observations in testicular tissue of chromium-treated rats, Reprod. Toxicol., 5, 443–447, 1991. 117. A. Vyskoÿcil, C. Viau, M. ÿCíÿzková, and G. Truchon, Kidney function in male and female rats chronically exposed to potassium dichromate, J. Appl. Toxicol., 13, 375–376, 1993. 118. T.P. Coogan, J. Motz, C.A. Snyder, K.S. Squibb, and M. Costa, Differential DNA-protein crosslinking in lymphocytes and liver following chronic drinking water exposure of rats to potassium chromate, Toxicol. Appl. Pharmacol., 109, 60–72, 1991. 119. U. Glaser, D. Hochrainer, H. Klöppel, and H. Kuhnen, Low level chromium (VI) inhalation effects on alveolar macrophages and immune functions in Wistar rats, Arch. Toxicol., 57, 250–256, 1985. 120. U. Glaser, D. Hochrainer, H. Klöppel, and H. Oldiges, Carcinogenicity of sodium dichromate and chromium (VI/III) oxide aerosols inhaled by male Wistar rats, Toxicology, 42, 219–232, 1986. 121. B. Trivedi, D.K. Saxena, R.C. Murthy, and S.V. Chandra, Embryotoxicity and fetotoxicity of orally administered hexavalent chromium in mice, Reprod. Toxicol., 3, 275–278, 1989. 122. R.A. Newman, A.R. Khokhar, B.A. Sunderland, E.L. Travis, and R.E. Bulger, A comparison in rodents of renal and intestinal toxicity of cisplatin and a new water-soluble antitumor platinum complex: Nmethyl-iminodiacetato-diaminocyclohexane platinum (II), Toxicol. Appl. Pharmacol., 84, 454–463, 1986. 123. J.M. Ward and K.A. Fauvie, The nephrotoxic effects of cis-diammine-dichloroplatinum (II) (NSC119875) in male F344 rats, Toxicol. Appl. Pharmacol., 38, 535–547, 1976. 124. R.S. Goldstein, B. Noordewier, J.T. Bond, J.B. Hook, and G.H. Mayor, cis-Dichlorodiammineplatinum nephrotoxicity: time course and dose response of renal functional impairment, Toxicol. Appl. Pharmacol., 60, 163–175, 1981. 125. C.L. Litterst, Alterations in the toxicity of cis-dichlorodiammineplatinum-II and in tissue localization of platinum as a function of NaCl concentration in the vehicle of administration, Toxicol. Appl. Pharmacol., 61, 99–108, 1981. 126. L.M. De Witt, T.W. Jones, and L. Moore, Stimulation of the renal endoplasmic reticulum calcium pump: a possible biomarker for platinate toxicity, Toxicol. Appl. Pharmacol., 92, 157–169, 1988. 127. R.D. Mayer and M.D. Maines, Promotion of trans-platinum in vivo effects on renal heme and hemoprotein metabolism by D,L-buthionine-S,R-sulfoximine: possible role of glutathione. Biochem. Pharmacol., 39, 1565–1571, 1990.


128. R. Safirstein, P. Miller, S. Dikman, N. Lyman, and C. Shapiro, Cisplatin nephrotoxicity in rats: defect in papillary hypertonicity. Am, J. Physiol., 241, F175–F185, 1981. 129. H.Th. Heidemann, St. Müller, L. Mertins, G. Stepan, K. Hoffmann, and the late E.E. Ohnhaus, Effect of aminophylline on cisplatin nephrotoxicity in the rat, Br. J. Pharmacol., 97, 313–318, 1989. 130. Y. Sadzuka, T. Shoji, and Y. Takino, Mechanism of the increase in lipid peroxide induced by cisplatin in the kidneys of rats, Toxicol. Lett., 62, 293–300, 1992. 131. G. Bompart and C. Orfila, Cisplatin nephrotoxicity in lead-pretreated rats: enzymatic and morphological studies, Toxicol. Lett., 50, 237–247, 1990. 132. E. Scherini, Permanent alterations of the dendritic tree of cerebellar Purkinje neurons in the rat following postnatal exposure to cis-dichlorodiammineplatinum, Acta Neuropathol., 81, 324–327, 1991. 133. G. Cavaletti, G. Tredici, P. Marmiroli, M.G. Petruccioli, I. Barajon, and D. Fabbrica, Morphometric study of the sensory neuron and peripheral nerve changes induced by chronic cisplatin (DDP) administration in rats, Acta Neuropathol., 84, 364–371, 1992. 134. P.M.A.B. Terheggen, R.G. Van Der Hoop, B.G.J. Floot, and W.H. Gispen, Cellular distribution of cisdiamminedichloroplatinum (II)-DNA binding in rat dorsal root spinal ganglia: effect of the neuroprotecting peptide ORG.2766, Toxicol. Appl. Pharmacol., 99, 334–343, 1989. 135. R.S. Goldstein, G.H. Mayor, R.L. Gingerich, J.B. Hook, B. Robinson, and J.T. Bond, Hyperglucagonemia following cisplatin treatment, Toxicol. Appl. Pharmacol., 68, 250–259, 1983. 136. R.S. Goldstein, G.H. Mayor, R.L. Gingerich, J.B. Hook, R.W. Rosenbaum, and J.T. Bond, The effects of cisplatin and other divalent platinum compounds on glucose metabolism and pancreatic endocrine function, Toxicol. Appl. Pharmacol., 69, 432–441, 1983. 137. H. Azouri, J.-M. Bidart, and C. Bohuon, In vivo toxicity of cisplatin and carboplatin on the Leydig cell function and effect of the human choriogonadotropin. Biochem. Pharmacol., 38, 567–571, 1989. 138. L.M. Pogach, Y. Lee, S. Gould, W. Giglio, M. Meyenhofer, and H.F.S. Huang, Characterization of cisplatinum-induced Sertoli cell dysfunction in rodents, Toxicol. Appl. Pharmacol., 98, 350–361, 1989. 139. D.D. Choie, D.S. Longnecker, and M.P. Copley, Cytotoxicity of cisplatin in rat intestine, Toxicol. Appl. Pharmacol., 60, 354–359, 1981. 140. K.A. Keller and S.K. Aggarwal, Embryotoxicity of cisplatin in rats and mice, Toxicol. Appl. Pharmacol., 69, 245–256, 1983. 141. M.L. Bajt and S.K. Aggarwal, An analysis of factors responsible for resorption of embryos in cisplatintreated rats, Toxicol. Appl. Pharmacol., 80, 97–107, 1985. 142. M. Satoh, A. Naganuma, and N. Imura, Deficiency of selenium intake enhances manifestation of renal toxicity of cis-diamminedichloroplatinum in mice, Toxicol. Lett., 38, 155–160, 1987. 143. U. Schaeppi, I.A. Heyman, R.W. Fleischman, H. Rosenkrantz, V. Ilievski, R. Phelan, D.A. Cooney, and R.D. Davis, cis-Dichlorodiammineplatinum (II) (NSC-119 875): preclinical toxicologic evaluation of intravenous injection in dogs, monkeys and mice, Toxicol. Appl. Pharmacol., 25, 230–241, 1973. 144. N.A. Boughattas, F. Lévi, C. Fournier, B. Hecquet, G. Lemaigre, A. Roulon, G. Mathé, and A. Reinberg, Stable circadian mechanisms of toxicity of two platinum analogs (cisplatin and carboplatin) despite repeated dosages in mice, J. Pharmacol. Exp. Ther., 255, 672–679, 1990. 145. R. Lerza, G. Bogliolo, C. Muzzulini, and I. Pannacciulli, Failure of N-acetylcysteine to protect against cis-dichlorodiammine-platinum (II)-induced hematopoietic toxicity in mice. Life Sci., 38, 1795–1800, 1986. 146. D. Wierda and M. Matamoros, Partial characterization of bone marrow hemopoiesis in mice after cisplatin administration, Toxicol. Appl. Pharmacol., 75, 25–34, 1984. 147. S.C. Apfel, J.C. Arezzo, L.A. Lipson, and J.A. Kessler, Nerve growth factor prevents experimental cisplatin neuropathy. Ann. Neurol., 31, 76–80, 1992. 148. G. Lasfargues, D. Lison, P. Maldague, and R. Lauwerys, Comparative study of the acute lung toxicity of pure cobalt powder and cobalt-tungsten carbide mixture in rat, Toxicol. Appl. Pharmacol., 112, 41–50, 1992. 149. C. Di Giulio, P.G. Data, and S. Lahiri, Chronic cobalt causes hypertrophy of glomus cells in the rat carotid body, Am. J. Physiol., 261, C102–C105, 1991. 150. J.R. Nation, A.E. Bourgeois, D.E. Clark, and M.F. Hare, The effects of chronic cobalt exposure on behavior and metallothionein levels in the adult rat, Neurobehav. Toxicol. Teratol., 5, 9–15, 1983.


151. K.A. Suarez and P. Bhonsle, The relationship of cobaltous chloride-induced alterations of hepatic microsomal enzymes to altered carbon tetrachloride hepatotoxicity, Toxicol. Appl. Pharmacol., 37, 23–27, 1976. 152. J.L. Paternain, J.L. Domingo, and J. Corbella, Developmental toxicity of cobalt in the rat, J. Toxicol. Environ. Health, 24, 193–200, 1988. 153. B.B. Muhoberac, T. Hanew, S. Halter, and S. Schenker, A model of cytochrome P-450-centered hepatic dysfunction in drug metabolism induced by cobalt-protoporphyrin administration, Biochem. Pharmacol., 38, 4103–4113, 1989. 154. M.B. Anderson, N.G. Pedigo, R.P. Katz, and W.J. George, Histopathology of testes from mice chronically treated with cobalt, Reprod. Toxicol., 6, 41–50, 1992. 155. S.M. Spaethe and D.J. Jollow, Effect of cobalt protoporphyrin on hepatic drug-metabolizing enzymes, Biochem. Pharmacol., 38, 2027–2038, 1989. 156. M. Lundborg and P. Camner, Lysozyme levels in rabbit after inhalation of nickel, cadmium, cobalt, and copper chlorides, Environ. Res., 34, 335–342, 1984. 157. S. Hirano, H. Ebihara, S. Sakai, N. Kodama, and K.T. Suzuki, Pulmonary clearance and toxicity of intratracheally instilled cupric oxide in rats, Arch. Toxicol., 67, 312–317, 1993. 158. S. Hirano, S. Sakai, H. Ebihara, N. Kodama, and K.T. Suzuki, Metabolism and pulmonary toxicity of intratracheally instilled cupric sulfate in rats, Toxicology, 64, 223–233, 1990. 159. I.C. Fuentealba, R.W. Davis, M.E. Elmes, B. Jasani, and S. Haywood, Mechanisms of tolerance in the copper-loaded rat liver, Exp. Mol. Pathol., 59, 71–84, 1993. 160. I.C. Fuentealba, S. Haywood, and J. Foster, Cellular mechanisms of toxicity and tolerance in the copper-loaded rat. III. Ultrastructural changes and copper localization in the kidney, Br. J. Exp. Pathol., 70, 543–556, 1989. 161. S. Toyokuni, S. Okada, S. Hamazaki, M. Fujioka, J.-L. Li, and O. Midorikawa, Cirrhosis of the liver induced by cupric nitrilotriacetate in Wister rats, Am. J. Pathol., 134, 1263–1274, 1989. 162. N. Shiraishi, T. Taguchi, and H. Kinebuchi, Copper-induced toxicity in macular mutant mouse: an animal model for Menkes’ kidney-hair disease, Toxicol. Appl. Pharmacol., 110, 89–96, 1991. 163. K. Agarwal, A. Sharma, and G. Talukder, Clastogenic effects of copper sulphate on the bone marrow chromosomes of mice in vivo, Mutat. Res., 243, 1–6, 1990. 164. M. Galleano and S. Puntarulo, Hepatic chemiluminescence and lipid peroxidation in mild iron overload, Toxicology, 76, 27–38, 1992. 165. M. Younes, I. Eberhardt, and R. Lemoine, Effect of iron overload on spontaneous and xenobioticinduced lipid peroxidation in vivo, J. Appl. Toxicol., 9, 103–108, 1989. 166. A. Pietrangelo, A. Cossarizza, D. Monti, E. Ventura, and C. Franceschi, DNA repair in lymphocytes from humans and rats with chronic iron overload, Biochem. Biophys. Res. Commun., 154, 698–704, 1988. 167. S. Okada, S. Hamazaki, S. Toyokuni, and O. Midorikawa, Induction of mesothelioma by intraperitoneal injections of ferric sacchrate in male Wister rats, Br. J. Cancer, 60, 708–711, 1989. 168. T.C. Iancu, R.J. Ward, and T.J. Peters, Ultrastructural observations in the carbonyl iron-fed rat, an animal model for hemochromatosis, Virchows Arch. B., 53, 208–217, 1987. 169. C.H. Park, B.R. Bacon, G.M. Brittenham, and A.S. Tavill, Pathology of dietary carbonyl iron overload in rats, Lab. Invest., 57, 555–563, 1987. 170. B.R. Bacon, A.S. Tavill, G.M. Brittenham, C.H. Park, and R.O. Recknagel, Hepatic lipid peroxidation in vivo in rats with chronic iron overload, J. Clin. Invest., 71, 429–439, 1983. 171. L. Golberg, L.E. Martin, and A. Batchelor, Biochemical changes in the tissues of animals injected with iron. 3. Lipid peroxidation. Biochem. J., 83, 201–298, 1961. 172. A.G. Smith and J.E. Francis, Genetic variation of iron-induced uroporphyria in mice, Biochem. J., 291, 29–35, 1993. 173. J.-L. Li, S. Okada, S. Hamazaki, Y. Ebina, and O. Midorikawa, Subacute nephrotoxicity and induction of renal cell carcinoma in mice treated with ferric nitrilotriacetate, Cancer Res., 47, 1867–1869, 1987. 174. P. Carthew, B.M. Dorman, R.E. Edwards, J.E. Francis, and A.G. Smith, A unique rodent model for both the cardiotoxic and hepatotoxic effects of prolonged iron overload, Lab. Invest., 69, 217–222, 1993. 175. P. Garthew, R.E. Edwars, B.M. Dorman, and J.E. Francis, Rapid induction of hepatic fibrosis in the gerbil after the paretenal administration of iron-dextran complex, Hepatology, 13, 534–539, 1991.


176. A.G. Smith, J.R.P. Cabral, P. Carthew, J.E. Francis, and M.M. Manson, Carcinogenicity of iron in conjection with a chlorinated environmental chemical, hexachlorobenzene, in C57BL/10ScSn mice, Int. J. Cancer, 43, 492–496, 1989. 177. N.N. Scharding and F.W. Oehme, The use of animal models for comparative studies of lead poisoning, Clin. Toxicol., 6, 419–424, 1973. 178. K. Kostial, T. Maljkovic, and S. Jugo, Lead acute toxicity in rats in relation to age and sex, Arch. Toxicol., 31, 265–269, 1974. 179. G.R. Hogan, Effects of ovariectomy and orchiectomy on lead-induced mortality in rats, Environ. Res., 21, 314–316, 1980. 180. B. Garbar and E. Wei, Lead toxicity in mice with genetically different levels of d-aminolevulic acid, Bull. Environ. Contam. Toxicol., 9, 80–83, 1973. 181. G.R. Hogan, Variation of lead-induced lethality in estradiol-treated mice, J. Toxicol. Environ. Health, 9, 353–357, 1982. 182. A.V. Lorenzo, M. Gewirtz, and D. Averill, CNS lead toxicity in rabbit offspring, Environ. Res., 17, 131–150, 1978. 183. K. Nagatoshi, Experimental chronic lead poisoning, Fol. Psychiatr. Neurol. Jpn., 33, 123–131, 1979. 184. M.V.S. Kumar and T. Desiraju, Regional alterations of brain biogenic amines and GABA/glutamate levels in rats following chronic lead exposure during neonatal development, Arch. Toxicol., 64, 305–314, 1990. 185. R. Kishi and E. Uchino, Effects of low lead exposure on neuro-behavioral function in the rat, Arch. Environ. Health, 38, 25–33, 1983. 186. L. Altman, F. Weinsberg, K. Sveinsson, H. Lilienthal, H. Wiegand, and G. Winneke, Impairment of long-term potentiation and learning following chronic lead exposure, Toxicol. Lett., 66, 105–112, 1993. 187. R. Sundström, K. Müntzing, H. Kalimo, and P. Sourander, Changes in the integrity of the blood-brain barrier in suckling rats with low dose lead encephalopathy, Acta Neuropathol., 68, 1–9, 1985. 188. D. Lorton and W.J. Anderson, The effects of postnatal lead toxicity on the development of cerebellum in rats, Neurobehav. Toxicol. Teratol., 8, 51–59, 1986. 189. S.R. Overmann, Behavioral effects of symptomatic lead exposure during neonatal development in rats, Toxicol. Appl. Pharmacol., 41, 459–471, 1977. 190. S.V. Chandra, M.M. Ali, D.K. Saxena, and R.C. Murthy, Behavioral and neurochemical changes in rats simultaneously exposed to manganese and lead, Arch. Toxicol., 49, 49–56, 1981. 191. J. Deluca, P.J. Donovick, and R.G. Burright, Lead exposure, environmental temperature, nesting and consummatory behavior of adult mice of two ages, Neurotoxicol. Teratol., 11, 7–11, 1989. 192. J.M. Donald, M.G. Cutler, and M.R. Moore, Effects of lead in the laboratory mouse development and social behavior after lifelong exposure to 12 μM lead in drinking fluid, Neuropharmacology, 26, 391–399, 1987. 193. E.E. Aiegler, B.B. Edwards, R.L. Jensen, K.R. Mahaffer, and S.J. Fomon, Adsorption and retention of lead by infants, Pediatr. Res., 12, 29–34, 1978. 194. H.M. Mykkanen, J.W.T. Dickerson, and M.C. Lancaster, Effects of age on the tissue distribution of lead in the rat, Toxicol. Appl. Pharmacol., 51, 447–454, 1979. 195. D.R. Brown, Neonatal lead exposure in the rat: decreased learning as a function of age and blood cencentrations, Toxicol. Appl. Pharmacol., 32, 628–637, 1975. 196. I. Kitchen, Lead toxicity and alterations in opioid systems, Neurotoxicology, 14, 115–124, 1993. 197. K. Tokoyama and S. Araki, Alterations nerve conduction velocity in low and high lead exposure: an animal study, Ind. Health, 24, 67–74, 1986. 198. H.E. Falke and W.C.M. Zwennis, Toxicity of lead acetate to female rabbits after chronic subcutaneous administration. 1. Biochemical and clinical effects, Arch. Toxicol., 64, 522–529, 1990. 199. G.M. Hass, D.V.L. Brown, R. Eisenstein, and A. Hemmens, Relations between lead poisoning in rabbit and man, Am. J. Pathol., 45, 691–715, 1964. 200. A. Oskarsson, Effects of perinatal treatment with lead and disculfiram on ALAD activity in blood, liver and kidney and urinary ALA excretion in rats, Pharmacol. Toxicol., 64, 344–348, 1989. 201. J.J. Barlow, J.K. Baruah, and A. Davison, δ-Aminolevulinic acid dehydratase activity and focal brain hemorrhages in lead-treated rats, Acta Neuropathol., 39, 219–223, 1977. 202. M. Hayashi, Lead toxicity in the pregnant rat, Environ. Res., 30, 152–160, 1983.


203. K. Tomokuni, M. Ichiba, and Y. Hirai, Species defference of urinary excretion of d-aminolevurinic acid and coproporphyrin in mice and rats exposed to lead, Toxicol. Lett., 41, 255–259, 1988. 204. S.U. Rehman, Effects of zinc, copper, and lead toxicity on d-aminolevulinic acid dehydratase activity, Bull. Environ. Contam. Toxicol., 33, 92–98, 1984. 205. S.K. Tandon and S.J.S. Flora, Dose and time effects of combine exposure to lead and ethanol on lead body burden and some neuronal, hepatic and haematopoietic biochemical induces in the rats, J. Appl. Toxicol., 9, 347–352, 1989. 206. E.K. Silbergeld, R.E. Hruska, D. Bradly, Lamon, J.M., and B.C. Frykholm, Neurotoxic aspects of porphyrinopathies: lead and succinylacetone, Environ. Res., 29, 459–471, 1982. 207. S.J.S. Flora, S. Singh, and S.K. Tandon, Chelation in metal intoxication XVIII: combined effects of thiamine and calcium disodium versenate on lead toxicity, Life Sci., 38, 67–71, 1986. 208. E. Schlick, K. Mengel, and K.D. Friedberg, The effect of low lead doses in vitro and in vivo on the d-ala-d activity of erythrocytes, bone marrow cells, liver and brain of the mouse, Arch. Toxicol., 53, 193–205, 1983. 209. S. Sassa, Toxic effects of lead, with particular reference to porphyrin and heme metabolism, in Handbook of Experimental Pharmacology, Vol. 44, Springer Verlag, Berlin, 1978, 333. 210. J. Maes and G.B. Gerber, Increased ALA dehydratase activity and spleen weight in lead-intoxicated rats. A consequence of increased blood cell destruction, Experimentia, 34, 381–382, 1978. 211. N. Karmakar, R. Saxena, and S. Ananda, Histopathological changes induced in rats tissues by oral intake of lead acetate, Environ. Res., 41, 23–28, 1986. 212. B.A. Fowler, C.A. Kimmel, J.S. Woods, E.E. McConnell, and L.D. Grant, Chronic low level lead toxicity in the rat. III. An integrated assessment of long-term toxicity with special reference to the kidney, Toxicol. Appl. Pharmacol., 56, 59–77, 1980. 213. B.J. Spit, A.A.E. Wibowo, V.J. Feron, and R.L. Zielhuis, Ultrasonic changes in the kidneys of rabbits treated with lead acetate, Arch. Toxicol., 49, 85–91, 1981. 214. F. Khalil-Manesh, H.C. Gonic, A. Cohen, E. Bergamaschi, and A. Mutti, Experimental model of lead nephropathy. II. Effect of removal from lead exposure and chelation treatment with dimercaptosuccinic acid (DMSA), Environ. Res., 58, 35–54, 1992. 215. R.A. Goyer, Lead toxicity: a problem in environmental pathology, Am. J. Pathol., 64, 167–181, 1971. 216. F.E. Hemphill, M.L. Kaerberle, and W.B. Buck, Lead suppression of mouse resistance to Salmonella typhimurium, Science, 172, 1031–1033, 1971. 217. B.R. Blakley and D.L. Archer, The effect of lead acetate on the immune response in mice, Toxicol. Appl. Pharmacol., 61, 18–26, 1981. 218. L.D. Koller, The immunotoxic effects of lead in lead-exposed laboratory animals. Ann. N. Y. Acad. Sci., 587, 160–167, 1990. 219. N. Kobayashi and T. Okamoto, Effect of lead oxide on the induction of lung tumors in Syrian hamsters, J. Natl. Cancer Inst., 52, 1605–1610, 1974. 220. N.I. Kerkvliet and L. Baecher-Steppan, Immunotoxicology studies on lead: effect of exposure on tumor growth and cell-mediated tumor immunity after synergic or allogenic stimulation, Immunopharmacology, 4, 213–224, 1982. 221. L.D. Koller and S. Kovacic, Decreased antibody response in mice exposed to lead, Nature, 250, 148–150, 1974. 222. L.D. Koller, J.H. Exon, and J.G. Roan, Humoral antibody response in mice after single dose exposure to lead or cadmium, Proc. Soc. Exp. Biol. Med., 151, 339–342, 1976. 223. R.A. Trejo, N.R. DiLuzio, L.D. Loose, and E. Hoffman, Reticuloendothelial and hepatic alterations following lead acetate administration, Exp. Mol. Pathol., 17, 145–158, 1972. 224. R.M. McClain and B.A. Becker, Teratogenicity, fetal toxicity, and placental transfer of lead nitrate in rats, Toxicol. Appl. Pharmacol., 31, 72–78, 1975. 225. R.Z. Sokol and N. Berman, The effects of age exposure on lead-induced testicular toxicity, Toxicology, 69, 269–278, 1991. 226. T.J. Walsh and H.A. Tilson, Neurobehavioral toxicology of the organoleads, Neurotoxicology, 5, 67–86, 1984. 227. W.J. Niklowitz, Ultrastructural effects of acute teraethyllead poisoning on nerve cells of the rabbit brain, Environ. Res., 8, 17–36, 1974.


228. W. Niklowitz, Neurofibrillary changes after acute experimental lead poisoning, Neurology, 25, 927–934, 1975. 229. H.A. Tilson, C.F. Mactutus, R. McLamb, and T.A. Burne, Characterization of triethyllead chloride neurotoxicity in adult rats, Neurobehav. Toxicol. Teratol., 4, 671–681, 1982. 230. T.J. Walsh, R.L. McLamb, and H.A. Tilson, Organometal-induced antinociception: a time and doseresponse comparison of triethyl and trimethyl lead and tin, Toxicol. Appl. Pharmacol., 73, 295–299, 1984. 231. J.S. Hong, H.A. Tilson, P. Hudson, S.F. Ali, W.E. Wilson, and V. Hunter, Corelation of neurochemical and behavioral effects of triethyl lead chloride in rats, Toxicol. Appl. Pharmacol., 69, 471–479, 1983. 232. T.J. Walsh, D.W. Schulz, H.A. Tilson, and D. Dehaven, Acute exposure to triethyl lead enhances the behavioral effects of dopaminergic agonists: involvement of brain dopamine in organolead neurotoxicity, Brain Res., 363, 222–229, 1986. 233. S.F. Ali and S.C. Bondy, Triethyllead-induced peroxidative damage in various regions on the rat brain, J. Toxicol. Environ. Health, 26, 235–242, 1989. 234. K. Kristensson, H. Eriksson, B. Lundh, L.-O. Plantin, L. Wachtmeister, M.L. Azazi, C. Morath, and E. Heilbronn, Effects of manganese chloride on the rat developing nervous system, Acta Pharmacol. Toxicol., 59, 345–348, 1986. 235. J.K. Malik and A.K. Srivastava, Studies on the interaction between manganese and fenitrothion in rats, Toxicol. Lett., 36, 221–226, 1987. 236. G.S. Shukla and S.V. Chandra, Manganese toxicity: lipid peroxidation in rat brain, Acta Pharmacol. Toxicol., 48, 95–100, 1981. 237. S.V. Chandra, G.S. Shukla, and R.C. Murthy, Effect of stress on the response of rat brain to manganese, Toxicol. Appl. Pharmacol., 47, 603–608, 1979. 238. K. Teramoto, F. Wakitani, S. Horiguchi, T. Jo, T. Yamamoto, H. Mitsutake, and H. Nakaseko, Comparison of the neurotoxicity of several chemicals estimated by the peripheral nerve conduction velocity in rats, Environ. Res., 62, 148–154, 1993. 239. S.V. Chandra and G.S. Shukla, Manganese encephalopathy in growing rats, Environ. Res., 15, 28–37, 1978. 240. H. Eriksson, S. Lenngren, and E. Heilbronn, Effect of long-term administration of manganese on biogenic amine levels in discrete striatal regions of rat brain, Arch. Toxicol., 59, 426–431, 1987. 241. J.P. Nachtman, R.E. Tubben, and R.L. Commissaris, Behavioral effects of chronic manganese administration in rats: locomotor activity studies, Neurobehav. Toxicol. Teratol., 8, 711–715, 1986. 242. S.V. Chandra and G.S. Shukla, Effect of manganese on synthesis of brain catecholamines in growing rats, Acta Pharmacol. Toxicol., 48, 349–354, 1981. 243. S.V. Chandra, M.M. Ali, D.K. Saxena, and R.C. Murthy, Behavioral and neurochemical changes in rats simultaneously exposed to manganese and lead, Arch. Toxicol., 49, 49–56, 1981. 244. E. Bonilla and A.L.N. Prasad, Effects of chronic manganese intake on the levels of biogenic amines in rat brain regions, Neurobehav. Toxicol. Teratol., 6, 341–344, 1984. 245. A.M. Scheuhammer, Chronic manganese exposure in rats: histological changes in the pancreas, J. Toxicol. Environ. Health, 12, 353–360, 1983. 246. C.L. Witzleben, P. Pitlick, J. Bergmeyer, and R. Benoit, A new experimental model of intrahepatic cholestasis, Am. J. Pathol., 53, 409–423, 1968. 247. D. Wassermann and M. Wassermann, The ultrastructure of the liver cell in subacute manganese administration, Environ. Res., 14, 379–390, 1977. 248. R.R. Rogers, R.J. Garner, M.M. Riddle, R.W. Luebke, and R.J. Smialowicz, Augmentation of murine natural killer cell activity by manganese chloride, Toxicol. Appl. Pharmacol., 70, 7–17, 1983. 249. B. Srisuchart, M.J. Taylor, and R.P. Sharma, Alteration of humoral and cellular immunity in manganese chloride-treated mice, J. Toxicol. Environ. Health, 22, 91–99, 1987. 250. J. Komura and M. Sakamoto, Effects of manganese forms on biogenic amines in the brain and behavioral alterations in the mouse: long-term oral administration of several manganese compounds, Environ. Res., 57, 34–44, 1992. 251. D.N. Cox, G.J. Traiger, S.P. Jacober, and R.P. Hanzlik, Comparison of the toxicity of methylcyclopentadienyl manganese tricarbonyl with that of its two major metabolites, Toxicol. Lett., 39, 1–5, 1987. 252. P.A. McGinley, J.B. Morris, R.J. Clay, and G. Gianutsos, Disposition and toxicity of methylcyclopentadienyl manganese tricarbonyl in the rat, Toxicol. Lett., 36, 137–145, 1987.


253. P.J. Hakkinen and W.M. Haschek, Pulmonary toxicity of methylcyclopentadienyl manganese tricarbonyl: nonciliated bronchiolar epithelial (Clara) cell necrosis and alveolar damage in the mouse, rat, and hamster, Toxicol. Appl. Pharmacol., 65, 11–22, 1982. 254. R.P. Hanzlik, R. Stitt, and G.J. Traiger, Toxic effects of methylcyclopentadienyl manganese tricarbonyl (MMT) in rats: role of metabolism, Toxicol. Appl. Pharmacol., 56, 353–360, 1980. 255. R.J. Clay and J.B. Morris, Comparative pneumotoxicity of cyclopentadienyl manganese tricarbonyl and methylcyclopentadienyl manganese tricarbonyl, Toxicol. Appl. Pharmacol., 98, 434–443, 1989. 256. B.E. Fishman, P.A. McGinley, and G. Gianutsos, Neurotoxic effects of methylcyclopentadienyl manganese tricarbonyl (MMT) in the mouse: basis of MMT-induced seizure activity, Toxicology, 45, 193–201, 1987. 257. R. Kishi, K. Hashimoto, S. Shimizu, and M. Kobayashi, Behavioral changes and mercury concentrations in tissues of rats exposed to mercury vapor, Toxicol. Appl. Pharmacol., 46, 555–566, 1978. 258. A. Fredriksson, L. Dahlgren, B. Danielsson, P. Eriksson, L. Dencker, and T. Archer, Behavioural effects of neonatal metallic mercury exposure in rats, Toxicology, 74, 151–160, 1992. 259. B.R.G. Danielsson, A. Fredriksson, L. Dahlgren, A.T. Gårdlund, L. Olsson, L. Dencker, and T. Archer, Behavioural effects of prenatal metallic mercury inhalation exposure in rats, Neurotoxicol. Teratol., 15, 391–396, 1993. 260. F. Livardjani, M. Ledig, P. Kopp, M. Dahlet, M. Leroy, and A. Jaeger, Lung and blood superoxide dismutase activity in mercury vapor exposed rats: effect of N-acetylcysteine treatment, Toxicology, 66, 289–295, 1991. 261. G.P. Daston, R.J. Kavlock, E.H. Rogers, and B. Carver, Toxicity of mercuric chloride to the developing rat kidney. I. Postnatal ontogeny of renal sensitivity, Toxicol. Appl. Pharmacol., 71, 24–41, 1983. 262. G.M. Kyle, R. Luthra, J.V. Bruckner, W.F. MacKenzie, and D. Acosta, Assessment of functional, morphological, and enzymatic tests for acute nephrotoxicity induced by mercuric chloride, J. Toxicol. Environ. Health, 12, 99–117, 1983. 263. E.M. McDowell, R.B. Nagle, R.C. Zalme, J.S. McNeil, W. Flamenbaum, and B.F. Trump, Studies on the pathophysiology of acute renal failure. I. Correlation of ultrastructure and function in the proximal tubule of the rat following administration of mercuric chloride, Virchows Arch. B Cell Pathol., 22, 173–196, 1976. 264. M.-A. Duran, D. Spencer, M. Weise, N.O. Kronfol, R.F. Spencer, and D.E. Oken, Renal epithelial amino acid concentrations in mercury-induced and postischemic acute renal failure, Toxicol. Appl. Pharmacol., 105, 183–194, 1990. 265. B.O. Lund, D.M. Miller, and J.S. Woods, Studies on Hg(II)-induced H2O2 formation and oxidative stress in vivo and in vitro in rat kidney mitochondria, Biochem. Pharmacol., 45, 2017–2024, 1993. 266. X. Liu, T. Jin, and G.F. Nordberg, Increased urinary calcium and magnesium excretion in rats injected with mercuric chloride, Pharmacol. Toxicol., 68, 254–259, 1991. 267. H. Fukino, M. Hirai, Y.M. Hsueh, S. Moriyasu, and Y. Yamane, Mechanism of protection by zinc against mercuric chloride toxicity in rats: effects of zinc and mercury on glutathione metabolism, J. Toxicol. Environ. Health., 19, 75–89, 1986. 268. H. Fukino, M. Hirai, Y.M. Hsueh, and Y. Yamane, Effect of zinc pretreatment on mercuric chlorideinduced lipid peroxidation in the rat kidney, Toxicol. Appl. Pharmacol., 73, 395–401, 1984. 269. L. Magos, S. Sparrow, and R. Snowden, The comparative renotoxicology of phenylmercury and mercuric chloride, Arch. Toxicol., 50, 133–139, 1982. 270. Y. Yamane and T. Koizumi, Protective effect of molybdenum on the acute toxicity of mercuric chloride, Toxicol. Appl. Pharmacol., 65, 214–221, 1982. 271. D. Holt and M. Webb, The toxicity and teratogenicity of mercuric mercury in the pregnant rat, Arch. Toxicol., 58, 243–248, 1986. 272. A.M. Bernard, C. Collette, and R. Lauwerys, Renal effects of in utero exposure to mercuric chloride in rats, Arch. Toxicol., 66, 508–513, 1992. 273. D. Pezerovic, P. Narancsik, and S. Gamulin, Effects of mercury bichloride on mouse kidney polyribosome structure and function, Arch. Toxicol., 48, 167–172, 1981. 274. T. Tanaka, A. Naganuma, and N. Imura, Role of γ-glutamyltranspeptidase in renal uptake and toxicity of inorganic mercury in mice, Toxicology, 60, 187–198, 1990. 275. J.B. Nielsen, H.R. Andersen, O. Andersen, and H. Starklint, Mercuric chloride-induced kidney damage in mice: time course and effect of dose, J. Toxicol. Environ. Health, 34, 469–483, 1991. Copyright © 2002 by Taylor & Francis

276. A. Yasutake, K. Hirayama, and M. Inouye, Sex difference in acute renal dysfunction induced by methylmercury in mice, Renal Failure, 12, 233–240, 1990. 277. A.R. Chowdhury, S. Makhija, K.D. Vachhrajani, and A.K. Gautam, Methylmercury- and mercuric chloride-induced alterations in rat epididymal sperm, Toxicol. Lett., 47, 125–134, 1989. 278. M.K. Lakshmana, T. Desiraju, and T.R. Raju, Mercuric chloride-induced alterations of levels of noradrenaline, dopamine, serotonin and acetylcholine esterase activity in different regions of rat brain during postnatal development, Arch. Toxicol., 67, 422–427, 1993. 279. M. Carmignani, V.N. Finelli, and P. Boscolo, Mechanisms in cardiovascular regulation following chronic exposure of male rats to inorganic mercury, Toxicol. Appl. Pharmacol., 69, 442–450, 1983. 280. T.M.C. Tan, Y.M. Sin, and K.P. Wong, Mercury-induced UDP glucuronyltransferase (UDPGT) activity in mouse kidney, Toxicology, 64, 81–87, 1990. 281. P. Hultman and S. Eneström, Dose-response studies in murine mercury-induced autoimmunity and immune-complex disease, Toxicol. Appl. Pharmacol., 113, 199–208, 1992. 282. M.P. Dieter, M.I. Luster, G.A. Boorman, C.W. Jameson, J.H. Dean, and J.W. Cox, Immunological and biochemical responses in mice treated with mercuric chloride, Toxicol. Appl. Pharmacol., 68, 218–228, 1983. 283. B.B. Hoskins and E.W. Hupp, Methylmercury effects in rat, hamster, and squirrel monkey: lethality, symptoms, brain mercury, and amino acids, Environ. Res., 15, 5–19, 1978. 284. S. Omata, H. Tsubaki, K. Sakimura, M. Sato, R. Yoshimura, E. Hirakawa, and H. Sugano, Stimulation of protein and RNA synthesis by methylmercury chloride in the liver of intact and adrenalectomized rats, Arch. Toxicol., 47, 113–123, 1981. 285. T.L.M. Syversen, G. Totland, and P.R. Flood, Early morphological changes in rat cerebellum caused by a single dose of methylmercury, Arch. Toxicol., 47, 101–111, 1981. 286. M.K. Cheung and M.A. Verity, Experimental methyl mercury neurotoxicity: locus of mercurial inhibition of brain protein synthesis in vivo and in vitro, J. Neurochem., 44, 1799–1808, 1985. 287. S.F. Ali, C.P. Lebel, and S.C. Bondy, Reactive oxygen species formation as a biomarker of methylmercury and trimethyltin neurotoxicity, Neurotoxicology, 13, 637–648, 1992. 288. J. De Ceaurriz and M. Ban, Role of γ-glutamyltranspeptidase and β-lyase in the nephrotoxicity of hexachloro-1,3-butadiene and methyl mercury in mice, Toxicol. Lett., 50, 249–256, 1990. 289. P.R. Sager, M. Aschner, and P.M. Rodier, Persistent, differential alterations in developing cerebellar cortex of male and female mice after methylmercury exposure, Dev. Brain Res., 12, 1–11, 1984. 290. K. Nomiyama, K. Matsui, and H. Nomiyama, Effects of temperature and other factors on the toxicity of methylmercury in mice, Toxicol. Appl. Pharmacol., 56, 392–398, 1980. 291. P. Salvaterra, E.J. Massaro, J.B. Morganti, and B.A. Lown, Time-dependent tissue/organ uptake and distribution of 203Hg in mice exposed to multiple sublethal doses of methyl mercury, Toxicol. Appl. Pharmacol., 32, 432–442, 1975. 292. A. Yasutake and K. Hirayama, Acute effects of methylmercury on hepatic and renal glutathione metabolisms in mice, Arch. Toxicol., 68, 512–516, 1994. 293. M. Sato, H. Sugano, and Y. Takizawa, Effects of methylmercury on zinc-thionein levels of rat liver, Arch. Toxicol., 47, 125–133, 1981. 294. M. Kabuto, Chronic effects of methylmercury on the urinary excretion of catecholamines and their responses to hypoglycemic stress, Arch. Toxicol., 65, 164–167, 1991. 295. P.A. Desnoyers and L.W. Chang, Ultrastructual changes in rat hepatocytes following acute methyl mercury intoxication, Environ. Res., 9, 224–239, 1975. 296. L. Magos, G.C. Peristianis, T.W. Clarkson, A. Brown, S. Preston, and R.T. Snowden, Comparative study of the sensitivity of male and female rats to methylmercury, Arch. Toxicol., 48, 11–20, 1981. 297. M.-F. Wu, J.R. Ison, J.R. Wecker, and L.W. Lapham, Cutaneous and auditory function in rats following methyl mercury poisoning, Toxicol. Appl. Pharmacol., 79, 377–388, 1985. 298. R.J. Hargreaves, B.P. Eley, S.R. Moorhouse, and D. Pelling, Regional cerebral glucose metabolism and blood flow during the silent phase of methylmercury neurotoxicity in rats, J. Neurochem., 51, 1350–1355, 1988. 299. O. Kawamata, H. Kasama, S. Omata, and H. Sugano, Decrease in protein phosphorylation in central and peripheral nervous tissues of methylmercury-treated rat, Arch. Toxicol., 59, 346–352, 1987. 300. M.A. Verity, W.J. Brown, M. Cheung, and G. Czer, Methyl mercury inhibition of synaptosome and brain slice protein synthesis: in vivo and in vitro studies, J. Neurochem., 29, 673–679, 1977.


301. W.E. Stroo and J.B. Hook, Renal functional correlates of methyl mercury intoxication: interaction with acute mercuric chloride toxicity, Toxicol. Appl. Pharmacol., 42, 399–410, 1977. 302. M. Yonaha, M. Saito, and M. Sagai, Stimulation of lipid peroxidation by methyl mercury in rats, Life Sci., 32, 1507–1514, 1983. 303. J.R. O’Kusky and E.G. McGeer, Methylmercury-induced movement and postural disorders in developing rat: high-affinity uptake of choline, glutamate, and γ-aminobutyric acid in the cerebral cortex and caudate-putamen, J. Neurochem., 53, 999–1006, 1989. 304. P.A. Desnoyers and L.W. Chang, Ultrastructual changes in the liver after chronic exposure to methylmercury, Environ. Res., 10, 59–75, 1975. 305. K. Saijoh, T. Fukunaga, H. Katsuyama, M.J. Lee, and K. Sumino, Effects of methylmercury on protein kinase A and protein kinase C in the mouse brain, Environ. Res., 63, 264–273, 1993. 306. A. Yasutake and K. Hirayama, Sex and strain differences of susceptibility to methylmercury toxicity in mice, Toxicology, 51, 47–55, 1988. 307. A. Yasutake, T. Adachi, K. Hirayama, and M. Inouye, Integrity of the blood-brain barrier system against methylmercury acute toxicity, Jpn. J. Toxicol. Environ. Health, 37, 355–362, 1991. 308. H. Lindström, J. Luthman, A. Oskarsson, J. Sundberg, and L. Olson, Effects of long-term treatment with methyl mercury on the developing rat brain, Environ. Res., 56, 158–169, 1991. 309. B. Moller-Madsen and G. Danscher, Localization of mercury in CNS of the rat. IV. The effect of selenium on orally administered organic and inorganic mercury, Toxicol. Appl. Pharmacol., 108, 457–473, 1991. 310. J.S. Woods, M.A. Bowers, and H.A. Davis, Urinary porphyrin profiles as biomarkers of trace metal exposure and toxicity: studies on urinary porphyrin excretion patterns in rats during prolonged exposure to methyl mercury, Toxicol. Appl. Pharmacol., 110, 464–476, 1991. 311. J.S. Woods, H.A. Davis, and R.P. Baer, Enhancement of γ-glutamylcysteine synthetase mRNA in rat kidney by methyl mercury, Arch. Biochem. Biophys., 296, 350–353, 1992. 312. K. Mitsumori, K. Takahashi, O. Matano, S. Goto, and Y. Shirasu, Chronic toxicity of methylmercury chloride in rats: clinical study and chemical analysis, Jpn. J. Vet. Sci., 45, 747–757, 1983. 313. K. Mitsumori, K. Maita, and Y. Shirasu, Chronic toxicity of methylmercury chloride in rats: pathological study, Jpn. J. Vet. Sci., 46, 549–557, 1984. 314. H.R. Andersen and O. Andersen, Effects of dietary α-tocopherol and β-carotene on lipid peroxidation induced by methyl mercuric chloride in mice. Pharmacol. Toxicol., 73, 192–201, 1993. 315. S.G. Gilbert and J.P.J. Maurissen, Assessment of the effects of acrylamide, methylmercury, and 2,5hexanedione on motor functions in mice, J. Toxicol. Environ. Health, 10, 31–41, 1982. 316. K. Mitsumori, M. Hirano, H. Ueda, K. Maita, and Y. Shirasu, Chronic toxicity and carcinogenicity of methylmercury chloride in B6C3F1 mice, Fundam. Appl. Toxicol., 14, 179–190, 1990. 317. J.A.G. Geelen, J.A.M.A. Dormans, and A. Verhoef, The early effects of methylmercury on the developing rat brain, Acta Neuropathol., 80, 432–438, 1990. 318. R. Cagiano, M.A. De Salvia, G. Renna, E. Tortella, D. Braghiroli, C. Parenti, P. Zanoli, M. Baraldi, Z. Annau, and V. Cuomo, Evidence that exposure to methyl mercury during gestation induces behavioral and neurochemical changes in offspring of rats, Neurotoxicol. Teratol., 12, 23–28, 1990. 319. A.M. Saillenfait, M.T. Brondeau, D. Zissu, and J. De Ceaurriz, Effects of prenatal methylmercury exposure on urinary proximal tubular enzyme excretion in neonatal rats, Toxicology, 55, 153–160, 1989. 320. M. Bornhausen, H.R. Müsch, and H. Greim, Operant behavior performance changes in rats after prenatal methylmercury exposure, Toxicol. Appl. Pharmacol., 56, 305–310, 1980. 321. N.H. Peckham and B.H. Choi, Abnormal neuronal distribution within the cerebral cortex after prenatal methylmercury intoxication, Acta Neuropathol., 76, 222–226, 1988. 322. M. Inouye and Y. Kajiwara, Strain difference of the mouse in manifestation of hydrocephalus following prenatal methylmercury exposure, Teratology, 41, 205–210, 1990. 323. T. Nobunaga, H. Satoh, and T. Suzuki, Effects of sodium selenite on methylmercury embryotoxicity and teratogenicity in mice, Toxicol. Appl. Pharmacol., 47, 79–88, 1979. 324. M. Inouye and Y. Kajiwara, Developmental disturbances of the fetal brain in guinea-pigs caused by methylmercury, Arch. Toxicol., 62, 15–21, 1988. 325. F.W. Sunderman, Jr., M.C. Reid, L.M. Bibeau, and J.V. Linden, Nickel induction of microsomal heme oxygenase activity in rodents, Toxicol. Appl. Pharmacol., 68, 87–95, 1983.


326. F.W. Sunderman, Jr., S.K. Shen, J.M. Mitchell, P.R. Allpass, and I. Damjanov, Embryotoxicity and fetal toxicity of nickel in rats, Toxicol. Appl. Pharmacol., 43, 381–390, 1978. 327. F.W. Sunderman, Jr., K.S. Kasprzak, T.J. Lau, P.P. Minghetti, R.M. Maenza, N. Becker, C. Onkelinx, and P.J. Goldblatt, Effects of manganese on carcinogenicity and metabolism of nickel subsulfide, Cancer Res., 36, 1790–1800, 1976. 328. R.J. Smialowicz, R.R. Rogers, D.G. Rowe, M.M. Riddle, and R.W. Luebke, The effects of nickel on immune function in the rat, Toxicology, 44, 271–281, 1987. 329. K.S. Kasprzak, M.P. Waalkes, and L.A. Poirier, Effects of magnesium acetate on the toxicity of nickelous acetate in rats, Toxicology, 42, 57–68, 1986. 330. F.W. Sunderman, Jr., S.M. Hopfer, S.-M. Lin, M.C. Plowman, T. Stojanovic, S.H-Y. Wong, O. Zaharia, and L. Ziebka, Toxicity to alveolar macrophages in rats following parenteral injection of nickel chloride, Toxicol. Appl. Pharmacol., 100, 107–118, 1989. 331. M. Misra, R.E. Rodriguez, and K.S. Kasprzak, Nickel induced lipid peroxidation in the rat: correlation with nickel effect on antioxidant defense systems, Toxicology, 64, 1–17, 1990. 332. M. Misra, R. Olinski, M. Dizdaroglu, and K.S. Kasprzak, Enhancement by L-histidine of nickel (II)induced DNA-protein cross-linking and oxidative DNA base damage in the rat kidney, Chem. Res. Toxicol., 6, 33–37, 1993. 333. J. Cartana and L. Arola, Nickel-induced hyperglycaemia: the role of insulin and glucagon, Toxicology, 71, 181–192, 1992. 334. M.K. Smith, E.L. George, J.A. Stober, H.A. Feng, and G.L. Kimmel, Perinatal toxicity associated with nickel chloride exposure, Environ. Res., 61, 200–211, 1993. 335. R.E. Rodriguez, M. Misra, S.L. North, and K.S. Kasprzak, Nickel-induced lipid peroxidation in the liver of different strains of mice and its relation to nickel effects on antioxidant systems, Toxicol. Lett., 57, 269–281, 1991. 336. G.L. Fisher, C.E. Chrisp, and D.A. McNeill, Lifetime effects of intratracheally instilled nickel subsulfide on B6C3F1 mice, Environ. Res., 40, 313–320, 1986. 337. R.J. Smialowicz, R.R. Rogers, M.M. Riddle, R.J. Garner, D.G. Rowe, and R.W. Luebke, Immunologic effects of nickel. II. Suppression of natural killer cell activity, Environ. Res., 36, 56–66, 1985. 338. G.R. Hogan, Nickel acetate-induced mortality in mice of different ages, Bull. Environ. Contam. Toxicol., 34, 446–450, 1985. 339. M.P. Dieter, C.W. Jameson, A.N. Tucker, M.I. Luster, J.E. French, H.L. Hong, and G.A. Boorman, Evaluation of tissue disposition, myelopoietic, and immunologic responses in mice after long-term exposure to nickel sulfate in the drinking water, J. Toxicol. Environ. Health, 24, 357–372, 1988. 340. J.K. Dunnick, J.M. Benson, C.H. Hobbs, F.F. Hahn, Y.S. Cheng, and A.F. Edison, Comparative toxicity of nickel oxide, nickel sulfate hexahydrate, and nickel subsulfide after 12 days of inhalation exposure to F344/N rats and B6C3F1 mice, Toxicology, 50, 145–156, 1988. 341. L.L. David and T.R. Shearer, State of sulfhydryl in selenite cataract, Toxicol. Appl. Pharmacol., 74, 109–115, 1984. 342. R.A. LeBoeuf and W.G. Hoekstra, Adaptive changes in hepatic glutathione metabolism in response to excess selenium in rats, J. Nutr., 113, 845–854, 1983. 343. M. Jacobs and C. Forst, Toxicological effects of sodium selenite in Sprague-Dawley rats, J. Toxicol. Environ. Health, 8, 575–585, 1981. 344. G.R. Hogan, Decreased levels of peripheral leukocytes following sodium selenite treatment in female mice, Bull. Environ. Contam. Toxicol., 37, 175–179, 1986. 345. C. Watanabe and T. Suzuki, Sodium selenite-induced hypothermia in mice: indirect evidence for a neural effect, Toxicol. Appl. Pharmacol., 86, 372–379, 1986. 346. M. Jacobs and C. Forst, Toxicological effects of sodium selenite in Swiss mice, J. Toxicol. Environ. Health, 8, 587–598, 1981. 347. M.A. Al-Bayati, O.G. Raabe, and S.V. Teague, Effect of inhaled dimethylselenide in the Fischer 344 male rat, J. Toxicol Environ. Health, 37, 549–557, 1992. 348. C. Rios and A. Monroy-Noyola, D-Penicillamine and Prussian blue as antidotes against thallium intoxication in rats, Toxicology, 74, 69–76, 1992. 349. D.B. Peele, R.C. MacPhail, and J.D. Farmer, Flavor aversions induced by thallium sulfate: importance of route of administration, Neurobehav. Toxicol. Teratol., 8, 273–277, 1986.


350. M. Hasan and S.S. Haider, Acetyl-homocysteine thiolactone protects against some neurotoxic effects of thallium, Neurotoxicology, 10, 257–262, 1989. 351. L. Osorio-Rico, S. Galvan-Arzate, and C. Rios, Thallium increases monoamine oxidase activity and serotonin turnover rate in rat brain regions, Neurotoxicol. Teratol., 17, 1–5, 1995. 352. J. Marwaha, R. Freedman, and B. Hoffer, Electrophysiological changes at a central noradrenergic synapse during thallium toxicosis, Toxicol. Appl. Pharmacol., 56, 345–352, 1980. 353. J.S. Woods and B.A. Fowler, Alteration of hepatocellular structure and function by thallium chloride: ultrastructural, morphometric, and biochemical studies, Toxicol. Appl. Pharmacol., 83, 218–229, 1986. 354. D. Appenroth, S. Gambaryan, K. Winnefeld, M. Leiterer, C. Fleck, and H. Braunlich, Functional and morphological aspects of thallium-induced nephrotoxicity in rats, Toxicology, 96, 203–215, 1995. 355. L.Formigli, R. Scelsi, P. Poggi, C. Gregotti, A.D. Nucci, E. Sabbioni, L. Gottardi, and L. Manzo, Thallium-induced testicular toxicity in the rat, Environ. Res., 40, 531–539, 1986. 356. A.P. de Groot, V.J. Feron, and H.P. Til, Short-term toxicity studies on some salts and oxides of tin in rats, Food Cosmet. Toxicol., 11, 19–30, 1973. 357. P.J.M. Janssen, M.C. Bosland, J.P. Van Hees, B.J. Spit, M.I. Willems, and C.F. Kuper, Effects of feeding stannous chloride on defferent parts of the gastrointestinal tract of the rat, Toxicol. Appl. Pharmacol., 78, 19–28, 1985. 358. H.C. Dreef-van der Meulen, V.J. Feron, and H.P. Til, Pancreatic atrophy and other pathological changes in rats following the feeding of stannous chloride, Pathol. Eur., 9, 185–192, 1974. 359. G. Zareba and J. Chmielnicka, Aminolevulinic acid dehydratase activity in the blood of rats exposed to tin and zinc, Ecotoxicol. Environ. Saf., 9, 40–46, 1985. 360. G.J. Harry, J.F. Goodrum, M.R. Krigman, and P. Morell, The use of synapsin I as a biochemical marker for neuronal damage by trimethyltin. Brain Res., 326, 9–18, 1985. 361. L.W. Chang, G.R. Wenger, D.E. McMillan, and R.S. Dyer, Species and strain comparison of acute neurotoxic effects of trimethyltin in mice and rats, Neurobehav. Toxicol. Teratol., 5, 337–350, 1983. 362. R.S. Dyer, T.L. Deshields, and W.F. Wonderlin, Trimethyltin-induced changes in gross morphology of the hippocampus, Neurobehav. Toxicol. Teratol., 4, 141–147, 1982. 363. T.J. Walsh, M. Gallagher, E. Bostock, and R.S. Dyer, Trimethyltin impairs retention of a passive avoidance task, Neurobehav. Toxicol. Teratol., 4, 163–167, 1982. 364. R.S. Dyer, W.E. Howell, and W.F. Wonderlin, Visual system dysfunction following acute trimethyltin exposure in rats, Neurobehav. Toxicol. Teratol., 4, 191–195, 1982. 365. R.C. MacPhail, Studies on the flavor aversions induced by trialkyltin compounds, Neurobehav. Toxicol. Teratol., 4, 225–230, 1982. 366. T.J. Walsh, R.L. McLamb, and H.A. Tilson, Organometal-induced antinociception: a time- and doseresponse comparison of triethyl and trimethyl lead and tin, Toxicol. Appl. Pharmacol., 73, 295–299, 1984. 367. P.R.S. Kodavanti, J.A. Cameron, P.R. Yallapragada, P.J.S.Vig, and D. Desaiah, Inhibition of Ca2+ transport associated with cAMP-dependent protein phosphorylation in rat cardiac sarcoplasmic reticulum by triorganotins, Arch. Toxicol., 65, 311–317, 1991. 368. R.S. Dyer and W.E. Howell, Acute triethyltin exposure: effects on the visual evoked potential and hippocampal afterdischarge, Neurobehav. Toxicol. Teratol., 4, 259–266, 1982. 369. H.A. Tilson and T.A. Burne, Effects of triethyl tin on pain reactivity and neuromotor function of rats, J. Toxicol. Environ. Health, 8, 317–324, 1981. 370. D.B. Miller, D.A. Eckerman, M.R. Krigman, and L.D. Grant, Chronic neonatal organotin exposure alters radial-arm maze performance in adult rats, Neurobehav. Toxicol. Teratol., 4, 185–190, 1982. 371. P. Mushak, M.R. Krigman, and R.B. Mailman, Comparative organotin toxicity in the developing rat: somatic and morphological changes and relationship to accumulation of total tin, Neurobehav. Toxicol. Teratol., 4, 209–215, 1982. 372. C.P. LeBel, S.F. Ali, M. McKee, and S.C. Bondy, Organometal-induced increases in oxygen reactive species: the potential of 2′,7′-dichlorofluorescin diacetate as an index of neurotoxic damage, Toxicol. Appl. Pharmacol., 104, 17–24, 1990. 373. G.R. Wenger, D.E. McMillan, and L.W. Chang, Behavioral toxicology of acute trimethyltin exposure in the mouse, Neurobehav. Toxicol. Teratol., 4, 157–161, 1982. 374. D.A. Fox, Pharmacological and biochemical evaluation of triethyltin’s anticonvulsant effects, Neurobehav. Toxicol. Teratol., 4, 273–278, 1982.


375. M. Ghoneum, A.E. Hussein, G. Gill, and L.J. Alfred, Suppression of murine natural killer cell activity by tributyltin: in vivo and in vitro assessment, Environ. Res., 52, 178–186, 1990. 376. A.D. Nucci, C. Gregotti, and L. Manzo, Triphenyl tin hepatotoxicity in rats, Arch. Toxicol., 59, 402–405, 1986. 377. K. Lehotzky, J.M. Szeberenyi, Z. Gonda, F. Horkay, and A. Kiss, Effects of prenatal triphenyl-tin exposure on the development of behavior and conditioned learning in rat pups, Neurobehav. Toxicol. Teratol., 4, 247–250, 1982. 378. M. Yoshizuka, K. Hara, N. Haramaki, M. Yokoyama, N. Mori, Y. Doi, A. Kawahara, and S. Fujimoto, Studies on the hepatotoxicity induced by bis (tributyltin) oxide, Arch. Toxicol., 66, 182–187, 1992. 379. M. Yoshizuka, N. Haramaki, M. Yokoyama, K. Hara, A. Kawahara, Y. Umezu, H. Araki, N. Mori, and S. Fujimoto, Corneal edema induced by bis (ributyltin) oxide, Arch. Toxicol., 65, 651–655, 1991. 380. W. Seinen and M.I. Willems, Toxicity of organotin compounds. I. Atrophy of thymus and thymusdependent lymphoid tissue in rats fed di-n-octyltindichloride, Toxicol. Appl. Pharmacol., 35, 63–75, 1976. 381. W. Seinen, J.G. Vos, I.V. Spanje, M. Snoek, R. Brands, and H. Hooykaas, Toxicity of organotin compounds. II. Comparative in vivo and in vitro studies with various organotin and organolead compounds in different animal species with special emphasis on lymphocyte cytotoxicity, Toxicol. Appl. Pharmacol., 42, 197–212, 1977. 382. K. Miller, J. Maisey, and S. Nicklin, Effect of orally administered dioctyltin dichloride on murine immunocompetence, Environ. Res., 39, 434–441, 1986. 383. G.R. Hogan, B.S. Cole, and J.M. Lovelace, Sex and age mortality responses in zinc acetate-treated mice, Bull. Environ. Contam. Toxicol., 39, 156–161, 1987. 384. J.M. Llobet, J.L. Domingo, M.T. Colomina, E. Mayayo, and J. Corbella, Subchronic oral toxicity of zinc in rats, Bull Environ. Contam. Toxicol., 41, 36–43, 1988. 385. G. Cosma, H. Fulton, T. Defeo, and T. Gordon, Rat lung metallothionein and heme oxygenase gene expression following ozone and zinc oxide exposure, Toxicol. Appl. Pharmacol., 117, 75–80, 1992. 386. S. Hirano, S. Higo, N. Tsukamoto, E. Kobayashi, and K.T. Suzuki, Pulmonary clearance and toxicity of zinc oxide instilled into the rat lung, Arch. Toxicol., 63, 336–342, 1989. 387. H.F. Lam, M.W. Conner, A.E. Rogers, S. Fitzgerald, and M.O. Amdur, Functional and morphologic changes in the lung of guinea pigs exposed to freshly generated ultrafine zinc oxide, Toxicol. Appl. Pharmacol., 78, 29–38, 1985.


23

Human Clinical Toxicology Jill Dolgin, Pharm. D., A.B.A.T

CONTENTS Section 1. Introduction Section 2. Clinical Research Design Table 23.1 The Three Main Tasks of Experimental Toxicology Table 23.2 Five Criteria that Test the Validity of Animal Models Section 3. Epidemiology of Poisoning A. General Characteristics Table 23.3 Substances Most Frequently Involved in Human Exposure Table 23.4 Categories with Largest Numbers of Deaths Table 23.5 Distribution of Route of Exposure for Human Poison Exposure Cases and 764 Fatalities Table 23.6 Distribution of Reason for Exposure by Age Table 23.7 Distribution of Reason for Exposure and Age for 764 Fatalities Table 23.8 Number of Substances Involved in Human Poison Exposure Cases B. Pediatric Poisonings Table 23.9 Reported Poison Exposures in Children Younger than 6 years of Age, 1985 through 1989 Table 23.10 Pediatric Pharmaceutical Ingestion Fatalities: 1983–1990 Table 23.11 Pediatric Nonpharmaceutical Ingestion Fatalities: 1983–1990 Table 23.12 Ingestions of Solid Prescription Drugs, by Age of Victim Table 23.13 Ingestions of Solid Prescription Drugs, by Container Type Table 23.14 Leading Solid Prescription Drugs Ingested, Resulting in Hospitalization Table 23.15 Owner of Medication Ingested Table 23.16 Container Type and Ownership of Medication Table 23.17 Risk Factors Involved in Drug Poisoning in Children 0 to 17 Years C. Adolescent Toxic Exposures Table 23.18 Drug-Related Fatalities by Drug Class among Adolescents 11 to 17 Years Old Reported to the American Association of Poison Control Centers in 1989, 1990, and 1991 D. Poisoning in the Elderly Table 23.19 Distribution of Age and Sex for 764 Fatalities Table 23.20 Annualized Poisoning Hospitalization Rates and Relative Risks, Massachusetts 1982–1985 Table 23.21 Annualized Poisoning-Related Death Rates and Relative Risks, Massachusetts 1983–1985


Section 4.

Section 5.

Section 6.

Section 7.

Table 23.22 Intentionality and Agent of Poisoning Deaths in Massachusetts Adults 60 Years and Older, 1983–1985 Management Trends Table 23.23 Therapy Provided in Human Exposure Cases Table 23.24 Ipecac Administration by Site and Age Table 23.25 Decontamination Trends Role of the Toxicology Laboratory Table 23.26 Common Drugs Included on Most Toxicology Screens Table 23.27 Types of Medical and Toxicological Test Situations Table 23.28 Some Common Clinical Presentations and Differential Diagnoses in Overdose Table 23.29 Toxicological Syndromes by Class of Drugs Table 23.30 Useful Laboratory Tests in Toxicological Diagnosis Table 23.31 Comparison of Generic Toxicological Methods Table 23.32 Potential Interferences for Quantitative Serum Drug Tests Used in Emergency Toxicology Role of the Poison Control Center Table 23.33 Site of Caller and Site of Exposure to Human Poison Exposure Cases Table 23.34 Symptom Assessment at the Time of Initial Call to Poison Center Table 23.35 Management Site of Human Poison Exposure Cases Table 23.36 Medical Outcome of Human Poison Exposure Cases by Patient Age Table 23.37 Distribution of Medical Outcome by Reason for Exposure for Human Poison Exposure Victims Contemporary Issues Table 23.38 Nationwide Poison Control Centers — Directory

References

SECTION 1. INTRODUCTION Nearly two million human poisonings are reported to poison information centers each year; however, there are an estimated 2 to 3 million additional unreported exposures.1 The purpose of this chapter is to review the epidemiological characteristics of human poisoning, clinical toxicology research designs, and general management techniques of the acutely poisoned patient. In addition, the role of the poison control centers in managing these patients and several contemporary issues in clinical toxicology are discussed. Although several excellent texts contain valuable information on the toxicity and treatment of poisoned patients,2,3 the most up-to-date information on both human and animal poisonings is provided by Poisindex® (MICROMEDEX, Medical Information Systems, Denver). It contains data on the chemical composition, toxicity, and the current medical management of more than 750,000 drugs, household chemicals, industrial and environmental toxins, and biologicals (including plant and animal toxins). Poisindex® also facilitates the identification of manufactured drugs by providing a description of the tablet/capsule shape, color, and the symbols imprinted on them. It also provides slang terminology, color, and shape for street drugs. Poisindex is edited and updated every 3 months. Another valuable source of information is a regional poison control center. Currently there are more than 100 regional poison control centers located throughout the United States; 38 have been certified by the American Association of Poison Control Centers (AAPCC). (See Section 6.)


SECTION 2. CLINICAL RESEARCH DESIGN Predicting the effects of toxic agents in animals are critical and mandatory facts of human risk management. The three main tasks of experimental animal studies in toxicology are listed in Table 23.1.4 Basic to understanding human toxicities is the assumption that information gained from animal models can be extrapolated to analogous human situations. This places great importance on the validations of extrapolations from animal data. However, extrapolation from an animal requires specification of the effects that will serve to test the validity of the model. A hierarchy of five criteria, shown in Table 23.2, can be used to determine the validity of animal models. Other relevant information, from epidemiology studies and clinical research, is then integrated with the animal data to make regulatory decisions regarding human safety. The principle of research design in clinical toxicology involves assessing causation in diseaseexposure associations, evaluating the appropriateness of the research design, and evaluating the validity of a particular research study. The various research designs available to study clinical problems include the randomized clinical trial (RCT), cohort studies, case-control studies, cross-sectional studies, case series, and case reports. The advantages and disadvantages of each method are beyond the scope of this chapter and are outlined in Chapter 24 and discussed in several excellent texts on epidemiology.6,7

TABLE 23.1 The Three Main Tasks of Experimental Toxicology 1. Spectrum of toxicity Detection of adverse effects of chemicals in selected laboratory animal species and description of the dose-effect relationship over a broad range of doses. 2. Extrapolation Prediction of adverse effects in other species, particularly in man. 3. Safety Prediction of safe levels of exposure in other species, particularly in man. From Zbinden (1991).4 Reprinted with permission.

TABLE 23.2 Five Criteria that Test the Validity of Animal Models 1. Face validity A model is superficially similar to the human condition. 2. Content validity Examining the characteristics shared in common by an animal model and the human condition it seeks to simulate, to determine whether the model represents the specific content which a study is designed to measure 3. Concurrent validity Multiple measures of toxic reactions within subjects can provide profiles distinctive to particular toxins or classes of toxins. 4. Construct validity Utilizing a theoretical model of the nature of living organisms and how they interact with their biosphere 5. Predictive validity Demonstration that extrapolation from animal models compares or can be predictive of human toxicity From Russell (1991).5 Reprinted with permission.


SECTION 3. EPIDEMIOLOGY OF POISONING A. GENERAL CHARACTERISTICS The AAPCC estimates that 4.4 million poisonings occurred nationwide in 1991. Of the 1.8 million exposures reported, 764 resulted in death. Although nonpharmaceuticals were involved in more than 50% of all poisonings (Table 23.3), pharmaceuticals were most frequently involved in fatalities (Table 23.4). Ingestion was the most common route of exposure (Table 23.5). Accidental exposures accounted for more than 87% of all poisonings (Table 23.6); they were most common in children younger than 6 years of age and in the elderly. Intentional exposures were most common in adolescents (>14 years of age) and the most frequent cause of death in the adult population (Tables 23.6 and 23.7). Most accidental poisonings involved only one substance; however, 50 to 60% of intentional poisonings in adults were poly-drug exposures (Table 23.8).

TABLE 23.3 Substances Most Frequently Involved in Human Exposure No.

%a

191,830 183,013 153,424 112,564 105,185 76,941 70,523 69,096 64,805 64,472 63,536 58,450 53,666 50,296 46,482 40,883

10.4 10.0 8.3 6.1 5.7 4.2 3.8 3.8 3.5 3.5 3.5 3.2 2.9 2.7 2.5 2.2

Substance Cleaning substances Analgesics Cosmetics Plants Cough and cold preparations Bites/envenomations Pesticides (includes rodenticides) Topicals Antimicrobials Foreign bodies Hydrocarbons Sedatives/hypnotics/antipsychotics Chemicals Alcohols Food poisoning Vitamins

Note: Despite a high frequency of involvement, these substances are not necessarily the most toxic, but rather often represent only ready availability. a

Percentages are based on the total number of human exposures rather than the total number of substances. From Litovitz et al., 1992.1 Reprinted with permission.


TABLE 23.4 Categories with Largest Numbers of Deaths Category

No.

% of All Exposures in Category

Analgesics Antidepressants Sedative/hypnotics Stimulants and street drugs Cardiovascular drugs Alcohols Gases and fumes Asthma therapies Chemicals Hydrocarbons Cleaning substances Pesticides (including rodenticides)

190 188 97 90 87 72 49 39 37 36 26 18

0.104 0.525 0.166 0.434 0.348 0.143 0.188 0.229 0.069 0.057 0.014 0.026

From Litovitz et al., 1992.1 Reprinted with permission.

TABLE 23.5 Distribution of Route of Exposure for Human Poison Exposure Cases and 764 Fatalities All Exposure Cases

Fatal Exposure Cases

Route

No.

%

No.

%

Ingestion Dermal Ophthalmic Inhalation Bites and stings Parenteral Other Unknown Total

1,473,361 143,196 119,027 107,634 72,331 5,217 4,917 6,423 1,932,106

76.3 7.4 6.2 5.6 3.7 0.3 0.3 0.3 100.0

622 8 2 116 5 37 3 29 822

75.7 1.0 0.2 14.1 0.6 4.5 0.4 3.5 100.0

Note: Multiple routes of exposure were observed in many poison exposure victims. Percentage is based on the total number of exposure mutes (1,932,106 for all patients, 822 for fatal cases) rather than the total number of human exposures (1,837,939) or fatalities (764). From Litovitz, 1992.1 Reprinted with permission.


TABLE 23.6 Distribution of Reason for Exposure by Age 18–64 Years

>64 Years

Unknown

Reason

No.

>6 Years %

6–12 Years No.

%

No.

%

No.

%

No.

%

No.

%

No.

%

Accidental Intentional Adverse reaction Unknown Total

1,093,264 2583 3437 784 1,100,068

59.5 0.1 0.2 0.0 59.9

93,941 6391 1812 556 102,700

5.1 0.3 0.1 0.0 5.6

39,874 40,286 1598 57 82,515

2.2 2.2 0.1 0.0 4.5

223,533 114,451 14,361 2877 355,222

12.2 6.2 0.8 0.2 19.3

25,409 3338 1564 419 30,730

1.4 0.2 0.1 0.0 1.7

130,486 27,148 7272 1798 166,704

7.1 1.5 0.4 0.1 9.1

1,606,507 194,197 30,044 7191 1,837,939

87.4 10.6 1.6 0.4 100.0



13–17 Years

Total

TABLE 23.7 Distribution of Reason for Exposure and Age for 764 Fatalities Reason

17 Years

Total

30 6 3 0 0 39

0 3 1 0 0 4

0 1 2 0 0 3

8 20 29 12 2 71

38 30 35 12 2 117

0 1 0 0 1 2 2 44

0 0 0 0 0 0 0 4

23 0 16 5 44 0 1 48

385 24 66 56 531 15 51 668

408 25 82 61 576 17 54 764

Accidental General Environmental Misuse Occupational Unknown Total Intentional Suicide Misuse Abuse Unknown Total Adverse reaction Unknown Total


TABLE 23.8 Number of Substances Involved in Human Poison Exposure Cases No. of Substances 1 2 3 4 5 6 7 8 9 ≥10 Unknown Total

No. of Cases

% of Cases

1,666,684 92,378 67,662 5693 2294 935 476 219 136 391 1071 1,837,939

90.7 5.0 3.7 0.3 0.1 0.1 0.0 0.0 0.0 0.0 0.1 100.0



B. PEDIATRIC POISONINGS According to the 1991 statistics, approximately 60% of exposures involved children younger than 6 years of age. The number of pediatric deaths due to poisonings increased from 25 deaths in 1990 to 44 deaths in 1991. A 5-year (1985–1989) retrospective analysis of pediatric deaths was conducted to aid poison prevention and educational efforts, guide new product formulation and aversive agent use, reassess over-the-counter status for selected pharmaceuticals, and identify areas of research for the treatment of pediatric poisonings.8 A hazard factor was devised to assess the risk of each agent to produce a major (residual disability) or life-threatening outcome when a child is involved in an overdose. This factor indicates a substance’s relative pediatric hazard by evaluating its packaging, accessibility (as a reflection of common storage practices in the home), availability (as a reflection of marketing), formulation, and closure types. The hazard score ranking allows for a comparison among categories. Of the 3.8 million pediatric exposures reported between 1985 and 1989, 2117 patients experienced a major outcome and 111 fatalities occurred. Table 23.9 shows the substance categories implicated in pediatric exposure calls, the total number of pediatric exposures that occurred in each category, the number of major effects, the number of fatal exposures, and the hazard factor. Tables 23.10 and 23.11 address unintentional pediatric ingestion fatalities reported to the AAPCC in 1983 through 1990. Iron supplements emerge as the single most frequent cause of unintentional death in pediatrics, representing more than 30% of all deaths. The ingestion hazards parallel the substances required to have child-resistant closures by the Poison Prevention Packaging Act. This demonstrates, however, that the requirement of child-resistant closures does not render a product “child-proof.” In 1989, King and Palmisana performed an epidemiological study to identify the risk factors responsible for the ineffectiveness of child-resistant closures. Although the Poison Prevention Packaging Act of 1970 has resulted in a 65% decline in the ingestion of products packaged in child-resistant containers, ingestion of prescription drugs by children has declined by only 36%. Reasons for these data include: 1. Availability of non-child-resistant packaging upon consumer request (i.e., consumer noncompliance) 2. Misuse of child-resistant closures by the consumer in the home 3. Transferring medicines from child-resistant packages to unsafe containers, or using no container at all 4. Unsafe storage practices, such as leaving containers within easy reach; and 5. Violations of the ACT by the dispensing pharmacist, physician, or Health Care Facility. Reprinted with permission (King and Palmisana, 1989).9 Pharmacies and health care facilities have also been shown to be noncompliant with the packaging act of 1970. Reports indicate that between 14% and 44% of pharmacies surveyed were in violation of federal packaging standards. Table 23.12 demonstrates that 2 year olds accounted for the majority (53.7%) of ingestions of prescription drugs. 9 Table 23.13 demonstrates that in more than 75% of cases surveyed, non-child-resistant packages or no containers were involved in the ingestion.9 Table 23.14 indicates the leading solid prescription drugs ingested that resulted in hospitalization.9 Table 23.15 demonstrates that the owner of each prescription was able to be identified in 80% of the cases surveyed. Although parents’ prescriptions accounted for 53.6% of the ingestions, nearly 30% involved grandparents’ medications. 9 Noncompliance with child-resistant packaging was a major reason for the exposure as indicated in Table 23.16.9 Other barriers to pediatric poisonings include the use of warning stickers designed to deter children from getting into containers. However, studies have failed to demonstrate any benefit from their use and, in fact, the warning stickers may attract children who otherwise would have ignored


the product.10 Woolf and Lovejoy10 discuss the epidemiology of drug overdose in children and the determinants that result in a high risk of drug poisoning in this group (Table 23.17).

TABLE 23.9 Reported Poison Exposures in Children Younger than 6 Years of Age, 1985 through 1989 Substance Categories and Subcategoriesa Nonpharmaceutical exposures Adhesives/glues Alcohols Ethanol (beverage) Arts/crafts/office supplies Auto/aircraft/boat products Ethylene glycol Methanol Batteries Bites/envenormations Copperhead Rattlesnake Unknown snake Other/unknown reptile Scorpion Black widow spider Brown recluse spider Building products Chemicals Acid: hydrochloric Alkali Dioxin Ethylene glycol Strychnine Cleaning substances Acid: drain cleaner Acid: industrial cleaner Aklali: drain cleaner Alkali: industrial cleaner Alkali: oven cleaner Oven cleaner: other/unknown Cosmetics/personal care Deodorizers (nonpersonal) Dyes Essential oils Fertilizers Fire extinguishers Food products/poisoning Foreign bodies/toys Fumes/gases/vapors Carbon monoxide Chlorine gas Hydrogen sulfide Methane and natural gas Gas:other Fungicides (nonmedicinal) Heavy metals


Total Pediatric Exposures (1985–1989)

No. of Major Effects

No. of Deaths

Hazard Factorb

37,986 80,443 2622 80,294 12,019 2321 1883 12,753 48,821 44 125 651 682 1585 1240 455 13,721 87,463 784 10,267 11 1002 32 386,052 208 438 1474 938 4619 224 395,985 39,408 10,369 6557 23,581 860 49,500 163,722 8436 3103 2208 228 700 1138 2714 11,926

15 46 11 3 11 4 3 7 100 4 19 10 3 43 4 2 2 72 3 24 1 6 1 205 6 3 19 17 10 4 57 1 0 5 1 0 7 21 55 42 6 2 3 1 1 14

0 5 2 0 4 1 3 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 0 4 0 0 1 0 0 0 3 0 0 0 0 0 0 0 21 18 0 0 1 2 0 0

0.7 1.0 8.0c 0.1 2.0 3.5c 5.1c 0.9 3.3c 146.4c 244.8c 24.7c 7.1c 43.7c 5.2c 7.1c 0.2 1.4c 6.2c 3.8c 146.4c 9.6c 50.3c 0.9 46.5c 11.0c 21.9c 29.2c 3.5c 28.8c 0.2 0.0 0.0 1.2 0.1 0.0 0.2 0.2 14.5c 31.1c 4.4c 14.1c 9.2c 4.2c 0.6 1.9c

TABLE 23.9 (Continued) Reported Poison Exposures in Children Younger than 6 Years of Age, 1985 through 1989 Substance Categories and Subcategoriesa Other Unknown Herbicides Paraquat Hydrocarbons Kerosene Lighter fluid/naphtha Mineral seal oil Insecticides/pesticides Chlorinated hydrocarbon alone Organophosphate alone Organophosphate with other pesticide Rotenone Lacrimators Matches/fireworks/explosive Moth repellants Mushrooms Paints/stripping agents Photographic products Plants Polishes/waxes Rodenticides Sporting equipment Gun bluing compound Swimming pool/aquarium Tobacco products Unknown nondrug substance Pharmaceutical exposures Analgesics Acetaminophen with propoxyphene Aspirin: unknown formulation Methadone Morphine Propoxyphene Other/unknown narcotic Anesthetics Anticholinergic Anticoagulants Anticonvulsants Carbamazepine Phenytoin Valproic acid Other anticonvulsant Antidepressants Arnitriptyline Amoxapine Desipramine Doxepin Imipramine Maprotiline




No. of Deaths

Hazard Factorb

1094 39 6488 66 129,024 10,751 8865 6564 100,105 9694 16,560 1806 284 4779 11,655 19,548 32,724 47,114 2688 375,649 12,249 41,261 2134 100 8067 36,742 20,318

4 1 3 1 168 51 24 13 122 29 56 4 2 1 1 6 9 10 1 33 4 2 0 0 1 14 14

0 0 0 0 5 2 0 0 6 0 2 0 0 0 0 0 0 0 0 1 0 1 4 3 0 0 0

5.9c 41.3c 0.7 24.4c 2.2c 7.9c 4.4c 3.2c 2.1c 4.8c 5.6c 3.6c 11.3c 0.3 0.1 0.5 0.4 0.3 0.6 0.1 0.5 0.1 3.0c 48.3c 0.2 0.6 1.1

325,539 2171 10,002 127 164 514 732 14,025 6516 1860 9198 4113 3619 1197 54 12,003 2897 200 935 887 2503 209

119 6 18 4 3 1 4 17 13 1 106 81 19 5 1 125 39 4 13 8 29 3

8 0 1 2 0 1 0 4 0 0 4 2 2 0 0 7 2 0 3 0 2 0

0.6 4.5c 3.1c 76.1c 29.5c 6.3c 8.8c 2.4 3.2c 0.9 19.3c 32.5c 9.3c 6.7c 29.8c 17.7c 22.8c 32.2c 27.6c 14.5c 20.0c 23.1c

TABLE 23.9 (Continued) Reported Poison Exposures in Children Younger than 6 Years of Age, 1985 through 1989 Substance Categories and Subcategoriesa Nortriptyline Other cyclic antidepressant Unknown cyclic antidepressant Cyclic antidepressant with benzodiazepine Cyclic antidepressant with phenothiazine Lithium Antihistamines Antimicrobials Antimalarials Isoniazid Rifampin Antineoplastics Asthma therapies Aminophylline Cardiovascular drugs Antiarrhythmics Antihypertensives Cardiac glycosides Nitroprusside Cough/cold preparations Diagnostic agents Diuretics Electrolytes/minerals Iron Eye/ear/nose/throat preparations Glaucoma medications Gastrointestinal preparations Antidiarrheals: diphenoxylate/atropine Hormones and antagonists Insulin Oral hypoglycemics Miscellaneous drugs Neuromuscular blocking agents Muscle relaxants Methocarbamol Other Sedative/hypnotics/antipsychotics Barbiturates: long acting Barbituarates: short acting Chloral hydrate Ethchlorvynol Glutethimide Methaqualone Phenothiazines Other Serums, toxoids, vaccines Stimulants/street drugs Amphetamines Cocaine Lysergic acid diethylamide




No. of Deaths

Hazard Factorb

347 179 142 288 832 1054 38,390 122,686 204 219 84 570 20,502 8622 37,385 1203 8099 3846 47 249,038 697 11,175 50,751 11,234 32,805 74 99,636 2500 6357 217 2609 17,650 8 3165 341 1012 33,048 4475 1051 443 81 30 66 7451 330 448 21,260 6409 546 117

2 2 5 2 10 7 20 28 2 3 2 0 43 38 182 3 139 24 1 72 1 8 57 52 17 1 48 18 20 2 11 10 2 7 2 3 153 37 4 18 1 2 1 57 2 0 84 18 20 2

0 0 0 0 0 0 4 3 2 0 0 1 3 3 7 0 0 2 1 4 0 0 8 7 0 0 3 1 1 1 0 2 0 0 0 0 2 0 0 0 0 0 0 2 0 0 1 0 0 0

9.3c 18.0c 56.7c 11.2c 19.4c 10.7c 1.0 0.4 31.6c 22.1c 38.4c 2.8 3.6c 7.7c 8.1c 4.0c 27.6c 10.9c 68.5c 0.5 2.3 1.2 2.1c 8.5c 0.8 21.8c 0.8 12.2c 0.5c 22.3c 6.8c 1.1 402.7c 3.6c 9.4c 4.8c 7.6c 13.3c 6.1c 65.5c 19.9c 107.4c 24.4c 12.8c 9.8c 0.0 6.4c 4.5c 59.0c 27.5c

TABLE 23.9 (Continued) Reported Poison Exposures in Children Younger than 6 Years of Age, 1985 through 1989 Substance Categories and Subcategoriesa



No. of Deaths

Hazard Factorb

694 325 177 175,378 44 4630 145,872 21,020

3 2 30 55 1 0 33 38

0 0 0 2 0 0 1 0

7.0c 9.9c 273.0c 0.5 36.6c 0.0 0.4 2.9c

3,852,618

2,270

Marijuana Mescaline/peyote Phencyclidine Topicals Silver nitrate Miscellaneous veterinary Vitamins Unknown drugs Total a b c

122

Subcategories with hazard factors ≥3 and statistical significancea are listed under each substance category. See text for explanation. p < .05, Fisher’s Exact Test comparing each individual category (or subcategory) with all other cases.

From Litovitz and Manoguerra, 1992.8 Reprinted with permission.

TABLE 23.10 Pediatric Pharmaceutical Ingestion Fatalities: 1983–1990 Substances Ingested Anticonvulsants Antidepressants Cardiovascular drugs Iron supplements Salicylates Miscellaneous

N

% Total (N-53)

3 10 8 16 6 11

5.7 18.9 13.2 30.2 11.3 20.7

From Litovitz and Manoguerra, 1992.8 Modified with permission.

TABLE 23.11 Pediatric Nonpharmaceutical Ingestion Fatalities 1983–1990 (Total N = 44) Substances Pesticides Hydrocarbons Alcohols and glycols Gun-bluing Cleaning substances Chemicals Cosmetics and personal care products Plants

N

%

12 12 7 4 3 3 2 1

27.3 27.3 15.9 9.1 6.8 6.8 4.5 2.3

From Litovitz and Manoguerra, 1992.1 Modified with permission.


1.0

TABLE 23.12 Ingestions of Solid Prescription Drugs, by Age of Victim Age Categories (months)

No.

Infant (< 12 mo) 1 (12–23) 2 (24–35) 3 (36–47) 4 (48–59) 5 (60–71) Unknown Total

(%)

1 205 456 124 37 25 1

(0.1) (24.2) (53.7) (14.6) (4.4) (2.9) (0.1)

849

(100.0)

9

From King and Palmisano, 1989. Reprinted with permission.

TABLE 23.13 Ingestions of Solid Prescription Drugs, by Container Type Container Type Child-resistant Non-child-resistant No container Other Total a

No.a

(%)

159 268 214 20 661

(24.1) (40.5) (32.4) (3.0) (100.0)

Number reporting container type.

From King and Palmisano, 1989.9 Reprinted with permission.

TABLE 23.14 Leading Solid Prescription Drugs Ingested, Resulting in Hospitalization Prescription Drug

No. of Cases

No. Hospitalized (%)

Ferrous sulfate Catapres (clonidine HCl) Ativan (lorazepam) Lanoxin (digoxin) Lomotil (diphenoxylate/atropine) Elavil (amitriptyline HCl) Tylox (oxycodone/acetaminophen)

32 26 22 18 11 10 5

12 (37.5) 12 (46.2) 3 (13.6) 4 (22.2) 7 (66.6) 7 (70.0) 3 (60.0)



TABLE 23.15 Owner of Medication Ingested Owner

No.

(%)

Cumulative %

Mother Father Grandmother Grandfather Sibling Self Dog Other (neighbor, relative)

294 70 143 51 10 4 4 103

(43.3) (10.3) (21.0) (7.5) (1.5) (0.6) (0.6) (15.2)

Total

679

(100.0)

43.3 53.6 74.6 82.1 83.6 84.2 84.8 100.0

9

From King and Palmisano, 1989. Reprinted with permission.

TABLE 23.16 Container Type and Ownership of Medicationa Father Child-resistant No container Non-child-resistant Totals (%) Total noncompliance (%) Parent’s noncompliance Grandparent’s noncompliance a

Mother

13 82 23 75 25 79 61 (13.1%) 236 (50.8%) 48 (78.6%) 154 (65.3%) 202 (68.0%)

Grandfather

Grandmother

Totals

7 13 25 45 (9.7%) 38 (84.4%)

27 40 56 123 (26.4%) 96 (78.0%)

129 151 185 465

134 (79.8%)

Excludes other owners of medication (i.e., neighbor, aunt, sibling, other).


TABLE 23.17 Risk Factors Involved in Drug Poisoning in Children 0 to 17 Years High-risk children Sex/age difference Developmental/behavioral determinants Family determinants Repetitive poisonings Adolescent psychiatric illness Intentional poisoning (Munchausen’s Syndrome by Proxy) Environmental determinants Locale and time of overdose Storage practices, child-resistant packaging Warning stickers Dispensing practices From Woolf and Lovejoy, 1993.10


C. ADOLESCENT TOXIC EXPOSURES Paulson (1988)11 indicated that intentional poisonings in adolescents is one of the 10 leading causes of death and potentially productive years of life lost in the U.S. Alcohol use and abuse plays a large role in fatal injuries in this age group. Approximately 8% of all intentional exposures resulting in death were in children aged 13 to 17 years as illustrated in Table 23.7. Drug-related fatalities by drug class among adolescents 11 to 17 years old reported to the AAPCC from 1989 to 1991 are listed in Table 23.18. Of 764 fatalities reported in 1991, 52 (6.8%) were in the adolescent age group; 92% were 13 to 17 years old. TABLE 23.18 Drug-Related Fatalities by Drug Class among Adolescents 11 to 17 Years Old Reported to the American Association of Poison Control Centers in 1989, 1990, and 1991 Drug Class Cyclic antidepressants Amitriptyline Desipramine Doxepine Imipramine Maprotaline Nortriptyline Calcium channel blockers Nifedipine Verapamil Salicylates Theophylline Propranolol Methamphetamine Cocaine Street drug (unknown type) Paracetamol (acetaminophen) Carbamazepine Haloperidol Glipizide +cyclobenzaprine Isoniazid Lidocaine (lignocaine) Methocarbamol Chlorpromazine Temazepam Colchicine/allopunnol/ibuprofen Amfebutamone (bupropion) +lithium Phenylpropanolamine/chlorphenamine Paracetamol +doxylamine + detromethorphan +pseudoephedrine Totals a b c d e f g h i j k l

1989

1990

1991

13 1 4 1c 5d 1 1e 2 0 2f 1 3i 3 1 0 1 1 1l 0 0 0 0 0 0 0 0 0 0 0 26

7 3a 2 0 1 0 1 2 0 2g 0 1 2j 0 0 0 0 0 1 1 0 1 0 0 0 0 1 1 1 18

11 1 7b 2 0 0 1 3 1 2h 5 1 1 0 1 0 2k 0 0 0 1 0 1 1 1 1 0 0 0 29

Totals 31

7

6 5 5 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 72

Ethanol also present in 1 case, thioridazine +alprazolam in another, methyldopa +perphenazine in the third case. Salicylate also present in 1 case. Verapamil +piroxicam also present. Tranylcypromine also present. Mesoridizine also present. Digoxin present in 1 case, meclizine present in the other. Propranolol also present in one case. Naproxen +propranolol also present in 1 case. Amoxicillin +cafalexin present in 1 case, ephedrine in another. Atenolol also present. Dextropropoxyphene also present in 1 case. Ampicillin +erythromycin also present.

From Woolf and Lovejoy, 1993.10 Reprinted with permission.


D. POISONING

IN THE

ELDERLY

Poisoning in the elderly is a continuing public health problem. Accidential poisoning, due to dementia and confusion, improper use or storage of a product, and therapeutic errors account for the most exposures of patients >64 years of age.12 However, approximately 11% were intentional with suicidal intent. The mortality rate from poisoning is much higher in the elderly than in other age groups. Of the 764 fatalities reported in 1991, 18% were Š64 years old (Table 23.19). Woolf et al.13 analyzed poisoning-related hospitalization and mortality rates among older adults in Massachusetts from 1983 to 1985. The poisoning hospitalization rates and poisoning-related death rates are listed in Tables 23.20 and 23.21. Table 23.22 lists intentionality and agent of poisoning deaths for 152 of the 275 total deaths.

TABLE 23.19 Distribution of Age and Sex for 764 Fatalities Age (yr)

Male

Female

Unknown

Total

%

3 months

2 weeks; 2 species 3 months; 2 species 6 months; 2 species 6 months; rodent 12 months; nonrodent Rat and mouse 24-month carcinogenicity

The following is a brief summary of the Segment reproductive toxicology studies. Refer to Chapters 11 and 12 for additional information. 1. Segment I: Reproductive and Fertility Studies in Rats • All reproductive parameters from exposure of gametes to F2 generation • Dosing: males and females before mating; females through end of lactation 2. Segment II: Teratology • • • •

Pregnant females dosed during period of organogenesis Rats: Gestational days 6–15 of 21 Rabbits: Gestational days 6–18 of 31 Evaluate the teratogenic effects on soft tissue and skeletal tissues of fetus

3. Segment III: Peri-Postnatal • Pregnant females dosed on gestational days 15 through postpartum day 21 • Examines late gestational, parturition, and lactation effects

SECTION 7. SUBSTANCES GENERALLY RECOGNIZED AS SAFE


0370_frame_C28 Page 1171 Thursday, July 12, 2001 12:29 PM

TABLE 28.2 Substances Generally Recognized as Safe (GRAS)6 Multiple Purpose GRAS Food Substances Citric acid Glutamic acid Glutamic acid hydrochloride Hydrochloric acid Phosphoric acid Sodium acid pyrophosphate Aluminum sulfate Aluminum ammonium sulfate Aluminum potassium sulfate Aluminum sodium sulfate Caffeine Calcium citrate Calcium phosphate Caramel Glycerin Methylcellulose Monoammonium glutamate Monopotassium glutamate Potassium citrate Silica aerogel Sodium carboxymethylcellulose Sodium caseinate Sodium citrate Sodium phosphate Sodium aluminum phosphate Sodium tripolyphosphate High fructose corn syrup Triethyl citrate Dietary Supplements Ascorbic acid Linoleic acid Biotin Calcium carbonate Calcium citrate Calcium glycerophosphate Calcium oxide Calcium pantothenate Calcium phosphate Calcium pyrophosphate Carotene Choline bitartrate Choline chloride Copper gluconate Ferric phosphate Ferric pyrophosphate Ferric sodium pyrophosphate Ferrous gluconate Ferrous lactate Ferrous sulfate Inositol Iron reduced

Dietary Supplements (Con’t) Magnesium oxide Magnesium phosphate Magnesium sulfate Magnesium chloride Manganese citrate Manganese gluconate Manganese glycerophosphate Sequestrants Citric acid Sodium acid phosphate Calcium citrate Calcium diacetate Calcium hexametaphosphate Monobasic calcium phosphate Dipotassium phosphate Disodium phosphate Isopropyl citrate Monoisopropy citrate Potassium citrate Sodium citrate Sodium gluconate Sodium hexametaphosphate Sodium metaphosphate Sodium phosphate Sodium pyrophosphate Tetra sodium pyrophosphate Sodium tripolyphosphate Stearyl citrate Stabilizers Chondrus extract Anticaking Agents Aluminum calcium silicate Calcium silicate Magnesium silicate Sodium aluminosilicate Sodium calcium aluminosilicate hydrated Tricalcium silicate Chemical Preservatives Ascorbic acid Erythorbic acid Sorbic acid Thiodipropionic acid Ascorbyl palmitate Butylated hydroxyanisole Butylated hydroxytoluene Calcium ascorbate Calcium sorbate Dilauryl thiodipropionate

Source: Section 21, CFR PART 182.


Chemical Preservatives (Con’t) Potassium bisulfite Potassium metabisulfite Potassium sorbate Sodium ascorbate Sodium bisulfite Sodium metabisulfite Sodium sorbate Sodium sulfite Sulfur dioxide Tocopherols Dietary Supplements Manganese sulfate Manganous oxide Niacin Niacinamide D-Pantothenyl alcohol Potassium chloride Potassium glycerophosphate Pyridoxine hydrochloride Riboflavin Riboflavin-5-phosphate Sodium pantothenate Sodium phosphate Thiamine hydrochloride Thiamine mononitrate Tocopherols α−Tocopherol acetate Vitamin A Vitamin A acetate Vitamin A, palmitate Vitamin B12 Vitamin D2 Vitamin D3 Zinc chloride Zinc gluconate Zinc oxide Zinc stearate Zinc sulfate Nutrients Ascorbic acid Biotin Calcium citrate Calcium phosphate Calcium pyrophosphate Choline bitartrate Choline chloride Manganese hypophosphite Sodium phosphate Tocopherols α-Tocopherol acetate Zinc chloride


SECTION 8. GLOSSARY ADME Absorption, Distribution, Metabolism and Excretion ANDA Abbreviated New Drug Company CANDA Computer-Assisted New Drug Application CBER FDA’s Center for Biologics Evalution and Research: division charged regulating biological products CDER FDA’s Center for Drug Evaluation and Research: division charged with developing and enforcing policy with regard to the safety, effectiveness, and labeling of all drug products for human use ELA Establishment License Application for a biologic FDA Food and Drug Administration: federal agency charged with issuing regulations on safety of foods, drugs, and cosmetics in United States IND Investigational New Drug Application: a request to initiate clinical study of a new drug product NDA New Drug Application: a request for approval to market new drugs PLA Product License Application for a biologic

REFERENCES 1. U.S. Government Manual, 1992/1993 Office of the Federal Register, National Archive and Records Administration. 2. Food and Drug Administration, Guideline for the Format and Content of the Nonclinical Pharmacology/Toxicology Section of an Application, Center for Drugs and Biologics, Department of Health and Human Services. 3. Food and Drug Administration, New Drug Evaluation Guidance Document, refusal to file, July 12, 1993. 4. Lumpkin, M. M., 45-day worksheets, participants at the commissioner’s industry exchange meeting, Philadelphia, PA, May 28, 1992. 5. 21 CFR Ch. 1 (4-1-87 Edition) 201.57. 6. 21 CFR Part 182, Substance Generally Recognized as Safe, Ch. 1 (4-1-92 Edition) 181.33.


29

Regulatory Toxicology: Medical Devices Steven J. Hermansky, Pharm.D., Ph.D., D.A.B.T.

CONTENTS Section 1. Introduction Section 2. Historical Perspective Table 29.1 Classification of Plastics Table 29.2 ISO 10993 — Biological Evaluation of Medical Devices Section 3. Regulation of Medical Devices A. United States Table 29.3 FDA Panels of Devices with Example Devices Table 29.4 Three Regulatory Classes Table 29.5 Areas to be Addressed in a Regulatory Submission to the FDA Table 29.6 Presentation of Data for Regulatory Submission to the FDA B. Europe Table 29.7 Hierarchy of European Regulation of Medical Devices Table 29.8 European Classifications as Defined by the Active Implantable Medical Device Directive and the Medical Device Directive Section 4. Determining Toxicology Testing Needs Table 29.9 The ISO Standard 10993-1 Guidance for Selection of Biocompatibility Tests as Modified by the FDA Table 29.10 Examples of Body Contact Categories as Determined by the ISO 10993-1 Guidance Matrix Figure 29.1 Biocompatability Flowchart for the Selection of Toxicity Tests for 510(k)s Section 5. Testing Methods A. Use of Extracts Table 29.11 Extraction Solutions — Common Examples Table 29.12 Extraction Ratios — Common Examples Table 29.13 Temperature and Time Conditions — Common Examples B. Biocompatibility Tests Table 29.14 Studies Often Conducted during the Safety Evaluation of Medical Devices Section 6. Glossary References


SECTION 1. INTRODUCTION Although the use of medical devices has a long history, modern medical use of devices only became realistic as an effective therapeutic intervention as the use of aseptic techniques expanded in the late 1800s. The first devices were, of course, fashioned from materials found in nature. Often, materials were obtained from domesticated animals. For example, leather was sculptured to form replacements for lost ears and noses and goose trachea was used as tubing in the measurement of arterial pressure. In all cases, key to the search for successful device materials was identifying materials with appropriate mechanical properties while minimizing the potential for adverse biological reactions. Although tissues obtained from domestic animals continue to be used for medical devices, material science has continued to develop and now includes many forms of metal, ceramic, plastics, and other synthetic polymers. The role of toxicology in medical device development is to ensure that the materials selected for use in devices are safe for their intended use. As in all disciplines of toxicology, the safety tests used to evaluate a device material must be appropriate to the route and duration of exposure. This chapter provides information, related to both testing and regulatory strategies, intended to be beneficial to the medical device researcher. Even though the difference between a medical device and a drug may seem obvious, the clarity of the distinction is becoming blurred as therapeutic interventions become more complex and disease specific. The difference between a drug and a device can generally be summarized by the simple observation that a drug is independent of its physical form as long as the physical form allows for systemic absorption of the active drug. However, a medical device is generally dependent upon its physical form to exert its therapeutic (biologic) effect. There are multiple examples of therapeutic medicaments that are not easily classified as either a drug or a device based upon their mechanism of action and intended therapeutic effect. The specific issues associated with the categorization of a treatment modality as either a device or drug is beyond the scope of this chapter. This chapter is divided into six sections including this introduction. The second section presents a historical perspective to medical device testing. The history of medical device toxicology continues to be important to the researcher since regulatory guidelines are continuing to evolve in this rapidly developing field. Furthermore, the “history” of this area of toxicology is recent memory to many researchers, and historical terminology is important in many current discussions. The third section focuses on the varied and often confusing regulatory arena for medical devices. The development of the European Community and publication of the International Standards Organisation (ISO) guideline documents have had significant impact on the medical device manufacturer. The regulatory arena continues to evolve and the information in this chapter presents a snapshot of the landscape. The fourth section attempts to provide the researcher with some basic references necessary to understand the rationale behind the selection of toxicity tests for any given material or medical device. The fifth section provides some basic information relevant to the nuances of testing solid materials and devices using models originally designed to evaluate the potential toxicity of soluble or semisoluble chemicals and drugs. The standard models have, therefore, been modified to provide information useful to the process of assessing the potential risk associated with materials/medical devices, and this section attempts to provide some information associated with the nature of the modifications. The sixth and final section provides some basic definitions for terms frequently used in the evaluation of the safety of medical devices.

SECTION 2. HISTORICAL PERSPECTIVE Because of the difficulties encountered when attempting to evaluate the safety of a solid test article, a historical description of the development of medical devices is considered useful to anyone evaluating medical devices for safety. The inherent safety of “natural” materials was often assumed. This, of course, was not always supported under use conditions.


Synthetic materials originally selected for medical use were chosen primarily for their physical attributes. These were materials first created for military, commercial, or industrial purposes. Since these materials were essentially considered to be inert and stable in the biological environment, the safety of medical device materials initially focused on sterility. However, as the developing device industry evolved and the devices were intended to treat increasingly complex conditions, materials were increasingly scrutinized for appropriateness for use in the biological environment. As researchers examined the biological effects of medical device materials, whether synthetic or natural, under use conditions, adverse events were increasingly noted.1 Therefore, a need for methods to determine the safety of materials was recognized by both the medical device industry and regulatory agencies, and the focus of medical device material identification shifted from purely performance to a combination of performance and safety. To that end, the industry became aware that a procedure was necessary for predicting which materials could be used safely.2 Since plastics were already a common material used in many varied devices, a set of tests designed by the U.S. Pharmacopoeia (USP) for classifying plastics using a series of biological tests was selected as a guidance.3 In this classification system, the proposed end use of the plastic, including the nature and extent of expected biological interaction, as well as possible solutions (e.g., drug vehicles) that may contact the plastic, is used as a guide to determine for which class the plastic should be qualified. Then, to determine whether the plastic is suitable for that class, a series of biological reactivity tests must be conducted. Representative samples of the plastic are used for the biological reactivity testing using specific methods prescribed by the USP. If the results of the tests are acceptable, as defined by the USP, for all the tests in the selected class, the plastic is considered to have met the requirements for that class (Table 29.1 presents a summary of the tests to be successfully conducted for each class). It is important to note that the class into which a plastic is placed is not related to its physical form or chemistry but is defined by the tests conducted and successfully completed. Although the classification of plastics by the USP methods are not generally considered to address adequately the safety of medical devices, a knowledge of the classes is important for the material researcher since some plastic raw materials may be identified using a plastic classification. This classification may therefore provide the researcher with a baseline indication of the appropriateness of the raw material for possible use in a device. It is important to note that the plastic class does not indicate safety of the plastic raw material in the final device and it is the responsibility of the device manufacturer to substantiate safety of the final device utilizing appropriate test methods. To that end, in 1976, the U.S. Congress passed the Medical Device Amendments to the Food, Drug, and Cosmetic Act. Critical wording of this Act necessitated an emphasis on the nature of the end use of a medical device in the assessment of safety parameters. Furthermore, the focus of safety testing was the final device, as intended for use, rather than individual raw materials to be used in the device. In part to address the requirements of this Act, the U.S. Food and Drug Administration (FDA), in collaboration with Canada and Great Britain, drafted a guidance document in 1987 for medical devices, known as the Tripartite Agreement. This agreement utilized a matrix approach of intimacy and duration of tissue contact to categorize a medical device based upon its intended use. This assumed that more critical medical devices (that is, those implanted into the body, life supporting, and/or life sustaining) would, by definition, have a greater intimacy of tissue contact and a longer duration of that contact.4 Based upon the category determined by the parameters of the Tripartite matrix, the necessary safety evaluations that had to be successfully completed before the device could be marketed were identified. The intended use of a device, specifically, the intimacy of tissue contact combined with the duration of this contact, as initially defined by the Tripartite Agreement would dominate the selection of safety tests for a specific device both then and now. However, other criteria for categorization of medical devices have been recognized, including whether the device has direct or indirect contact with biological tissues (see below), and today considerations for medical device Copyright © 2002 by Taylor & Francis

TABLE 29.1 Classification of Plasticsa Class I • Saline extract of sample evaluated using the systemic injection and intracutaneous reactivity tests Class II • Saline extract of sample evaluated using the systemic injection and intracutaneous reactivity tests • Ethyl alcohol in saline (5% v/v) extract evaluated using the systemic injection and intracutaneous reactivity tests Class III • Saline extract of sample evaluated using the systemic injection and intracutaneous reactivity tests • Ethyl alcohol in saline (5% v/v) extract evaluated using the systemic injection and intracutaneous reactivity tests • PEG 400 extract of sample evaluated using the systemic injection test (ip) • Vegetable oil extract of sample evaluated using the systemic injection test (ip) Class IV • Saline extract of sample evaluated using the systemic injection and intracutaneous reactivity tests • Ethyl alcohol in saline (5% v/v) extract evaluated using the systemic injection and intracutaneous reactivity tests • Vegetable oil extract of sample evaluated using the systemic injection (ip) and intracutaneous reactivity tests • Sample (not extract) implanted into the muscle of an animal and evaluated for macroscopically visible signs of inflammation Class V • Saline extract of sample evaluated using the systemic injection and intracutaneous reactivity tests • Ethyl alcohol in saline (5% v/v) extract evaluated using the systemic injection and intracutaneous reactivity tests • PEG 400 extract of sample evaluated using the systemic injection (ip) and intracutaneous reactivity tests • Vegetable oil extract of sample evaluated using the systemic injection (ip) and intracutaneous reactivity tests Class VI • Saline extract of sample evaluated using the systemic injection and intracutaneous reactivity tests • Ethyl alcohol in saline (5% v/v) extract evaluated using the systemic injection and intracutaneous reactivity tests • PEG 400 extract of sample evaluated using the systemic injection (ip) and intracutaneous reactivity tests • Vegetable oil extract of sample evaluated using the systemic injection (ip) and intracutaneous reactivity tests • Sample (not extract) implanted into the muscle of an animal and evaluated for macroscopically visible signs of inflammation a

If a plastic raw material is considered to meet the requirements of one of these classes, testing of a representative sample of that plastic would have met the acceptable standards for all of the very specific, biological reactivity tests listed below that class. See the USP, 1994, for the specific testing parameters and criteria for meeting the individual test procedures. Notes: All systemic tests are dosed by the intravenous route of administration unless noted in the table as being dosed by the intraperitoneal (ip) route of administration. PEG 400 = polyethylene glycol 400, which may be diluted with saline prior to conducting the biological assay. Source: USP.8

testing may include inductive and inflammatory responses, general toxicity, specific organ toxicity, immunotoxicity, reproductive toxicity, genetic toxicity, and carcinogenicity. The Tripartite Agreement has recently been replaced by a more international series of guidance documents that continue to be prepared and refined. The ISO has led the current efforts to establish a series of international medical device guidelines. The ISO guidance documents maintained the matrix approach of the Tripartite Agreement and proposed a guidance document in 1992. The focal point of the guidance was ISO 10993-1: Biological Evaluation of Medical Devices — Part 1: Guidance on Selection of Tests. Additional supporting documents have been and continue to be developed. The title of other parts of the ISO 10993 guidance are shown in Table 29.2 although not all guidance documents are finalized. The ISO 10993 guidance documents have become the standard for manufacturers desiring to market medical devices in Europe, Japan, and the United States. The ISO 10993 series of documents were formally adopted by the FDA, with modification,


TABLE 29.2 ISO 10993 — Biological Evaluation of Medical Devices Part

Title

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 ISO/DIS 14155 ISO/DIS 12891

Guidance on selection of tests Animal welfare requirements Tests for genotoxiciity, carcinogenicity, and reproductive toxicity Selection of tests for interaction with blood Tests for cytotoxicity: in vitro methods Tests for local effects after implantation Ethylene oxide sterilization residuals Clinical investigation Degradation of materials related to biological testing Tests for irritation and sensitization Tests for systemic toxicity Sample preparation and reference materials Identification and quantification of degredation products from polymers Identification and quantification of degredation products from ceramics Identification and quantification of degradation products from metals and alloys General guidance on toxicokinetic study design for degradation products and leachables Establishment of allowable limits for leachable substance Chemical characterization of materials Clinical investigation of medical devices Implants for surgery — retrieval and analysis of surgical implants

in 1995. Therefore, the ISO guidance documents will be the focus of information presented in this chapter. However, the ISO 10993 guidance documents are dynamic and the reader is encouraged to ensure that the most up-to-date information is available before initiating any testing.

SECTION 3. REGULATION OF MEDICAL DEVICES A. UNITED STATES There are several methods of classification of devices in the United States. The method used will depend upon the specific use for the device and, importantly, by whom and how the device is intended to be used. The FDA has established specific classifications for nearly 2000 different generic classes of devices. These different types of devices are further grouped into 16 “panels” based upon medical specialties. The 16 panels and some representative classes of devices are presented in Table 29.3. The panels and generic class description (name) of the device helps the manufacturer to identify whether its proposed device has already been assessed and placed into a generic classification by the FDA. The FDA has further assigned each of these generic classes of devices to one of three regulatory classes. The assignment of a regulatory class is based upon the level of control the FDA considers to be necessary to ensure the safety and effectiveness of the device when manufactured and marketed as intended. The three FDA regulatory classes and requirements that apply to each, along with some examples, are shown in Table 29.4. To market a device in the United States, the manufacturer must determine whether a regulatory submission is necessary. Generally, a submission to FDA will be necessary to market a medical device in the United States unless the FDA has published a notice indicating that this generic class of device is exempt from the premarket notification procedure. Devices exempted by FDA for submission are generally Class I devices for which the FDA considers general manufacturing controls to be sufficient to ensure the safety and efficacy of the device for the patient and/or user.


When it has been determined that a submission to FDA is necessary prior to marketing a device in the United States, the manufacturer must then determine the type of submission. In the United States, the two forms of submission are the premarket notification, or 510(k), and the premarket approval, or PMA. Most devices on the market in the United States are Class II and, therefore, most devices are cleared for commercial distribution in the United States by the 510(k) process. Most class I devices are exempt from the need for a regulatory submission and most class III devices require a premarket approval application (see below) prior to marketing in the United States. Although it is beyond the scope of this chapter to describe the specific issues associated with these submissions, a brief description of each is provided. Areas that generally must be addressed in a regulatory submission required for either the 510(k) or PMA are shown in Table 29.5. Preferred organization of the data for a regulatory submission in the United States is shown in Table 29.6. The 510(k) is a submission to the FDA demonstrating that the proposed noncritical and nonexempt device (generally Class II) is as safe and as effective (that is, substantially equivalent) to a legally marketed device that was or is currently on the U.S. market. Therefore, an important aspect of the 510(k) process is identification of the previously marketed, substantially equivalent device for use in comparison with the proposed device. Substantial equivalence of the marketed and proposed device is established based upon a number of factors, but the two devices do not have to be identical. The need for toxicological/safety testing will be, at least partially, based on the similarity of the two devices including materials and manufacturing practices. Clearly, the manufucturer must provide, at the least, adequate safety information on any new materials in the new device. The PMA process is used by the FDA to evaluate the safety and effectiveness of noncritical and nonexempt devices (usually Class II) for which a currently marketed, substantially equivalent device cannot be identified as well as most critical devices (Class III). Because of the level of risk associated with the use of critical devices, the FDA determines that these devices require a rigorous regulatory submission prior to marketing. To substantiate safety and efficacy, the PMA process requires more information than is presented during a comparison to a preexisting device. Therefore, each critical device marketed in the United States must be individually shown to be safe and effective.


TABLE 29.3 FDA Panels of Devices with Example Devices Clinical chemistry and clinical toxicology devices Urinary pH (nonquantitative) test system Blood specimen collection device High-pressure liquid chromatography system for clinical use Pipetting and diluting system for clinical use Breath alcohol test system Codeine test system Hematology and pathology devices Cytocentrifuge Occult blood test Prothrombin time test Automated slide stainer Immunology and microbiology device Automated colon counter Enriched culture medium Anesthesiology devices Pressure regulator Emergency airway needle Oxygen mask Ventilator tubing Cardiovascular devices Blood pressure alarm Blood pressure cuff Stethoscope Ear oximeter Pacemaker lead adapter Vascular clamp Defribrillator (including paddles) Dental devices Caries detection device Resin applicator Preformed plastic denture tooth Root canal post Dental injecting needle Dental floss Manual toothbrush Ear, nose, and throat devices Hearing aid Ossicular replacement device Tympanostomy tube Otoscope External nasal splint


Gastroenterology–urology devices Stomach pH electrode Hemorrhoidal ligator Ostomy pouch and accessories General and plastic surgery devices Chin prosthesis Occlusive wound dressing Surgeon’s glove Skin marker Sutures General hospital and personal use device Patient scale Elastic bandage Suction snakebite kit Tongue depressor Neurological devices Cutaneous electrode Neurosurgical headrest Aneurysm clip Implanted neuromuscular stimulator Obstetrical and gynecological devices Endometrial brush Fetal blood sampler Obstetric forceps Condom Menstrual tampon Ophthalmic devices Visual acuity chart Ophthalmoscope Artificial eye Contact lens and solutions Orthopedic devices Joint prosthesis (many) Cast components Physical medicine devices Cane Mechanical chair Arm sling Hot or cold disposable pack Radiology devices Bone densitometer Ultrasonic pulsed doppler imaging system

TABLE 29.4 Three Regulatory Classes Class I. General Controls (with or without exemptions) Least perceived risk to device user and/or patient Stethoscope Canes and crutches Class II. General Controls and Performance Standards (with or without exemptions) Mercury thermometer Condom Blood pressure cuff Class III. General Controls and Premarket Approval Most perceived risk to device user and/or patient Pacemaker Implanted neuromuscular stimulator Replacement heart valve

TABLE 29.5 Areas to be Addressed in a Regulatory Submission to the FDA Intended use Indications for use Target population Design Materials Performance Sterility Biocompatibility Mechanical safety Chemical safety Anatomical sites Human factors Energy used and/or delivered Compatibility with the environment and other devices Where used Standards met Electrical safety Thermal safety Radiation safety For 510(k): To market a noncritical and nonexempt device (generally Class II) Selection of predicate device that is as safe and as effective (that is, substantially equivalent) to a legally marketed device that was or is currently on the U.S. market


TABLE 29.6 Presentation of Data for Regulatory Submission to the FDA To facilitate FDA review of the data, analysis, and conclusions in the application, the manufacturer and contract laboratory, if used, should check the following: • Logical presentation of the biocompatibility data • Scientific soundness of the test method and data analysis • Relevance of the test program to the device and the intended use • Completeness of the summary report of the tests or studies The summary of test results should be presented in a table format in each report whenever possible. Each study or test attachment report should contain sufficient and well-organized information in reasonable detail so that the FDA reviewer can determine: • What exact material or device was tested • What tests were performed • How the tests were performed • What the test results were A description of the tests and the results obtained are essential; and reasonable and sufficient details of all test procedures and results should be submitted to the FDA. For biocompatibility studies, manufacturers should use a standard scoring system for each test method, if a standard scoring system exists. Each test report should include the following: • Name and address of the manufacturer of the item tested • Name and technical description of the item tested • Name and address of the laboratory where the tests were conducted • Test methods including the scoring method • Number of samples and replicates tested • Any control data needed to establish the validity of the test • The date when the tests were conducted • Summary report(s) of results obtained • Analysis, interpretation of results, and conclusions

B. EUROPE In Europe, the 15 original member states (along with several more recently added member states) of the European Community have authored, reviewed, and accepted a common method by which manufacturers must evaluate and qualify their proposed devices prior to marketing. The Active Implantable Medical Device Directive and the Medical Device Directive define the legal framework accepted by the member states through which medical devices are regulated (see Table 29.7 for the components of this basic framework). Through the directives and system of Notified and Competent Bodies, many devices must meet essential requirements prior to being marketed. As in the United States, devices must be evaluated for safety and efficacy to ensure that they do not harm the patient, clinician, or any third party. Also similar to the United States, there is a system of classification determined by the Medical Device Directives as shown, with examples, in Table 29.8. For most medical devices, the independent Notified Body often signifies that the device may be marketed in the European Community by granting the manufacturer the right to label the product or its packaging with a “CE marking,” which signifies that the manufacturer has adequately proved the safety and efficacy of the proposed device. In theory, a CE marking obtained through a Notified Body appointed by the government agency of one of the member states gives the manufacturer the right to market the subject device in any one of the member states. However, any of the individual member states may, at any time, implement a safeguard clause and require additional testing to evaluate the device further to ensure that the CE marking was not incorrectly granted.


TABLE 29.7 Hierarchy of European Regulation of Medical Devices European Commission Multicountry body consisting of weighted membership from member countries CEN/CENELEC Committees for European Standards (CEN: European Committee for Standardization) (CENELEC: European Committee for Electrotechnical Standardization) Competent Authorities Government agency that approves and monitors Notified Bodies Coordinates complaint management and device noncompliance issues Qualified to issue the final assessment certificate once an adequate technical file has been submitted and reviewed Accreditation Bodies Agency/group that works with Notified Bodies to provide assurance (“accreditation”) to further ensure testing standards have been met Review quality systems Notified Bodies Address quality systems and may perform some testing; must be “notified” for each product line for which they are responsible The Notified Bodies must remain a legally separate entity from manufacturers Manufacturer

TABLE 29.8 European Classifications as Defined by the Active Implantable Medical Device Directive and the Medical Device Directive (MDD) • Placement of a device into a class is based upon risk and duration of use • The intended use determines the class placement • For combination devices and devices with accessories, each part of the device must be placed into a class and the highest class placement applies to the entire device • There is no connection between the MDD classifications and FDA classifications Class I Most basic classification for medical devices Wheelchairs Surgical instruments Class IIa Short-term surgically invasive devices Some active, noninvasive devices Class IIb Energy/substance delivering Utilizes or emits ionizing radiation Long-term surgically invasive devices Class III Most cardiovascular and CNS devices


SECTION 4. DETERMINING TOXICOLOGY TESTING NEEDS Once toxicology testing has been determined to be necessary, the specific tests to be performed must be identified by the manufacturer. The FDA and European Community both generally use the ISO 10993 Biological Evaluation of Medical Devices in the evaluation of manufacturers’ biological safety testing program for medical devices regardless of the specific regulatory submission made — 510(k), PMA, or for a CE marking. As noted above, the testing recommended by the ISO guidelines utilize a categorization of the device based on the specific nature of the interaction of the device and component parts with the body. The ISO standard 10993-1 as modified by the FDA (Table 29.9) summarizes the initial toxicology tests necessary for a proposed device based on the “Nature of Body Contact”: noncontact, surface-contacting (skin, mucosal membranes, and breached or compromised surfaces), external communicating (blood path, indirect; tissue/bone/dentris communicating and circulating blood), or implant devices and by the duration of contact: limited exposure (ð24 h), prolonged exposure (> 24 h to 30 days), or permanent contact (>30 days). Testing requirements increase as intimacy and duration of contact of the device increase. Using the expected intimacy of contact and duration of contact for the proposed device, the intimacy and duration categories can be determined and, therefore, the required tests for biological evaluation identified. Effects on reproduction (including developmental effects) and biodegradation may be considered for specific materials/devices depending upon the intended end use. Additional tests for specific target organ toxicity such as immunotoxicity or neurotoxicity may be necessary, depending on the characteristics of the intended use of the device including the intimacy and duration of contact. Table 29.10 provides examples of some medical devices and the corresponding body contact categories. The intended use of the device as well as the experience with and knowledge of the materials used in its manufacture must be considered during the determination of which tests to conduct. To that end, the ISO 10993 standard is intended to be used as a guide, and, therefore, the final decision on which tests to be conducted must be made by individuals qualified in material biology and/or toxicology. The establishment of the safety assessment plan for a device should be made by the appropriate professionals, qualified by training and experience using interpretation and judgment, when considering the factors relevant to the device or its materials, the intended use, and current knowledge of the device and its materials provided by scientific literature and previous clinical experience.5 There are several specific situations for which ISO 10993 is not applicable and should not be used. Even though many of the materials used in these applications are the same, it is important that the researcher be familiar with these cases to ensure that the ISO guidance documents are not inappropriately applied to these situations. For example, packaging materials for drugs and biologics are not considered medical devices. Furthermore, several special standards for specific categories of devices are available for guidance. The researcher must ensure that the proposed device would not be included in one of these specific guidance documents before proceeding with the selection of testing methods. For all toxicological end points, a stepwise approach to selection of tests is recommended. The researcher should first conduct a comprehensive literature review to ensure that animal studies are not inappropriately conducted. The literature search should be followed, as appropriate, by relevant in vitro assays and, finally, in vivo assays in animals if the end point has not been resolved. To aid the manufacturer, the FDA has assembled a biocompatability flowchart for the selection of toxicity tests for 510(k)s (Figure 29.1).


TABLE 29.9 The ISO Standard 10993-1 Guidance for Selection of Biocompatibility Tests as Modified by the FDA

Blood path, indirect External communicating devices

Tissue/bone/dentin communicating+

Circulating blood

Tissue/bone Implant devices Blood

a b c

o x

x

o o

o x

x

o o

o x

x

o x x

o x

x x o^ x x

o o

x x

o o

o x

x x

Carcinogenicity

o o o o o x x x o o o x x x o o o x x x

Chronic Toxicity

x x x x x x x x x x x o x o o x x x x o o x x x

Hemocompatability

x x x x x x x x x x x x x x x x x x x x x x x x

Implantation

x x x x x x x x x x x x x x x x x x x x x x x x

Genotoxicity

Breached or compromised surfaces

Systemic Toxicity (Acute)

Mucosal membrane

Irritation or Intractuaneous

Surface devices

A B C A B C A B C A B C A B C A B C A B C A B C

Sensitization

Skin

Contact Durationb

Cytotoxicity

Body Contacta

Subchronic Toxicity

Biological Effectc

Device Categories

o

o x x x

o o

x x x

x x x x x

x x x

x

x

o

x

x

x

x

x

x

x

See text. A = limited (24 h); B = prolonged (24 h to 30 days); C = permanent (>30 days). See text. x = ISO Evaluation Tests for Consideration; o = additional tests that may be applicable.

Note: + Tissue includes tissue fluids and subcutanous spaces; ^ for all devices used in extracorporial circuits. Source: Adapted from FDA General Program Memorandum G95-1, Tables 1 and 2.


TABLE 29.10 Examples of Body Contact Categories as Determined by the ISO 10993-1 Guidance Matrix Body Contact Category Surface Devices

External communicating devices

Implant devices

Examples

Skin

Stethoscope head Electrodes Compression bandages Surgical gowns

Mucosal membrane

Examination gloves Inhalers Endotracheal tubes Urinary catheters External feeding tubes

Breached or compromised surface

Bandages, dressings Wound patches Occlusive tapes

Tissue/bone/dentin communicating

Surgical gloves Laparoscopes Endoscopes

Blood path indirect

Hypodermic needles Extension sets Transfer sets Blood transfusion sets

Circulating blood

IV catheters Dialysis tubing Hemodialyzers Oxygenator

Tissue/bone

Bone/dental cements Cerebrospinal drains Implanted drug delivery port Orthopedic joints, pins, plates, screws Pacemakers

Blood

Blood monitors Heart valves Vascular grafts Internal drug delivery catheter Permanent pacemaker electrodes


FIGURE 29.1 Biocompatibility flowchart for the selection of toxicity tests for 510(k)s. (Adapted from FDA General Program Memorandum G95-1, Attachment C.)


SECTION 5. TESTING METHODS A. USE

OF

EXTRACTS

Devices, unlike drugs and chemicals, cannot be easily administered to animals in carefully measured, exact doses in varying amounts so that a dose–response relationship can be established. Device materials can include metal, fabric, silicone, and ceramics — none of which is easily administered in its final form to biological systems. Furthermore, these materials are not generally considered to be bioavailable under conditions of normal use in that they do not typically fully break down in a biological environment or they break down at a such a slow rate that biologically significant changes may not be detected for many years.4 However, the potential for the effects of any biological components of the material as well as other chemicals that may be more bioavailable must be evaluated prior to final use of the material in a device. Chemicals other than the primary constituent of the material that may be bioavailable may include contaminants, processing aids, unreacted monomer, etc. The task presented to the manufacturer/material scientist is to prepare the material and any other chemicals in a way that introduces the relevant chemicals to the biological system such that any potential biological effects can be observed and measured. It can be argued that total dissolution of the material using an aggressive solvent and/or physical pulverization would not generally be relevant for a material that is not intended to dissolve under normal use as a medical device component. Thus, an approach to safety evaluation that is both relevant and sufficiently challenging to the biological system must be designed. To be available to cause a systemic effect, a chemical must migrate away from the site of tissue contact. The biological environment is a complex mixture of hydrophobic (nonpolar) and hydrophilic (polar) microenvironments. Therefore, the incubation of a material/device in a hydrophobic solution (e.g., vegetable oil) and a hydrophilic solution (e.g., saline) should dissolve or “leach” the potentially bioavailable chemicals out of the material. The potentially bioavailable chemicals should remain in the solutions after incubation and the administration of the resulting incubation solutions to a biological system would expose that system to the chemicals that may cause systemic effects. These chemicals, which may diffuse away from the device when in contact with a biological environment, are often called “leachables.” Examples of extraction solutions are provided in Table 29.11. To ensure that the leachables are present in the saline or oil incubation solutions at relevant concentrations, the incubation conditions must be considered. The ratio of material to incubation solution should be sufficient to ensure that the amount of leachables is likely to expose the biological test system to the chemical adequately so that an effect, if it occurs, will be detected. Of course, the appropriate ratio for each material under every exposure condition is impossible to calculate since the specific leachable chemicals are generally unknown. However, as a general rule, the ratio of material to solution should greatly exceed the anticipated ratio to be encountered under finaluse conditions. The relevance of the safety testing program could well be challenged if, during regulatory review, it is discovered that the ratio of material to solution is less than the anticipated exposure under final-use conditions. The device industry and regulatory agencies have designed a specific set of ratios that are considered to be appropriate for most materials under most conditions. Examples of these standard ratios are provided in Table 29.12. In addition to the ratio of material to solution, the length of time that the material is exposed to the biological system must also be considered. It can be assumed that the laws of physics apply to biological systems in that the longer the material is in contact with the biological system, an increasing percentage of the leachable chemical will diffuse away from the material and become biologically available. Unfortunately, it is not practical for the development of products nor the advancement of science to incubate materials and/or devices in saline and oil for 20 years before a material can be used in a device intended for chronic use in a patient. Therefore, the device industry and regulatory agencies


have applied another law of physics in which the diffusion rate of a chemical toward equilibrium is increased at higher temperaturers. As with ratios of material to solution extraction, a specific set of temperatures to be used during extraction have been designed. The standard extraction conditions are presented in Table 29.13. In general, extraction temperatures approximating the normal human body temperature (37°C) should only be used when the body contact duration for the device is anticipated to be brief or when the integrity of the material may be compromised by using higher temperatures. The selection of the specific conditions to be used during extraction for any material or device is dependent upon many factors, including the potential for the material to degrade physically at the selected incubation temperature, the proposed end use of the material in the device, the anticipated time of exposure under routine-use conditions of the device, and several others. The material scientist is encouraged to select these conditions carefully.

TABLE 29.11 Extraction Solutions — Common Examples Polar (hydrophilic) liquid Saline Culture media (without sera) Distilled water Nonpolar (hydrophobic) liquid Cottonseed oil Sesame oil Other extraction liquids (may be used for specific issues and “Class” testing) Polyethylene glycol 400 (diluted to physiological osmotic pressure) Ethanol/saline (5% v/v)

TABLE 29.12 Extraction Ratios — Common Examples Thicknes s (mm)

Extraction Ratio (± 10%)

Examples of Materials

≤ 0.5 > 0.5 ≤ 1.0 > 1.0 Irregular

6 cm2/ml 3 cm2/ml 3 cm2/ml 1.25 cm2/ml 0.1–0.2 g/ml; 6 cm2/ml

Metal; synthetic polymer; ceramic; film, sheet, and tubing wall Metal; synthetic polymer; ceramic; tubing wall; slab; molded items Elastomer Elastomer Pellets, molded parts

TABLE 29.13 Temperature and Time Conditions — Common Examples 37 37 50 70 121


± ± ± ± ±

1°C 1°C 2°C 2°C 2°C

for for for for for

24 ± 2 h 72 ± 2 h 72 ± 2 h 24 ± 2 h 1.0 ± 0.2 h

B. BIOCOMPATIBILITY TESTS The potential toxicity associated with the use of the device/material is often evaluated by dosing the incubation solutions (extracts), now rich with leachable compounds, to appropriate biological systems. The methods used to detect possible toxicity associated with the solutions are generally standard toxicological methods slightly modified for the use of extracts. Most important of these modifications is the obvious need to conduct two tests for each toxicology end point since two extracts exist for each device/material (hydrophobic and hydrophilic). Therefore, each test must be run in duplicate to evaluate both extracts for each end point. A second modification to standard toxicology tests is necessary because of the dilute nature of the extracts. In spite of attempts to use high material-to-solution ratios and increased temperatures of incubation, the concentration of leachable chemicals within the solution will likely be quite low. Therefore, the dose volume of solution administered to the test system must be as high as feasible to ensure detection of any potential effects. A third modification of standard tests is the need to adapt the study to the physical nature of the extract. Thus, both the hydrophilic and hydrophobic extracts from a device that will be in contact with the blood should be evaluated for the potential to cause systemic toxicity. However, the hydrophobic (a vegetable oil) extract cannot be injected directly into the bloodstream at a sufficiently high dose without immediately killing the animals via lipid emboli. Therefore, this extract is administered to the animals by an alternative route. Obviously, the oral or dermal routes of exposure would be inappropriate because of significant differences in absorption and metabolism potential and, therefore, the intraperitoneal route of exposure is used as the most relevant alternative. Similarly, a saline extract cannot be added directly to many in vitro systems because of toxicity, and the extract medium must be altered to ensure successful dosing. The risk assessment process must also be modified to adapt to these differences between standard toxicology tests and the methods used to evaluate the extracts of medical devices. It is important to recognize that the level of uncertainty is increased when extracts are used since the actual test article (the material/device) is one more level removed from the human exposure condition. Because of this added level of uncertainty accompanying the use of extracts, physical pieces or, better yet, miniature devices can be prepared and evaluated in toxicology tests individually designed to evaluate the material/device. However, the time and cost associated with the preparation of miniature devices may be prohibitive even though the miniature “device” need not be an exact duplicate, nor does it have to be functional to be used for evaluation of toxicology. The miniature device only needs to maintain the same basic ratios of materials to be used in the device and to be of appropriate size to provide a sufficient “dose” of the materials to the test animals. If a miniature device can be devised and produced in a timely and cost-effective manner, the difficulty of adapting toxicology tests to evaluate a physical piece or pieces of plastic, fabric, or other solid material may eliminate the possibility of conducting the study. In spite of these complexities, which necessitate the use of extracts for most toxicology evaluations of medical devices, the researcher is encouraged to consider the use of solid materials since this approach eliminates the uncertainty introduced into the risk assessment process by the use of extracts. As a final note to the preparation of materials for evaluation of materials/devices, the researcher must recognize that the procedures used to sterilize devices are generally accepted to have the potential to change the physical characteristics of the component materials. These physical changes also have the potential to affect the biocompatibility of the material/device.6 Therefore, materials to be used in toxicology testing, whether they are extracted, used to create miniature devices, or sectioned into implantable aliquots, should be sterilized using the procedures to be used to sterilize the final device prior to use in humans. Therefore, a final modification to standard toxicology testing procedures is the possible need to evaluate a material/device in a multiple series of tests to ensure that all forms of sterilization considered to be acceptable for the device do not negatively


impact the biocompatability of the device or component parts. A guidance document regarding sterilization procedures has been prepared (ISO 10993-12). In determining testing requirements for medical devices, as for other test articles to be evaluated for toxicity, it is important to consider previously conducted study results or published literature before initiating a study using animals.7 To that end, the Animal Welfare Act has defined a responsibility for the individual researcher and, therefore, the manufacturer to consider all information before initiating animal testing. Studies often conducted during the safety evaluation of medical devices are presented in Table 29.14.

TABLE 29.14 Studies Often Conducted during the Safety Evaluation of Medical Devices Cytotoxicity • In vitro test that utilizes cell culture techniques • Used primarily to evaluate the potential for local toxic effects • Often used as an initial biological screen for new materials or changes to existing formulations (for example, existing materials sterilized by a new method) Sensitization • Methods used most frequently utilize guinea pigs as the test organism • Study duration of guinea pig assays is approximately 6 weeks • Evaluates the sensitization component of the immunological response of the biological system to foreign materials • Delayed hypersensitivity (Type IV) allergic reaction is the specific biological reaction evaluated in this study • Both natural and synthetic materials can produce sensitization reactions and the potential morbidity that may be caused by sensitization of an individual to a device is significant • All materials considered for use in medical devices should be evaluated for the potential to cause sensitization • The local lymph node assay (LLNA) utilizing mice is becoming more widely accepted as a method (study duration is approximately 1 week) • Using human volunteers to evaluate sensitization should be considered for some applications • Study duration of human studies is approximately 6 weeks Skin Irritation (Topical) • Generally uses albino rabbits as the test model because of high potential for observable skin reaction in this model • Most obviously used to evaluate potential for a topical product to produce skin irritation at the site of application • Evaluation of treatment sites uses standardized scoring criteria to allow for a calculation of a “Primary Irritation Index” (combined severity of erythema and edema scores) • For materials/devices intended to be in contact with abraded skin and/or open wounds, testing for skin irritation needs to be expanded to include animals in which the epidermis has been abraded using appropriate methods • Study duration is approximately 3 days Skin Irritation (Intracutaneous Reactivity) • Uses albino rabbits as the test model because of high potential for observable skin reaction in this model • Extracts injected into the surface layers of the skin (approximately between the epidermis and dermis) • Evaluation of injection sites uses standardized scoring criteria similar to topical testing • Has become a standard test to help evaluate the potential for many materials/devices, regardless of their intended final use, to produce a local reaction at the site of tissue contact • Study duration is approximately 3 days Acute Systemic Toxicity • Utilizes mice injected with extracts by either the intravenous or intraperitoneal route • Designed to evaluate the potential for harmful effect(s) to occur distant from the site of tissue contact • Evaluation for toxic effects generally follows standard toxicological methods including death, weight loss, and clinical signs • Study duration is approximately 3 days Implantation • Used as a direct measure of the local irritation potential of materials/devices in direct contact with living tissue


TABLE 29.14 (Continued) Studies Often Conducted during the Safety Evaluation of Medical Devices • Implanted test article may be a small piece of a device component, a specific part of a final device, or an entire device • Design often includes implantation of a standard control material into same animals (at distant sites) as the test article to serve as an irritant control • Site of implantation is generally a large muscle of a laboratory animal (often an albino rabbit) due to ease of placement and subsequent gross and histopathological scoring of any tissue changes • Care must be taken during test article preparation to avoid the inadvertent presence of any sharp edges/corners on the test article that may bias the results of the test by causing inflammation due to the shape of the test article • Basic muscle implantation techniques do not absolutely require microscopic evaluation of the implantation site, but histology should be used as frequently as possible to maximize the potential to detect biologically significant reactions to the test article • Attempts should be made to evaluate the tissue response macroscopically and microscopically without disturbing the test article location in the tissue since removal of the test article may also remove some inflammatory tissue from the site • Specialized studies using implantation into the intended site of use of the device should be considered • Method of inserting (implanting) the test article into the tissue has the potential to impact the results of the study • Study duration can be 5 days to several months depending on proposed end use of the device Hemocompatibility • Evaluates the potential for a material/device to effect the formed cellular elements of the blood (cells/platelets) as well as to activate the coagulation and/or complement systems of the circulation • Frequently conducted using in vitro assays in which the end point is red blood cell lysis, coagulation, etc. • Assays generally use the blood from laboratory animals although this has been challenged due to possible differences in cell friability and, therefore, use of donated human blood may be considered • Potential biological significance of hematological changes caused by materials/devices in contact with the blood suggests that in vivo testing should be considered when standard in vitro studies are inconclusive • Study duration is generally very brief (less than 1 day) Genotoxicity • Assays evaluate the potential for test article to induce genetic changes including mutations and/or altered chromosomal structure • In vitro studies including the Ames assay and chromosomal aberration are frequently used • In vivo studies such as the mouse micronucleas assay may also be utilized • Study duration varies with the specific assay Subchronic/Chronic Systemic Toxicity • These assays should only be conducted for devices used repeatedly (same device frequently on the same patient or different devices used frequently on the same patient) • Studies are typically individually designed for specific devices • Test system may be virtually any laboratory animal model and should be selected based upon the desired end point of the study • Evaluation for toxic effects generally follows standard toxicological methods including death, weight loss, clinical signs, clinical pathology, hematology, and histopathology (of local tissue effects as well as possible target organs) • These assays may be conducted by evaluating implanted materials (obviously, test and control articles must be placed into different animals for these assays) or by repeatedly dosing animals with freshly prepared extracts • Study duration may be as short as 2 weeks or as long as several years depending upon the desired end point of the study Carcinogencity • Rarely required for device materials • Test system is generally mice or rats • Evaluation for carcinogenicity end point follows standard toxicological methods and is dependent upon hematology and histopathology • These assays may be conducted by evaluating implanted materials (obviously, test and control articles must be placed into different animals for these assays) or by repeatedly dosing animals with freshly prepared extracts • Study duration (including significant time to complete histopathological evaluation) is at least 2 years and is often close to 3 years


SECTION 6. GLOSSARY Agar Diffusion Cytotoxicity Assay A cytotoxicity assay (also called the agar overlay method) using cultured mammalian cells in which the material/device is placed in contact with a layer of agar which, in turn, is in direct contact with the cells, and, after a prescribed period of incubation, the cells are examined for signs of toxicity resulting from exposure to the test article. Annex Quality conformity assessment as defined by the European Medical Device Directives. Annex I through VII are defined. Biocompatibility The science of determining the suitability of a material for a proposed contact with biological tissues. It is important to note that a material determined to be “biocompatible” for one application (e.g., for a device intended to be in contact only with the intact surface of the skin) may not be biocompatible for another application in which it will be implanted into a body cavity. Therefore, the term biocampatibility is dependent upon the suitability of the safety evaluation as it relates to the intended use of the material. Biomaterial A material that has direct or indirect patient contact. A biomaterial (also termed a biomedical material) may be composed of any synthetic or natural rubber or fiber, polymeric or elastomeric formulation, alloy, ceramic, bonding agent, ink, or other nonviable substance, including tissue rendered nonviable, used as a device or any part thereof. Biomedical material Synonymous with biomaterial. CE marking A “passport” that can allow a manufacturer to circulate its products freely within the European marketplace. The marking applies only to products regulated by European health, safety, and environmental protection legislation (product directives). This is estimated to include more than 50% of the goods currently exported from the United States to Europe including medical devices. CE is short for a French phrase, Confomite Europeene. Class testing The testing of plastics for biological reactivity according to predetermined testing requirements defined by the USP. Combination product A product containing both a drug and device component that are physically, chemically, or otherwise combined to result in a medical product that is used therapeutically as a single entity. In the case of a combination product, the medical device component of the product must be evaluated for safety according to device requirements (as addressed in this chapter) while the drug component must be fully evaluated as necessary for drugs. The final, combined finished product will also need to be evaluated for safety. Design dossier Documentation similar to a Technical File that is submitted to a Notified Body. Defined in Annex II, Section 4 of the Medical Device Directives. Direct contact When the materials of a device are in direct (that is, intimate) contact with the surface or tissues of the body (adhesive bandages, pacemaker leads, as well as dialysis chambers are examples). Direct Contact Cytotoxicity Assay A cytotoxicity assay using cultured mammalian cells in which the material/device is placed in direct contact with the cells and, after a prescribed period of incubation, the cells are examined for signs of toxicity resulting from exposure to the test article. Elution Cytotoxicity Assay A cytotoxicity assay using cultured mammalian cells in which the material/device is extracted in culture media before the media is used to culture the cells. The cells are then incubated in the extracted media for a prescribed period and the cells examined for signs of toxicity resulting from exposure to the test article. Essential requirements The basic documentation requirements for a manufacturer to obtain a CE marking. Many devices require additional documentation to obtain a CE marking.


Requirements are based upon perceived risk. Defined in Annex I of the Medical Device Directives. Extract A solution produced by the incubation of a material/medical device in an appropriate vehicle. After incubation, the vehicle contains the soluble chemicals (or leachables) that have dissolved out of or off of the material/medical device and this combination of soluble chemicals in the vehicle is considered to be an extract. Indirect contact When the materials of a device do not contact the surface or tissues of the body but the materials of the device may influence the body. That is, a solution or material contacts the device, may become contaminated with leachables from the device, and then contacts the body. An example is an intravenous (iv) bag. ISO International Standards Organisation. Leachable A chemical that may, under anticipated-use conditions, dissolve away from a material or device when in contact with a biological system and, therefore, has the potential to produce a biological effect that may be distant from the site of tissue contact. Medical device Any instrument, apparatus, appliance, material, or other article, including software, whether used alone or in combination, intended by the manufacturer for use by human beings solely or principally for the purpose of diagnosis, prevention, monitoring, or treatment; alleviation of disease, injury, or handicap; investigation, replacement, or modification of the anatomy or of a physiological process; control of conception; and that which does not achieve its principal intended action of the human body by pharmaceutical, immunological, or metabolic means, but may be assisted in its function by such means (ISO 10993-1). Predicate device A previously marketed device that is substantially equivalent to a proposed device. The predicate device is used as a comparison to the proposed device to establish safety and efficacy. Processing aid A material that contacts the product during the manufacturing process and, therefore, has a potential for affecting product quality and/or may elicit a biological response following the use of a medical device. Solvents, cleaning products, lubricants, and mold-release agents are examples of processing aids. Technical documentation All documents supporting a European CE Marking. Technical file European documentation for a medical device including the essential requirements data, product specifications, and manufacturing data. Type examination Testing of a device by a European Notified Body. Defined in Annex II, Section 3 of the Medical Device Directives. USP Negative Control Plastic RS A standardized plastic produced by the USP for use as a control material in some biocompatibility assays.

REFERENCES 1. Rubin, J.P. and Yaremchuk, M.J., Complications and toxicities of implantable biomaterials used in facial reconstructive and aesthetic surgery: a comprehensive review of the literature, Plast. Reconstr. Surg., 100, 1336, 1997. 2. Gotman, I., Characteristics of metals used in implants, J. Endourol., 11, 383, 1997 3. Northup, S.J., Strategies for biological testing of biomaterials, J. Biomater. Appl., 2, 132, 1987. 4. Pinchuk, L., A review of the biostability and carcinogenicity of polyurethanes in medicine and the new generation of “biostable” polyurethanes, J. Biomater. Sci. Polym. Ed., 6, 225, 1994. 5. ANSI/AAMI, 10993-1: Biological evaluation of medical devices — Part 1, Guidance on selection of tests, 1997. 6. Nair, P.D., Currently practiced sterilization methods — some inadvertent consequences, J. Biomater. Appl., 10, 121, 1995.


7. Schwindaman, D., Federal regulation of experimental animal use in the United States of America, Rev. Sci. Tech., 13, 247, 1994. 8. U.S. Pharmacopoeia 23/National Formulary 18, 1995 edition, United States Pharmacopeial Convention, Inc., Rockville, MD, 1994, chap. 88.


30

Regulatory Toxicology: Consumer Products Dennis J. Naas, B.S.

CONTENTS Section 1. Overview Table 30.1

Section 2. Section 3. Section 4.

Section 5.

Federal Regulations and Regulatory Agencies for Consumer Products in the United States Table 30.2 General Consumer Product Classes and Corresponding Regulations/Regulatory Agencies Table 30.3 General Consumer Product Class Definitions Consumer Product Safety Commission When to Perform Animal Safety Testing Design, Conduct, and Monitoring of Safety/Toxicity Studies in Animals A. Good Laboratory Practices B. Animal Welfare C. Study Protocol D. Laboratory Selection E. Study Design Considerations Safety Assessment of Consumer Products A. Household Products Table 30.4 Federal Hazardous Substances Act Methods for Assessing Dermal and Eye Toxicity Table 30.5 Federal Hazardous Substances Act Primary Dermal Irritation Scoring Method Table 30.6 Example Calculation of a Primary Dermal Irritation Score According to the Method Described in the Federal Hazardous Substances Act Table 30.7 Recommended Scoring System for Primary Eye Irritation Table 30.8 Federal Hazardous Substances Act Criteria for Classification as an Eye Irritant Table 30.9 Recommended Methods for Assessing Oral and Inhalation Toxicity Table 30.10 Hazard Classification under the Federal Hazardous Substances Act B. Art Materials C. Cosmetics Table 30.11 Cosmetic Product Categories D. Disinfectant Household Products E. Dietary Supplements


Section 6. Resources for Regulatory and Toxicty Information Table 30.12 Resources for Regulatory and Toxicity Information References

SECTION 1. OVERVIEW When a chapter on consumer products was first contemplated, its scope seemed quite straightforward and limited. The interest was to develop a chapter or section that related to the Consumer Product Safety Commission (CPSC), its makeup and authority, and the requirements and guidelines for toxicity safety testing of consumer products to aid the practicing industrial toxicologist. The chapter was intended to provide general information on typical testing programs a consumer products company might follow in developing health and toxicity safety information on such consumer products as household and personal hygiene products. However, when one fully considers the term consumer product in a broad sense, the scope of this subject rapidly expands. For example, certainly, in the traditional sense and use of the term, standard household cleaning products are consumer products that are regulated under the Consumer Product Safety Act (CPSA) via the CPSC. However, a household cleaning product that contains an antimicrobial agent and is labeled as a disinfectant is regulated under the Federal Insecticide, Fungicide, and Rhodenticide Act (FIFRA), as administered by the Environmental Protection Agency (EPA), because it contains a pesticide. Also consider the area of personal hygiene products, i.e., “cosmetics.” This general category includes such diverse products as shaving creams, shampoos, antibacterial liquid soaps, sunscreens, and athlete’s foot treatments, certainly all available directly to the consumer, i.e., consumer products. However, the first two types of products are regulated under the Food, Drug and Cosmetic Act (FDCA) as cosmetics, while the last three, because they contain an “active” ingredient, are regulated via the Over-the-Counter (OTC) Drug Monograph. And although both the FDCA and the OTC are administered by the Food and Drug Administration (FDA), the requirements for marketing these two types of products are quite different. And the above product classes are not included in the special regulatory environments that exist for two other types of consumer products, art materials and dietary supplements. At least ten major U.S. federal regulations exist under which consumer products may be regulated (Table 30.1). As illustrated above, consumer products represent an extremely broad and diverse collection of substances, materials, and products, even if considered only within the context of the U.S. market and regulatory environment, as will be the case in this chapter. For the purposes of this discussion, a consumer product is considered to be any product customarily produced or distributed for sale to or for consumption by the individual customer. Exceptions to this definition are foods and food additives, which are specially regulated by the FDCA/FDA, fuels, which are regulated primarily based on physiochemical hazard rather than toxicity, FIFRA/EPA-regulated pesticides available directly to the consumer, and prescription drugs, which are subjected by the FDA to the most comprehensive safety assessments in existence. General consumer product classes and the corresponding federal regulations and administering agencies are presented in Table 30.2. Consumer product class definitions are presented in Table 30.3. The primary focus of this chapter is on safety/toxicity testing of consumer products that are regulated under CPSA/CPSC, i.e., household products and art supplies (as defined herein). The CPSA/CPSC also regulates consumer products on the basis of physiochemical properties such as explosive potential, flammability, corrosivity, etc., as well as common household items (beds, clothing, toys, etc.) primarily on the basis of physical safety. These aspects of CPSA/CPSC regulation will not be addressed. While a comprehensive discussion of all intricacies related to consumer product testing, safety, labeling, and other regulatory considerations is beyond the scope of this chapter, some limited discussion of these other considerations is provided, as well as


references on resources from which further information can be obtained. Aside from recognizing OTC drug products as a type of consumer product and briefly describing the responsible regulatory acts and agencies, the topic of OTC drug product safety evaluation, registration, and regulation will not be further discussed in this chapter.

TABLE 30.1 Federal Regulations and Regulatory Agencies for Consumer Products in the United States Regulation Federal Trade Commission Act Food, Drug, and Cosmetic Act The Investigational New Drug/New Drug Application Process Federal Insecticide, Fungicide, and Rodenticide Act Federal Hazardous Substances Act Color Additives Amendments to the FDCA Fair Packaging and Labeling Act Poison Prevention and Packaging Act Over-the-Counter Drug Monograph Process Labeling of Hazardous Art Materials Act Dietary Supplement Health and Education Act

Acronym

Year Promulgated

FTCA FDCA IND/NDA FIFRA FSHA CAA FPLA PPPA OTC LHAMA DSHEA

1914 1938 1938 1947 1960 1960, 1962 1966 1970 1972 1988 1994

Administrating Agency FTC FDA FDA EPA CPSC FDA FDA CPSC FDA CPSC FDA

Note: FTC = Federal Trade Commission.

TABLE 30.2 General Consumer Product Classes and Corresponding Regulations/Regulatory Agencies Product Class Household products Art supplies Cosmetics

Disinfectant household products Dietary supplements OTC drug products

a b

Regulation/Agency FHSA/CPSC PPPA/CPSC LHAMA/CPSC PPPA/CPSC FDCA/FDA CAA/FDA FPLA/FDA FTCA/FTC FIFRA/EPA PPPA/CPSC DSHEA/FDA FTCA/FTC OTC/FDA IND-NDA/FDA

Product Class Examples Window cleaner, silicone caulk, floor wax, antifreeze Children’s coloring books, crayons, glues Shaving cream, shampoo, deodorants

Lysol® products,a Tilex®,b Ephedrine, ginkoba, ginseng Treatments for athlete’s foot, antibacterial soap, sunscreen

Registered trademark of Reckitt & Coleman, Inc., Montvale, NJ. Registered trademark of the Clorox Company, Oakland, CA.


TABLE 30.3 General Consumer Product Class Definitions Class Household product

Art material

Cosmetic Disinfectant household product Dietary supplement OTC drug product

Definition This may be viewed generally as a default category of products customarily manufactured or distributed for sale for consumption or use, or customarily stored by, individuals in or about the household that do not fall into other classes. Items such as toys, bunk beds, matches, charcoal briquettes, lighters, electrical appliances, etc. are household products regulated under CPSA/CPSC; however, their regulation is based primarily on physical safety of the consumer and not on chemical toxicity hazard. For the purposes of this discussion, household products may be considered as chemicals or chemical mixtures to which consumers are exposed that do not fall into any of the other classes listed below. “Any raw or processed material, or manufactured product, marketed or represented by the producer or repackager as intended for and suitable for [use by] artists or crafts people of any age who create, or recreate in a limited number, largely by hand, works which may or may not have a practical use, but in which aesthetic considerations are paramount” (16 CFR 1500.14). “A product which enhances appearance, aids in personal hygiene and does not affect the structure or function of the skin.”1 Household products containing an active ingredient present to produce a specific biocidal (usually antimicrobial) effect that are marketed directly to the consumer. These materials are not intended for direct human application/exposure. Any product that is not a food or either a direct or indirect food additive, that is intended for ingestion as a supplement to the diet. Nonprescription drug or cosmetic-like products marketed directly to the consumer, deliberately ingested or applied, containing an active ingredient present to produce a specific desired (usually biological) effect.

SECTION 2. CONSUMER PRODUCT SAFETY COMMISSION The CPSC was formed as an independent commission (agency) on May 14, 1973 under the provisions of the Consumer Product Safety Act (CPSA). The complete text pertaining to the CPSC is located in the Code of Federal Regulation, CFR 16 Parts 1000 through 1750. The stated purposes of the commission are (16 CFR 1000.1): • To protect the public against unreasonable risks of injury associated with consumer products; • To assist consumers in evaluating the comparative safety of consumer products; • To develop uniform safety standards for consumer products and to minimize conflicting state and local regulation; and • To promote research and investigation into the causes and prevention of product-related deaths, illnesses, and injury. In addition to administering the CPSA, the CPSC administers three legislative acts pertinent to consumer product safety as defined herein, the Federal Hazardous Substances Act (FHSA, 16 CFR 1500.1 through 1500.13), the Labeling of Hazardous Art Materials Act [LHAMA, CFR 1500.14(b)(8)], and the Poison Prevention Packaging Act (PPPA, 16 CFR 1700 through 1750), passed initially in 1960, 1988, and 1970, respectively. Products regulated under CPSC do not have to be “registered” prior to marketing, unlike chemicals and products regulated by other agencies, i.e., the EPA under both the Toxic Substances Control Act (TSCA) and FIFRA, and drugs and food additives regulated by the FDA. Thus,


responsibility for appropriate safety stewardship of CPSC-regulated products falls almost exclusively to the producers and manufacturers. The CPSC is empowered to develop uniform safety standards for consumer products. In the event that a standard to “protect the public from the unreasonable risk of injury” adequately cannot be developed, the CPSC is further empowered to ban the product or impose specific labeling or packaging requirements. However, some previous attempts to ban products containing specific materials or to require special labeling have been successfully challenged in the courts.

SECTION 3. WHEN TO PERFORM ANIMAL SAFETY TESTING Perhaps the most confounding question facing the industrial toxicologist today is when to perform animal safety testing on a consumer product. No clear-cut formula exists to help make this determination. The testing considered, required, or recommended to satisfy one regulatory body may impact many other aspects of product commercialization (Material Safety Data Sheet contents, product labeling, U.S. Department of Transportation packaging classification, EPA TSCA Section 8e Significant Risk Notification, etc.). Increasing pressures to conserve laboratory animal resources must be balanced against the obligation to bring products to market responsibly. International regulatory considerations, in an increasingly global commercial environment, further confound the issue. The CPSC has well-defined labeling requirements and warning phrases based on the results of specific acute toxicity testing in animals. Undesirable warning phrases on package labels, i.e., “over-warning,” can clearly represent a competitive disadvantage. However, “under-warning” can be equally or more disadvantageous in this highly litigious society. The actual engagement of safety testing in animals can be a labor- and capital-intensive endeavor and should not be undertaken lightly. The toxicologist and company must be prepared to potentially accept and subsequently deal with an unexpected result. Internal testing resources have greatly diminished in recent years. Therefore, the use of external contract research organizations has become increasingly common. Considerations on laboratory selection and study design, conduct, and monitoring are presented in a subsequent section of this chapter. The following guidelines are offered to the industrial toxicologist evaluating the need for animal safety testing on a “new” consumer product: • Determine as precisely as possible, by ingredient material and their quantities, the chemical composition of the product. Most are mixtures. • Determine if, in the mixing of the ingredient materials combined to make the product, any reactions have or may have occurred to create a substance not originally present. The identification of a reaction product at this point could have several outcomes, depending upon the material that was formed. Inadvertent synthesis of a highly toxic reaction product would almost certainly halt further commercialization efforts, although this can sometimes be resolved via process refinement. Formation of a previously unknown substance or new chemical entity (NCE) could trigger testing, halt commercialization, cause process refinement, or require the toxicologist to perform a structure–activity relationship type of assessment on the new chemical. A reaction to form a known substance would simply result in this material being considered as one of the components in the final product, assessed in the same manner as one of the original ingredient materials. In a reactive process, the relative quantities of the original components would change; such changes should also be taken into account when assessing the toxicity hazard potential of the final product. CPSC addresses the assessment/testing of potentially hazardous mixtures as follows: “It may not be possible to reach a fully satisfactory decision concerning the toxic, irritant, corrosive, flammable, sensitizing, or


pressure-generating properties of a substance [mixture] from what is known about its components or ingredients. The mixture itself should be tested.”(16 CFR 1500.5). • Determine the relevant route or routes of human exposure. For example, a liquid household cleaner will or may come into contact with the skin, eyes, and possibly be ingested, and therefore data by these routes of exposure should be available. However, virtually no opportunity exists for inhalation exposure; therefore, data by this route would not necessarily be required. A spray oven cleaner does pose an inhalation exposure risk, as well as potential for exposure by the other routes. Therefore, information regarding the toxicity hazard by the inhalation route should be available for such a product. In general, data need to be available or testing needs to be performed only by relevant exposure routes. A new application for an existing product, e.g., a liquid cleaner made available in spray form, could trigger testing by a route of exposure not previously evaluated. • The total potential exposure to the product should be assessed, in terms of both the frequency of exposure and the dose received at each exposure. This information may be used to determine the adequacy of existing toxicity data and/or in the design of any studies that may be performed. Single-exposure, short-term (acute) data or studies are the minimum needed to classify a substance and in most cases will satisfy CPSC requirements with respect to hazard identification and labeling. • Evaluate the toxicity information available for the chemical or substances that comprise the product. Physiochemical factors such as pH, volatility, physical form, viscosity, etc., should be taken into account. The data should be reviewed for consistency, completeness, currency, and general quality. The reliability of the source of the information or data must be carefully considered; some resources are generally more reliable than others. A peer-reviewed publication of a study conducted under Good Laboratory Practice standards (GLPs) should be given much greater credence than a single sentence or two from a Material Safety Data Sheet (MSDS) obtained from a second party. Full study reports, when available, are preferable to abstracts. More recent studies tend to be of higher quality and thus more reliable than older ones because of the now-routine conduct of toxicity/safety studies under GLPs. A multitude of resources exists from which to obtain toxicity data as well as other physiochemical and hazard information on chemicals of commercial interest. A listing of examples of such resources is provided at the end of this chapter (see Table 30.12). Thus, the ultimate question that the industrial toxicologist must answer is “Does sufficient toxicity data of adequate type and quality exist to responsibly, safely, and efficiently bring this product to market without an unreasonable risk of injury to the consumer?” If the answer to this question is no, appropriate studies to address the data inadequacies must be performed.

SECTION 4. DESIGN, CONDUCT, AND MONITORING OF SAFETY/TOXICITY STUDIES IN ANIMALS A. GOOD LABORATORY PRACTICES Animal toxicity/safety studies conducted in support of consumer product marketing and/or registration should be conducted under GLPs. Several such federal standards exist; the EPA has promulgated two sets of GLPs, one for TSCA (40 CFR Part 792) and one for FIFRA (40 CFR Part 160). In addition, the FDA has issued Good Laboratory Practice for Nonclinical Laboratory Studies (21 CFR Part 58). For practical intents and purposes, these standards are essentially identical. Studies to support products regulated exclusively by CPSA/CPSC may be conducted under any of these sets of GLPs. Studies intended to support product registration under the jurisdiction of other regulatory agencies should follow the appropriate respective set of GLPs.


An important note to manufacturers/producers who may sponsor or conduct GLP studies concerns test substance/article characterization. All the above GLP standards require test substance/article characterization prior to starting the test. Although it is not explicitly stated that this characterization be carried out under GLPs, the presence of this passage within the standards certainly implies this to be the case.

B. ANIMAL WELFARE It is strongly recommended that the test facility be accredited by the Association for Assessment and Accreditation of Laboratory Care International (AAALAC International). Participation in this accreditation program is voluntary. Accreditation is based on thorough triannual inspection by a team of animal welfare experts of all facilities, equipment, procedures, and practices relating to the laboratory’s care, maintenance, and management of its animal resources, in accordance with federal animal welfare laws. AAALAC International accreditation helps provide assurance of the welfare and humane treatment of the animals used for testing.

C. STUDY PROTOCOL The study protocol is the controlling document with respect to study conduct. In accordance with the applicable GLPs, it should contain all information necessary to execute the project according to the stated study objective.

D. LABORATORY SELECTION With respect to externally contracted studies, all major regulatory agencies hold the study sponsor legally responsible for its conduct and results, not the testing facility. From this aspect, some level of monitoring external studies, including on-site inspections, is both prudent and common practice. Literally dozens of testing facilities able to conduct the studies to support consumer product marketing/registration are in operation. Unfortunately, no objective mechanism exists for evaluating their capabilities, competence, or quality. Laboratory selection factors may include personal knowledge, the laboratory’s reputation, professional references, prestudy site inspections, proximity, personnel, governmental agency inspections, study fees, and other factors. In the absence of knowledge in this area, utilization of the services of an independent professional consultant familiar with the contract research organization industry may be highly valuable.

E. STUDY DESIGN CONSIDERATIONS Animal toxicity/safety studies are not often conducted solely to support marketing of a consumer product in the United States under CPCA/CPCS/FHSA. The potential for product penetration into other, especially international, markets may require more rigorous testing than that described in FHSA. Some types of studies performed in accordance with other federal and/or international testing guidelines, while providing more information than required by the FHSA test methods, could yield results perfectly acceptable for arriving at an FHSA classification. For example, an acute oral toxicity study done to comply with either the EPA Office of Prevention, Pesticides and Toxic Substances (OPPTS) Guideline No. 870.1100 or the Organisation for Economic Cooperation and Development (OECD) Test Guideline No. 401 should produce an LD50 value that can be used to assign an FHSA classification. The same can be said for determination of acute dermal toxicity (EPA OPPTS Guideline No. 870.1200 or OECD Test Guideline No. 402). However, this is not always the case. Other major regulatory bodies usually require determination of the acute inhalation LC50 based on a 4-h exposure duration, while the FHSA is based on a 1-h exposure. Whereas extrapolation is possible, a 4-h exposure to the same concentration is obviously more likely to cause acute toxicity than a 1-h exposure. Similarly, primary dermal irritation is assessed under EPA



OPPTS and OECD test guidelines following a 4-h rather than a 24-h exposure. So while a study of a substance that caused severe irritation or corrosion in an OPPTS or OECD design could certainly be used to classify under FHSA, a minimally to moderately irritating result in such a study would not be useful for FHSA classification. Similar logic can be applied to primary eye irritation testing, with the difference being the OPPTS and OECD designs utilize only three rabbits whereas the FHSA design requires at least six. With proper planning and foresight, studies can be designed to meet or exceed the requirements of multiple regulatory agencies, thereby responsibly utilizing laboratory animal resources, maximizing the information obtained from the studies and its subsequent use, while minimizing product development time and costs without compromising consumer safety. Ultimately, the design of animal toxicity/safety studies must be such that previously identified data inadequacies are addressed, future data needs are anticipated and addressed to greatest extent reasonably possible, and the studies are acceptable to the regulatory body (or bodies) of interest.

SECTION 5. SAFETY ASSESSMENT OF CONSUMER PRODUCTS A. HOUSEHOLD PRODUCTS Toxicity/safety test methods, evaluation methods, classification criteria, and definitions for household products are presented and described in Tables 30.4 through 30.10.

TABLE 30.4 Federal Hazardous Substances Act Methods for Assessing Dermal and Eye Toxicity Acute Dermal Toxicitya Test species n Dose levels

Albino rabbits 10 (per dose level)d Sufficient to enable the calculation of an LD50

Chemical exposure duration Observation period (following exposure) Comments

24 h 14 days

Classification

Fully occlusive, impervious wraps are usedh The skin of the test site is abraded Result: The LD50 is the median dose at which 50% mortality is observed. See Table 30.10

a

Primary Dermal Irritancyb

Eye Irritationc

Albino rabbits 6 0.5 ml/site (liquids) 0.5 g/site (solids; semisolids)e 24 h 24 and 72 h

Albino rabbits 6 0.1 ml (liquids) 100 mg (solids; pastes)f

Fully occlusive, impervious wraps are usedh At least one intact and one abraded site are employed per animal Scoring: see Table 30.5

One eye is treated; contalateral eye serves as control Eyes are not washed for at least 24 h after exposure Scoring: see Table 30.7

See Tables 30.6 and 30.10

See Tables 30.8 and 30.10

24–72 hg 24, 48, and 72 h

16 CFR 1500.40. 16 CFR 1500.41. c 16 CFR 1500.42. d Normally five animals/sex/dose level are employed. e Solids should be dissolved in an appropriate solvent and the solution applied as directed for liquids. f For powders, flakes, granular materials, or other particulate, 0.1 ml of compacted material shall be used whenever this volume weighs 1 mm and extending beyond the area of exposure) a b

0 1 2 3 4 0 1 2 3 4

Taken from 16 CFR 1500.41. The “value” recorded is the average value for six or more animals subjected to the test.

TABLE 30.6 Example Calculation of a Primary Dermal Irritation Score According to the Method Described in the Federal Hazardous Substances Acta Skin Reaction Erythema and eschar Intact Skin Abraded Skin Subtotal Edema Intact Skin Abraded Skin Subtotal Total Score Primary Irritation Scorec: 16 ÷ 4 = 4 a

Exposure Time, h

Valueb

24 72 24 72

3 2 4 3 12

24 72 24 72

0 1 1 2 4 16

Classificationd: Not an irritant

Taken from 16 CFR 1500.42. The “value” recorded is the average value for six or more animals subjected to the test. c To calculate the primary irritation score, intact and abraded sites are evaluated, at 24 and 72 h after exposure, according to the scoring method presented in Table 30.5. The four values for erythema and eschar are added together for a subtotal (12 in this example). The four values for edema are added together for a second subtotal (4 in this example). The two subtotals are added together to give an overall total score (16 in this example). This total score is then divided by 4 to give the primary irritation score. The material can then be classified. d Substances/products whose Primary Irritation Score is ≥ 5 are classified as “irritants” under FHSA. b



TABLE 30.7 Recommended Scoring System for Primary Eye Irritationa,b Scorec

Ocular Reaction Cornea Opacity — Degree of density (area most dense taken for reading) No ulceration or opacity Scattered or diffuse areas of opacity (other than slight dulling of luster), details of iris still clearly visible Easily discernible translucent area, details of iris slightly obscured Nacreous area, no details or iris visible, size of pupil barely discernible Opaque cornea, iris not discernible through opacity Iris Normal Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperemia, or injection (any of these or combination of any thereof), iris still reacting to light (sluggish reaction is positive) No reaction to light, hemorrhage, gross destruction (any or all of these) Conjunctivae Redness (refers to palpebral and bulbar conjunctivae, excluding cornea and iris) Blood vessels normal Some blood vessels definitely hyperemic Diffuse crimson color with individual vessels not discernible Diffuse, beefy red Chemosis (refers to lids and/or nictitating membranes) No swelling Any swelling above normal (includes nictitating membranes) Obvious swelling with partial eversion of lids Swelling with lids about half closed Swelling with lids more than half closed

0 1* 2* 3* 4* 0 1* 2*

0 1 2* 3* 0 1 2* 3* 4*

a

Grading scale for scoring ocular lesions, as published in EPA-OPPTS Health Effects Test Guideline 870.2400 (1998). Reading of reactions is facilitated by the use of a binocular loupe, handheld slit lamp, or other means. After recording the scores at 24 h, the eyes may be further examined through the use of sodium fluoride solution and ultraviolet light to aid in the detection of corneal damage. c Starred (*) scores indicate a positive response. If any animal receives one or more scores that are starred, the animal is considered to exhibit a positive response for eye irritation. b

TABLE 30.8 Federal Hazardous Substances Act Criteria for Classification as an Eye Irritanta Ocular Reactions for Individual Animals Corneal ulceration (other than stippling) Corneal opacity (other than slight dulling of luster) Inflammation of the iris (other than deepening of the folds or a slight circumcorneal injection of the blood vessels) Conjunctival swelling with partial eversion of the eyelids or a diffuse crimson-red coloration with individual vessels not discernible Group Result (n = 6) 4–6 animals in the test group exhibit a positive reaction 2–3 animals in the test group exhibit a positive reaction 0–1 animals in the test group exhibit a positive reaction a

Test Result Positive Positive Positive Positive FHSA Classification Eye irritant Inconclusiveb Nonirritant

16 CFR 1500.42. If two to three animals exhibit a positive response, the test is repeated using new test animals. If three or more animals in the second test exhibit positive reactions, the material is classified as an eye irritant under FHSA. If one to two animals in the second test exhibit a positive response, a third test is conducted using new test animals. If one or more animals in the third test exhibit a positive response, the material is classified as an eye irritant under FHSA.

b


Test species n (per dose level) Dose levels Chemical exposure duration Observation period (following exposure) Comments Classification a

Acute Oral Toxicity

Acute Inhalation Toxicity (Gases and Vapors)

Acute Inhalation Toxicity (Dusts and Mists)

Albino rats (200–300 g) 10b 5000, 500, and 50 mg/kgc Single oral gavage 14 days Rats should be fasted approximately 18 h before dosing See Table 30.10

Albino rats (200–300 g) 10b 200,000, 20,000, or 200 ppmc,d Single 1-h exposure 14 days Either whole-body or nose-only exposure methods may be used See Table 30.10

Albino rats (200–300 g) 10b 2 mg/le Single 1-h exposure 14 days Either whole-body or nose-only exposure methods may be used See Table 30.10

Adapted from 16 CFR 1500.3. LD50/LC50 data from previously conducted acceptable studies may be used to assign a classification; however, some end points routinely included in studies for other regulatory agencies (clinical observation, gross necropsy, etc.) need not necessarily be included in an LD50/LC50 study done solely for FHSA classification purposes. LD50/LC50 studies can be designed to satisfy multiple regulatory agencies; refer to EPA OPPTS Series 870 — Health Effects Test Guidelines (870.1100 for oral and 870.1300 for inhalation) or OECD Guidelines for Testing of Chemicals, No. 401 (oral) and No. 403 (inhalation). b Normally five animals/sex/dose level are employed. c Testing is initiated using the highest dose level only. If less than 50% mortality occurs at this (or any subsequent) level, no further testing is done and the LD /LC 50 50 is estimated accordingly. If mortality is ≥ 50%, the next lower level is tested until the appropriate LD50/LC50 estimation can be made. d The highest exposure level may be limited to the saturated vapor concentration. e 16 CFR 1500.3 discusses testing results up to 200 mg/l in the labeling classification of dusts and mists based on acute inhalation toxicity. Dusts or mists with an estimated or calculated 1-h LC50 ≤ 2 mg/l are classified under FHSA as highly toxic. Dusts or mists with an estimated or calculated LC50 > 2 mg/l and ≤ 200 mg/l are classified as toxic. It is presumed that dusts and mists having an LC50 > 200 mg/l are considered nontoxic, but this is not explicitly stated. All major federal and international testing guidelines specify maximum limit test exposure levels of 2 to 5 mg/l (for 4 h). Exposure duration aside, the toxicological relevance of a 200 mg/l exposure concentration is highly questionable. Achieving relevant airborne concentrations 40 times below this level is technically challenging. Further, physical laws for aerosol particle behavior limit minimum (respirable) particle size, even in experimental test atmospheres.2 Based on these considerations, and in the absence of a clear benefit in doing so, inhalation testing of dusts and mists above 2 mg/l is not recommended.



TABLE 30.9 Recommended Methods for Assessing Oral and Inhalation Toxicitya


TABLE 30.10 Hazard Classification under the Federal Hazardous Substances Acta Term

Definition

Hazardous substance

“Any substance or mixture of substances which is toxic, corrosive, an irritant, a strong sensitizer, flammable or combustible, or generates pressure through decomposition, heat or other means, if such substance or mixture of substances may cause substantial personal injury or substantial illness during or as a proximate result of any customary or reasonably foreseeable handling or use, including reasonably foreseeable ingestion by children.” Definition includes: • Any substance that, by regulation, the CPSC finds meets the requirements noted in the definition above; • Radioactive substances, with respect to the way these substances are used in a particular class of article or as packaged, that the CPSC deems sufficiently hazardous to require labeling in accordance to the FHSA to protect public health; • Toys or articles intended for use by children that, by regulation the CPSC finds, in accordance with section 3(e) of the FHSA, presents an electrical, mechanical, or thermal hazard;. • Any article which is not itself a pesticide within the meaning of FIFRA, but which is a hazardous substance within the meaning of FHSA by reason of bearing or containing such a pesticide. Definition excludes: • Pesticides subject to regulation under FIFRA; • Foods, drugs, cosmetics subject to the FDCA; • Substances intended as fuels when stored in containers and used in the heating, cooking, or refrigeration system of a house; • Source materials, special nuclear material, or by-product material as defined by the Atomic Energy Act of 1954, as amended, and regulations issued pursuant thereto by the Atomic Energy Commission. Any substance that has the capacity to produce personal injury or illness through ingestion, inhalation, or absorption through body surfaces. The definition is expanded to include the specifics listed below. This classification also applies to any substance that is “toxic” (but not “highly toxic”) on the basis of human experience. Unless specified, the studies listed below require a sufficient number of rats to give a statistically significant result and are to be in conformity with good scientific practices. In the acute studies, rats are exposed once and are observed for 14 days. Acute Oral Toxicity LD50 > 50 mg/kg and < 5000 mg/kg Substances with an LD50 > 500 mg/kg and < 5000 mg/kg, may be considered for some labeling exemptions if it can be shown that the labeling is not needed because of the physical form of the material, the size or closure of the container, human experience with the article, or other relevant factors. Acute Inhalation Toxicity 1-h LC50 in rats is > 200 ppm and ≤ 20,000 ppm (gas or vapor), or 1-h LC50 in rats is > 2 mg/l and ≤ 200 mg/l (dust or mist), if such concentration is likely to be encountered by humans when the substance is used in any reasonably foreseeable manner Acute Dermal Toxicityb LD50 > 200 mg/kg and ≤ 2000 mg/kg A substance is toxic because it presents a chronic hazard if it falls into one of the following categories: Carcinogen — Substance is or contains a known or probable human carcinogen Neurotoxicant — Substance is or contains a known or probable human neurotoxicant Developmental or reproductive toxicant — Substance is or contains a known or probable human developmental or reproductive toxicant

Toxic

Acute toxicity

Chronic toxicityc



TABLE 30.10 (Continued) Hazard Classification under the Federal Hazardous Substances Acta Term Highly toxic

Corrosive

Irritant

Strong sensitizer

Sensitizer

Normal living tissue

Strong

Severity

Significant potential

a b c

Definition Any substance that falls into any one of the categories listed below. If the CPSC finds that available data on human experience with any substance indicate results different from those obtained on animals in the dosages and concentrations specified below, the human data take precedence. These studies involve a single exposure of 10 rats (oral, inhalation) or 10 rabbits (dermal) to the test substance followed by a 14-day observation period. Acute Oral Toxicity LD50 ≤ 50 mg/kg Acute Inhalation Toxicity 1-h LC50 ≤ 200 ppm (gas or vapor), or 1-h LC50 ≤ 2 mg/l (dust or mist), provided such concentration is likely to be encountered by man when the substance is used in any reasonably foreseeable manner. Acute Dermal Toxicityb LD50 ≤ 200 mg/kg Any substance that in contact with living tissue will cause visible destruction or irreversible alterations in the tissue by chemical action at the site of exposure. This does not include the effect of chemical action on inanimate surface (e.g., corrosive to aluminum or steel). A substance would be considered corrosive if tested as described in 16 CFR 1500.41 and visible destruction or irreversible alteration of the tissue at the site of contact was observed. Any substance not corrosive within the meaning above, which on immediate, prolonged, or repeated contact with normal living tissue will induce a local inflammatory response. This includes substances classified as primary skin irritants and eye irritants according to 16 CFR 1500.41 and 16 CFR 1500.42, or where there are human data to indicate the material is an irritant to the skin or eyes. A substance that will cause, on normal living tissue through an allergic or photodynamic process, a hypersensitivity that becomes evident on reapplication of the same substance and that is designated as such by the CPSC. Before designating the substance as a strong sensitizer, the CPSC will consider the frequency of occurrence and the severity of reaction, and from these data will conclude that the substance has a significant potential to produce hypersensitivity. A substance that will induce an immunologically-mediated (allergic) response, including photosensitivity, that becomes evident upon re-exposure to the same substance. Occasionally it may induce a response on first exposure by virtue of active sensitization. The allergic response occurs in normal living tissues, including skin and other organs such as the respiratory system or gastrointestinal tract, either singularly or in combination, following sensitization by contact, ingestion, or inhalation. The CPSC shall consider the available data for a number of factors, including quantitative or qualitative risk assessment, frequency of occurrence and range of severity of reactions in healthy or susceptible populations, experimental animal and human data (considering dose–response relationships) with human data taking precedence, potency or bioavailability data, crossreactivity, threshold of human sensitivity, epidemiological studies, case histories, occupational studies, and other appropriate in vivo or in vitro studies. The minimal severity reaction required for classification as a “strong” sensitizer is a clinically important allergic reaction. This may include: substantial illness, physical discomfort, distress, hardship, and functional or structural impairment. These reactions may require medical treatment or may produce loss of functional activities. A relative determination that must be made separately for each substance. It may be based on the chemical or functional properties of the substance, documented medical evidence of allergic reactions obtained from epidemiological studies or individual case reports, controlled in vitro or in vivo experiments, or susceptibility profiles in normal or allergic subjects.

16 CFR 1500.3. Method according to 16 CFR 1500.40. Detailed criteria for assigning a chronic toxicity classification to a material are located in 16 CFR 1500.135.



B. ART MATERIALS The Labeling of Hazardous Art Materials Act (LHAMA), promulgated in 1988, set standards for the evaluation and labeling of art materials (as defined earlier) for chronic toxic hazard potential. A key component of this act is that the producer or repackager of art materials is required to have the product formulation reviewed by a certified physician or toxicologist for potential chronic toxicity hazards. The reviewer would normally perform a component-based assessment on the product, which is then used to arrive at appropriate precautionary labeling. The Act indicates that the reviewer shall make the labeling recommendation(s). The requirements for labeling are set forth in the standard of the American Society for Testing and Materials identified as ASTM D-4236, the provisions of which are contained in 16 CFR 1500.2. In order for a thorough review to be performed, it is essential that a complete and accurate description of the product formula be available. The regulations specify that (with certain exceptions) this be held in confidence by the reviewer; however, unless the reviewer is an employee of the manufacturer or repackager, additional contractual assurances of confidentiality should be considered. As for many consumer products, art materials are usually mixtures of many chemicals and toxicity test results on the final formulation or product are rarely available. Therefore, not only the individual components, but also the amount (percentage, usually by weight) of each component must be known to perform a full evaluation. The physical form of the final product, use exposure scenarios, potential for chemical reaction in the mixture during processing and sensitive populations, especially children, must all be taken into account when assessing the product. On occasion, component information or final product formulation information is not available or is extremely difficult to obtain. In such cases, it may be appropriate to perform an empirical determination of the product composition analytically. It is important to recognize that the LHAMA labeling requirements do not diminish the effect of required acute toxicity hazard warnings. The focus of LHAMA is on requiring precautionary warnings for chronic adverse health effects, defined as “a persistent toxic effect that develops over time from a single, prolonged or repeated exposure to a substance” (16 CFR 1500.14). Under this definition, such obvious hazards as carcinogenicity, developmental or reproductive toxicity, and neurotoxicity are considered chronic adverse effects. In addition, such less obvious toxicities as permanent skin scarring, irreversible ocular damage (especially to the cornea), sensitization or a permanent adverse effect to any other organ system meet the LHAMA definition of a chronic adverse effect. Further, the Act specifically cites a concern for harm to a nursing infant; therefore, the potential for any of the substances present to be expressed in human milk must also be assessed.

C. COSMETICS In 21 CFR 720.4, the FDA describes 13 categories of cosmetics that contain at total of 66 different product types. In addition, each category contains provision for “other” products that do not specifically fit one of the preassigned product types. A listing of these categories and product types is presented in Table 30.11. Premarket approval of cosmetics (as previously defined) is not required, and, in fact, there is no regulatory statute that requires any product safety testing for cosmetics. The manufacturer is responsible for assuring the safety of its cosmetic products and no product can be marketed if it contains “a poisonous or deleterious substance which may render it injurious to health” (FDCA, Section 601). [Note: The safety of color additives must be demonstrated with reasonable certainty according to the 1960 Color Additives Amendment to the FDCA.] The industrial toxicologist is responsible for determining the level of testing necessary and appropriate for the safe introduction of cosmetic products into commerce. For many decades, this consisted of a fairly standard battery of acute tests for oral toxicity in rats, dermal toxicity in rabbits, primary dermal and ocular irritancy in rabbits, and dermal sensitization testing in guinea pigs. If appropriate, based on the intended-use pattern, acute inhalation testing in rats, or longer-term studies Copyright © 2002 by Taylor & Francis


by the appropriate route of exposure may also have been conducted. While the merits of such animal testing and use shall likely forever remain in debate, this historical testing has provided an enormous body of safety and toxicity data for cosmetic ingredients and final products against which current ingredients, products, and reformulations can be compared. Since relatively few new chemicals or substances are entering the cosmetic product chain, the need for new safety studies in animals has continued to diminish. Cosmetic producers now rely more on historical safety data, structure–activity assessments, component assessments, and certain validated in vitro testing to document and demonstrate the safety of their products. If testing is needed, standard toxicity testing protocols appropriate for the route of administration and potential exposure are used. Aside from the standard resources that can provide toxicity information on chemicals, several excellent resources targeted to the cosmetic industry are available. The International Journal of Toxicology (Taylor & Francis, publishers), the official journal of the American College of Toxicology, periodically dedicates an entire issue to the in-depth evaluation of a few to several cosmetic ingredients. These reports are issued by the Cosmetic Ingredient Review (CIR) Expert Panel as supported by the Cosmetic, Toiletry, and Fragrance Association, Inc. The CIR also publishes reports independently. The International Journal of Toxicology also periodically dedicates an entire issue to the publication of acute toxicity data. To date, data on approximately 500 Chemical Abstract Services-registered chemicals have been published. In vitro alternatives to animal testing continue to hold promise and to be developed for use in the safety assessment of cosmetics, cosmetic ingredients, and other materials and products. However, to date, only one in vitro test system has gained wide and formal acceptance in the U.S. regulatory community. The Corrositex® (InVitro International, Irvine, CA) has been accepted by the FDA, EPA, and CPSC as a suitable substitute for dermal corrosion data developed in animals, provided a positive result is obtained. Cosmetic manufacturers routinely file their cosmetic product ingredient composition (21 CFR 720) and register their production/manufacturing sites (21 CFR 710) with the FDA. Purported adverse reactions to cosmetics are reported to the FDA by the manufacturer (21 CFR 730). These are technically considered to be voluntary programs, but participation within the cosmetic industry is essentially complete.

TABLE 30.11 Cosmetic Product Categoriesa Category Baby products

Bath preparations

Eye makeup preparations


Product Types Shampoos Lotions Oils Powders Creams Other baby products Bath oils, tablets, salts Bubble baths Bath capsules Other bath preparations Eyebrow pencil Eyeliner Eye shadow Eye lotion Eye makeup remover Mascara Other eye makeup preparations


TABLE 30.11 (Continued) Cosmetic Product Categoriesa Category Fragrance preparations

Hair preparations (noncoloring)

Hair coloring preparations

Makeup preparations (not eye)

Manicuring preparations

Oral hygiene products

Personal cleanliness


Product Types Colognes and toilet waters Perfumes Powders (dusting and talcum; excluding aftershave talc) Sachets Other fragrance preparations Hair conditioners Hair sprays (aerosol fixatives) Hair straighteners Permanent waves Rinses (noncoloring) Shampoos (noncoloring) Tonics, dressings, and other hair grooming aids Wave sets Other hair preparations Hair dyes and colors (all types requiring caution statement and patch test) Hair tints Hair rinses (coloring) Hair shampoos (coloring) Hair color sprays (aerosol) Hair lighteners with color Hair bleaches Other hair coloring preparations Blushers (all types) Face powders Foundations Leg and body paints Lipstick Makeup bases Rouges Makeup fixatives Other makeup preparations Basecoats and undercoats Cuticle softeners Nail creams and lotions Nail extenders Nail polish and enamel Nail polish and enamel removers Other manicuring preparations Dentifrices (aerosol, liquid, pastes, and powders) Mouthwashes and breath fresheners (liquids and sprays) Other oral hygiene products Bath soaps and detergents Deodorants (underarm) Douches Feminine hygiene deodorants Other personal cleanliness products


TABLE 30.11 (Continued) Cosmetic Product Categoriesa Category Shaving preparations

Skin care preparations (creams, lotions, powders, and sprays)

Suntan preparations

a

Product Types Aftershave lotions Beard softeners Men’s talcum Preshave lotions (all types) Shaving cream (aerosol, brushless, and lather) Shaving soap (cakes, sticks, etc.) Other shaving preparations Cleansing (cold creams, cleansing lotions, liquids, and pads) Depilatories Face and neck (excluding shaving preparations) Body and hand (excluding shaving preparations) Foot powders and sprays Moisturizing Night Paste masks (mud packs) Skin fresheners Other skin preparations Suntan gels, creams, and liquids Indoor tanning preparations Other suntan preparations

Taken from 21 CFR 720.4.

D. DISINFECTANT HOUSEHOLD PRODUCTS Because they contain a pesticide, usually an antimicrobial agent, disinfectant household products bearing a label claim for antimicrobial activity are subject to regulation and registration via the EPA FIFRA. Data development and registration for approval to market as a disinfectant must be sought following the standard EPA FIFRA process described elsewhere in this book. Data required for submission may include, but are not necessarily limited to (as appropriate for the intended use), product chemistry, residue chemistry, environmental fate, toxicology, reentry protection, aerial drift evaluation, toxicity to wildlife and aquatic organisms, protection of plant and nontarget insects. Assessments of the pure and technical grades of the active substance may need to be performed, as well as information on intentionally added inert ingredients, metabolites of active or inert ingredients, the end-use product, the end-use product plus recommended vehicles or adjuvants, or any additional substance that could act as a synergist to the product for which registration is sought (40 CFR 158.75). Previously developed data may be used provided they were done under GLPs, meet the purposes of the regulation, and permit sound scientific judgments to be made. The Pesticide Assessments Guidelines contain the standards for conducting acceptable tests (although results from studies conducted using other appropriate protocols are acceptable), guidance on evaluation and reporting of data, definitions of terms, further guidance on when data are required, and examples of acceptable protocols. These guidelines are available through the National Technical Information Service, 5285 Port Royal Road, Springfield, VA 22161 (703-487-4650).



E. DIETARY SUPPLEMENTS The regulatory environment for dietary supplements is relatively new and evolving. The Dietary Supplement Health and Education Act (DSHEA) was enacted in 1994, primarily under the jurisdiction of the FDA. For dietary supplements, the FDA oversees safety, manufacturing, and product information, including claims, while the FTC regulates advertising. DSHEA applies only to “new” dietary supplements, i.e., those containing an ingredient not marketed in the United States before 1994. Manufacturers desiring to market such a product have two choices. One is to submit to FDA information that supports the conclusion that the new ingredient can reasonably be expected to be safe, i.e., that it does not present a significant or unreasonable risk of illness or injury under the recommended conditions of use. This information must be submitted at least 75 days before the product is expected to go on the market and is made available to the public 90 days after the FDA receives it. The second option for manufacturers is to ask the FDA to establish conditions under which the new ingredient would reasonably be expected to be safe. As of January 1999, no such petitions had been received. Contrast the above with food additives, which, if not generally recognized as safe (GRAS), are subject to the FDA approval process for new food ingredients before marketing. The food additive manufacturers must conduct extensive, often expensive, and long-term safety studies to meet this regulatory burden of proof of safety. This is further contrasted against the regulatory and approval requirements for drugs. Dietary supplements, which often make curative-, preventative-, or treatment-like claims, are not required to undergo clinical studies for effectiveness, safety, interactions with other substances, or determination of appropriate dosages. Issues concerning the safety assessment of dietary supplements via epidemiological types of studies are numerous. In the absence of controlled studies, an uncertain history of use must be relied upon. Many dietary supplements are derived from plants. Plant types, identification, and nomenclature vary regionally as well as internationally. The source, or origin, of the material used to prepare or manufacture the dietary supplement can be a source of variability and contamination. Biomarkers for exposure have not generally been established and therefore estimates of exposure are difficult to obtain. The potential effects of mixtures of dietary supplements and possible interactive effects must also be considered. Existing dietary supplements may or may not be manufactured under Good Manufacturing Practices (GMPs). DSHEA authorizes the FDA to establish GMPs for dietary supplements. The FDA has sought public comment on establishing GMPs for dietary supplements if conventional food GMPs are inadequate. Certain dietary supplement manufacturers trade associations are striving toward self-regulation. For example, industry-specific GMPs have been devised by the Council for Responsible Nutrition, which are voluntarily followed by some manufacturers. Limited safety testing of dietary supplements, using studies similar to the “upper limit protocols” for vitamins and minerals proposed by the National Academy of Sciences, has some support in the industry.



SECTION 6. RESOURCES FOR REGULATORY AND TOXICTY INFORMATION TABLE 30.12 Resources for Regulatory and Toxicity Information Name American College of Government Industrial Hygienists (ACGIH) Cosmetic Ingredient Review (CIR) Code of Federal Regulations (CFR) Consumer Product Safety Commission (CPSC) Environmental Protection Agency (EPA) EPA Integrated Risk Information System (IRIS) EPA Office of Pesticide Programs (OPP) EPA Technical Documents Food and Drug Administration (FDA) Grateful Med V2.6.2 International Agency for Research on Cancer (IARC) International Journal of Toxicology Medline Medscape MSDSs Organisation for Economic Cooperation and Development (OECD) Office of Prevention, Pesticides, and Toxic Substances (OPPTS) Patty’s Industrial Hygiene and Toxicology Toxline® Toxic Substances Control Act Test Submissions (TSCATS), via The Right to Know Network

Source of Availability http://www.acgih.org [email protected] http://www.access.gpo.gov/nara/cfr/ http://www.cpsc.gov http://www.epa.gov http://www.epa.gov/iris/ http://www.epa.gov/pesticides/ http://www.epa.gov/epahome/techdoc.htm http://www.fda.gov http://igm.nlm.nih.gov http://www.iarc.fr/index.html Taylor & Francis, Philadelphia, PA (publisher) http://igm.nlm.nih.gov http://www.medscape.com/ http://www.ilpi.com/msds/index.chtml http://www.oecd.org http://www.epa.gov/internet/oppts/ Available in book form or on CD-ROM from John Wiley & Sons, New York (publisher) Available via Grateful Med V2.6.2 http://www.rtk.net/tscatsinputstandard.html

REFERENCES 1. Jackson, E.M., Consumer products: cosmetics and topical over-the-counter drug products, in Regulatory Toxicology, Chengelis, C., Holson, J.F., and Gad, S.C., Eds., Raven Press, New York, 1995, chap. 5. 2. Anonymous, Commentary — recommendations for the conduct of acute inhalation limit tests (prepared by Technical Committee of the Inhalation Specialty Section, Society of Toxicology), Fundam. Appl. Toxicol., 18, 321–327, 1992.


31

Regulatory Toxicology: Notification of New Substances in the European Union Michael J. Derelanko, Ph.D., D.A.B.T., F.A.T.S.

CONTENTS Section 1. Introduction Table 31.1 Table 31.2 Table 31.3 Table 31.4 Table 31.5 Table 31.6

Exemptions from Notification Notification of a New Substance: Who Makes the Notification and to What Country Notification of a New Substance: Information and Test Data Required Information Common to All Notifications Data Requirements for Notification Comparison of the Notification Requirements of the EU and Selected Countries Selected OECD Guidelines for Testing of Chemicals Risk (R) Phrases Used in the European Community (EU)

Table 31.7 Table 31.8 Section 2. Glossary References Additional Related Information Table 31.9 OECD Screening Information Data Set (SIDS) Studies

SECTION 1. INTRODUCTION Requirements for notification of new substances in the European Union are established under Council Directive 92/32/EEC of April 30, 1992 amending for the seventh time Directive 67/548/EEC on the approximation of the laws, regulations, and administrative provisions relating to the classification, packaging, and labeling of dangerous substances.1 The purposes of the 7th Amendment are to approximate laws, regulations, and administrative provisions of the Member States on: a) the notification of substances; b) the exchange of information on notified substances; c) the assessment of the potential risk to humans and the environment of notified substances; d) classification, packaging, and labeling of dangerous substances. Testing requirements are linked to quantities of substances marketed with yearly production and cumulative thresholds triggering the type of tests. There are reduced notification requirements for substances placed on the market in quantities of less than 1 tonne per year per manufacturer. Production at greater than 1 tonne per year or 5 tonnes cummulative requires base set testing.


Once a notification has been made at more than 1 tonne, any quantity of substance may be supplied. However, the competent authority must be informed when the 10, 100, or 1,000 tonnes per year thresholds have been exceeded or the 50, 500, or 5,000 tonnes cumulative thresholds have been reached. All tests (including physiochemical) must be conducted to GLP and European Community (EC) test guidelines. The adequacy of existing test data which were generated by methods other than stated in the EC guidelines will be decided on a case-by-case basis. In the absence of any indication from the Competent Authority to the contrary, substances notified at less than 1 tonne per year may be placed on the market no sooner than 30 days after receipt by the Authority of a dossier in conformity with the Directive. In the absence of any indications from the Competent Authority to the contrary, substances notified at greater than 1 tonne per year may be placed on the market no sooner than 60 days after receipt by the Authority of a dossier in conformity with the Directive. The following information is intended as a brief overview of the requirements for notification of new substances in the European Union. Readers are referred to the referenced legislation for a detailed description of the requirements. Those unfamiliar with the notification procedure are advised to contact consultants specializing in notifications. Several European contract laboratories offer such services including advice on legislation, conduct of studies, preparation and submission of the notification in the relevant language, conduct of discussions and negotiations with competent authorities, and advice on and negotiation of higher level testing programs.

TABLE 31.1 Exemptions from Notification • • • • • • • • • a

Medicinal productsa Veterinary products Cosmetic products Waste Foodstuffs Animal feedstuffs Pesticides Radioactive substances Other substances or preparations covered by equivalent European Community (EC) notification or approval procedures Active ingredients only, chemical intermediates are not exempt.

Source: Brooker.2

TABLE 31.2 Notification of a New Substance: Who Makes the Notification and to What Country a. Substances manufactured in the European Community • Notification is made by the manufacturer to the Competent Authority in the country of manufacture. • A change of manufacturer or a change of country of manufacture will require a new notification. b. Substances manufactured outside of the European Community • A single notification is made to the Competent Authority of the country in which they are resident by a person designated by the manufacturer as its ‘sole’ representative. This person must be established within the community, need not be the importer, but must list all importations and quantities introduced onto the European market. or • A separate notification is made by each importer. Source: Brooker. 2


TABLE 31.3 Notification of a New Substance: Information and Test Data Required Annual Total < 10 kg 10–100 kg 100–1,000 kg > 1,000 kg (1 tonne) > 10 tonnes > 100 tonnes > 1,000 tonnes

Cumulative Total

Data Requirements (Refer to Table 31.5)

500 kg 5,000 kg 50 tonnes 500 tonnes 5,000 tonnes

Exempt Annex VII C Annex VII B Annex VII A Level 1 (may be requireda) Level 1 Level 2

a Testing at 10/50 tonnage thresholds will depend on the nature of the chemical, its uses, and the results of existing tests.

Source: Brooker.2

TABLE 31.4 Information Common to All Notifications Identity • Identity of manufacturer and notifier • Location of production site • Identity of addresses of importers • Name(s) of substance • Molecular and structural formula • Composition of substance • Methods of detection and determination • Physical state of the substance at 20°C and 101.3 kPa Substance information • Production — sufficient information to allow an approximate but realistic estimation of human and environmental exposure. Precise details (e.g., those of a commercially sensitive nature) are not required. • Technological process • Exposure estimates • Proposed uses • Quantities • Methods and precautions for handling, storage, and transport • Emergency measures • Packaging • Ways of rendering the substance harmless • Classification and proposed labeling Source: Brooker.2


TABLE 31.5 Data Requirements for Notification Annex VII C (Supply at 10–100 kg/yr) Flash point/flammability Acute toxicity (oral or inhalation) Annex VII B (Supply at 100–1,000 kg/yr or 500 kg cumulative) Melting point/boiling point Eye irritation Water solubility Skin sensitization Partition coefficient (n-octanol/water) Ames Flashpoint/flammability Vapor pressure (may be required) Biodegradation Daphnia acute toxicity test (may be required) Acute toxicity (oral or inhalation) Skin irritation Annex VII A “The Base Set” (Supply at > 1,000 kg/yr or 5,000 kg cumulative) Melting point/boiling point Flash point flammability Relative density Explosive properties Vapor pressure Self-Ignition temperature Surface tension Oxidizing properties Water solubility Granulometry Partition/coefficient (n-octanol/water) Ames test Acute toxicity (2 routes) In vitro cytogenetics Skin irritation Reproductive toxicity screen Eye irritation Toxicokinetic assessment (derived from base set data) Skin sensitization 28-day repeat dose toxicity Biodegradation Acute toxicity for fish Hydrolysis as a function of pH Acute toxicity for Daphnia Soil adsorption/desorption screen Algal growth inhibition Bacterial inhibition Level 1 Studiesa Annex VIII (Supply at > 10b or 100 tonnes/yr or 50b tonnes cumulative) Analytical method development 21-day Daphnia toxicity Physiochemical properties of thermal decomposition Further fish toxicity studies products Bioaccumulation study Test on higher plants Fertility study (one generation) Earthworm toxicity Teratology study Inherent biodegradation Subchronic/chronic toxicity study Further adsorption/desorption Additional mutagenicity studies Basic toxicokinetics

Chronic toxicity study Carcinogenicity study

Level 2 Studiesc Annex VIII (Supply at > 1,000 tonnes/yr or 5,000 tonnes cumulative) Additional test for accumulation, degradation, and mobility


TABLE 31.5 (Continued) Data Requirements for Notification (Level 2 continued) Fertility study (2-generation) Additional test for adsorption/desorption Developmental toxicity (peri- and postnatal) Further fish toxicity studies Teratology study (different species from level 1) Bird toxicity studies Toxicity studies with other organisms Biotransformation Pharmacokinetics Additional test to investigate organ or system toxicity a Studies required at level 1 are on a negotiated basis. Negotiations begin once a trigger tonnage has been exceeded. Studies chosen will be based on 1) the quantity supplied, 2) the results of the Base-Set Tests, and 3) the degree of exposure to humans and the environment. b Testing at the 10/50 tonnage thresholds will depend on the nature of the chemical, its uses, and the results of earlier tests. c Studies required at level 2 are on a negotiated basis. Negotiations begin once a trigger tonnage has been exceeded. Studies chosen will be based on: 1) the quantity supplied, 2) the results of earlier tests, and 3) the degree of exposure to humans and the environment.

Source: Brooker.2

TABLE 31.6 Comparison of the Notification Requirements of the EU and Selected Countries Study Spectra Melting point Boiling point Relative density Vapour pressure Surface tension Water solubility Partition coefficient Fat solubility Dissociation constant Granulometry Henry’s Law constant Volatility from water Complex formation constants Stability Viscosity Permeability Flash point (liquids) Flammability tests Explosivity Oxidizing properties Autoflammability Acute oral toxicity Acute dermal toxicity Acute inhalation toxicity Skin irritation Eye irritation Skin sensitization


OECDa

ECb

⻫ √g √g ⻫⻫

⻫⻫⻫⻫⻫⻫⻫⻫

⻫⻫⻫⻫⻫

⻫⻫⻫⻫⻫⻫

⻫

⻫⻫⻫⻫⻫⻫ √k √k ⻫⻫⻫

Requirement for Supply in: Switzerlandc Austriad Canadae ⻫⻫⻫⻫⻫⻫⻫⻫ √h ⻫ —i —i —i —i —i —i —i

—o —o —o —o —o —o

⻫⻫⻫⻫⻫⻫⻫⻫⻫

⻫⻫⻫⻫⻫⻫ √k √k ⻫⻫⻫

Australiaf

⻫⻫⻫⻫⻫

⻫ √g √g ⻫⻫

⻫⻫⻫⻫⻫

⻫⻫

⻫ √k √k ⻫⻫

⻫⻫

⻫⻫⻫⻫⻫⻫ √k √k ⻫⻫⻫

TABLE 31.6 (Continued) Comparison of the Notification Requirements of the EU and Selected Countries Study Subacute toxicity Ames test In vitro chromosome aberration test Mouse micronucleus test Mouse lymphoma, assay Acute fish toxicity Acute Daphnia toxicity Algal growth inhibition Daphnia reproduction study Fish bioaccumulation Earthworm toxicity Ready biodegradability Activated sludge respiration inhibition Abiotic degradation by hydrolysis Soil adsorption/desorption screening test Anaerobic biodegradation Soil biodegradation Photolysis a

Requirement for Supply in: Switzerlandc Austriad Canadae

OECDa

ECb

⻫ —l —l —l —l ⻫

⻫⻫⻫ —n —q ⻫⻫⻫

—j ⻫⻫ —j —j ⻫⻫ —i ⻫ —i

⻫ √s √t √u

⻫

⻫⻫

⻫⻫⻫

⻫ —i —i —i —i

Australiaf

⻫⻫ √m —m

⻫⻫⻫ √o

⻫⻫⻫ √p

⻫⻫

⻫⻫

⻫⻫⻫⻫ —r

⻫⻫

⻫

⻫

⻫⻫

⻫⻫

⻫

The MPD is recommended by the OECD for adequate hazard assessment of new chemical substances. (MPD = minimum premarketing data set). b Full notification for supply in the EC at 1 tonne per annum (or 5 tonnes cumulative) under the “Seventh Amendment” Council Directive 92/32/EEC. Note that a screening test for toxicity to reproduction will also be required as part of the Base Set when a suitable method has been developed. c These are the minimum data requirements for notification under the Swiss Ordinance on Environmentally Hazardous Substances. d Full notification for supply in Austria at 1 tonne per annum under the Austrian Chemicals Law. e These are the data of Schedule III of the Imminent Canadian New Substances Notification Regulations to permit supply at above 10 tonnes per annum (or over 50 tonnes cumulative). f Full notification for supply in Australia at 1 tonne per annum under the National Industrial Chemicals Notification and Assessment Scheme (NICNAS). g It is adequate to determine either the melting point or boiling point, whichever is most appropriate. h Solubility in an organic solvent is adequate as an alternative to fat solubility. i These additional studies may be required if the minimum data are inadequate for full environmental assessment. j Available toxicity studies are evaluated for notification under the Swiss Ordinance on Environmentally Hazardous Substances and also under the Order relating to Toxic Substances. k The choice of exposure route for the second acute toxicity study depends on the respirability of the substance evaluated from the granulometry test and the likely human exposure route. l The OECD MPD specifies that mutagenicity should be evaluated. m The second mutagenicity test for notification in Austria can be either the in vitro chromosome aberration test or an in vivo study such as the mouse micronucleus test, although the former may be preferred from an animal welfare viewpoint and be consistent with other notification schemes. n The mouse micronucleus test or an in vivo chromosome aberration test will normally be required immediately after notification in the EC if any of the in vitro Base Set mutagenicity tests are positive. o The third mutagenicity study required for notification in Canada can be either the mouse micronucleus test or the in vivo chromosome aberration test. p The mouse micronucleus test has been agreed with the Australian regulatory authorities as an alternative to the dominant lethal assay suggested in the official guidelines. q The mouse lymphoma assay or HPRT locus test is required as part of the EC Base Set if the Ames test is positive. r A fish bioaccumulation study may be needed if the substance is not “readily biodegradable” and has a high partition coefficient. s The activated sludge respiration inhibition test is conducted on nonbiodegradable substances to establish whether the lack of biodegradation is caused by toxicity to microorganisms, and also to predict if adverse effects on sewage treatment plants could occur. t Required for substances which are not “readily biodegradable.” u A soil adsorption/desorption screening test is part of the Base Set, but notifications will be accepted without this until the proposed reverse-phase high-performance liquid chromatography method is finalized, as an alternative to the OECD screening test. Source: Knight.3


TABLE 31.7 Selected OECD Guidelines for Testing of Chemicals Mammalian: #401 acute oral toxicity, LD50 #402 acute dermal toxicity #403 acute inhalation toxicity #404 acute dermal irritation/corrosion #405 acute eye irritation #406 contact sensitization #407 repeated dose 28-day oral toxicity (rodent) #408 repeated dose 90-day oral toxicity (rodent) #409 repeated dose 90-day oral toxicity (non-rodent) #410 repeated dose 28-day dermal toxicity #411 repeated dose 90-day dermal toxicity #412 repeated dose 28-day inhalation toxicity #413 repeated dose 90-day inhalation toxicity #414 developmental toxicity study #415 one generation reproduction #416 two generation reproduction #417 metabolism and pharmacokinetics #418 acute exposure delayed neurotoxicity of organophosphorus substances #419 repeated exposure delayed neurotoxicity of organophosphorus substances #420 acute oral toxicity, fixed dose procedure #421 reproductive/developmental toxicity screen #422 combined repeated dose 28-day oral toxicity/ developmental toxicity screen #423 acute oral toxicity, acute toxic class #424 neurotoxicity screening battery #425 acute oral toxicity, up and down method #451 carcinogenicity #452 chronic toxicity #453 combined chronic toxicity/carcinogenicity Genetox: #471 reverse mutation assay/Salmonella (Ames test) #472 reverse mutation assay/E. coli #473 In vitro mammalian chromosome aberration test #474 micronucleus test #475 In vivo bone marrow mammalian chromosome aberration test #476 In vitro mammalian cell gene mutation test (mouse lymphoma) #477 sex-linked recessive lethal test (Drosophila) #478 rodent dominant lethal test (mouse) #479 in vitro sister chromatid exchange (SCE) assay #480 gene mutation assay/Saccharomyces #481 mitotic recombination assay/Saccharomyces #482 In vivo unscheduled DNA synthesis (UDS) #483 mammalian spermatogonial chromosome aberration assay #484 mouse spot test #485 mouse heritable translocation assay #486 mitotic recombination assay/Saccharomyces Ecotox/Aquatic: #106 absorption/desorption #201 algal growth inhibition #202 acute toxicology/Daphnia


TABLE 31.7 (Continued) Selected OECD Guidelines for Testing of Chemicals #203 #204 #205 #206 #207 #208 #209 #210 #211 #301 #302 #305

acute toxicity/fish 14-day prolonged toxicity/fish avian dietary toxicity avian reproduction acute toxicity/earthworm terrestrial plant growth test activated sludge respiration inhibition fish early life-stage toxicity 21-day Daphnia reproduction ready biodegradability inherent biodegradability bioaccumulation/fish

TABLE 31.8 Risk (R) Phrases Used in the European Community (EU) R1: R2: R3: R4: R5: R6: R7: R8: R9: R10: R11: R12: R14: R15: R16: R17: R18: R19: R20: R21: R22: R23: R24: R25: R26: R27. R28: R29: R30: R31: R32: R33: R34: R35:

Explosive when dry Risk of explosion by shock, friction, fire or other sources of ignition Extreme risk of explosion by shock, friction, fire or other sources of ignition Forms very sensitive explosive metallic compounds Heating may cause an explosion Explosive with or without contact with air May cause fire Contact with combustible material may cause fire Explosive when mixed with combustible material Flammable Highly flammable Extremely flammable Reacts violently with water Contact with water liberates extremely flammable gases Explosive when mixed with oxidizing substances Spontaneously flammable in air In use may form flammable/explosive vapor-air mixture May form explosive peroxides Harmful by inhalation Harmful in contact with skin Harmful if swallowed Toxic by inhalation Toxic in contact with skin Toxic if swallowed Very toxic by inhalation Very toxic in contact with skin Very toxic if swallowed Contact with water liberates toxic gas Can become highly flammable in use Contact with acids liberates toxic gas Contact with acids liberates very toxic gas Danger of cumulative effects Causes burns Causes severe burns


TABLE 31.8 (Continued) Risk (R) Phrases Used in the European Community (EU) R36: R37: R38: R39: R40:

Irritating to the eyes Irritating to the respiratory system Irritating to the skin Danger of very serious irreversible effects Possible risk of irreversible effects*

* (As this book went to press, it seemed likely that the R40 phrase would be changed to “Limited evidence of a carcinogenic effect.” The current phrase above would be allocated to R68.) R41: R42: R43: R44: R45: R46: R48: R49: R50: R51: R52: R53: R54: R55: R56: R57: R58: R59: R60: R61: R62: R63: R64: R68:

Risk of serious damage to the eyes May cause sensitisation by inhalation May cause sensitisation by skin contact Risk explosion if heated under confinement May cause cancer May cause heritable genetic damage Danger of serious damage to health by prolonged exposure May cause cancer by inhalation Very toxic to aquatic organisms Toxic to aquatic organisms Harmful to aquatic organisms May cause long term adverse effects in the aquatic environment Toxic to flora Toxic to fauna Toxic to soil organisms Toxic to bees May cause long term adverse effects to the environment Dangerous for the ozone layer May impair fertility May cause harm to the unborn child Possible risk of impaired fertility Possible risk of harm to the unborn child May cause harm to breastfed babies Possible risk of irreversible effects*

* (As this book went to press, it seemed likely that the R40 phrase above would be allocated under R68.) Combination of Particular Risks R14/15: Reacts violently with water, liberating extremely flammable gases R15/29: Contact with water liberates toxic, extremely flammable gas R20/21: Harmful by inhalation and in contact with skin R20/21/22: Harmful by inhalation, in contact with skin and if swallowed R20/22: Harmful by inhalation and if swallowed R21/22: Harmful in contact with skin and if swallowed R23/24: Toxic by inhalation and in contact with skin R23/24/25: Toxic by inhalation, in contact with skin, and if swallowed R23/25: Toxic by inhalation and if swallowed R24/25: Toxic in contact with skin and if swallowed R26/27: Very toxic by inhalation and in contact with skin R26/27/28: Very toxic by inhalation, in contact with skin and if swallowed R26/28: Very toxic by inhalation and if swallowed R27/28: Very toxic in contact with skin and if swallowed R36/37: Irritating to eyes, respiratory system


TABLE 31.8 (Continued) Risk (R) Phrases Used in the European Community (EU) R36/37/38: R36/38: R37/38: R39/23: R39/23/24: R39/23/24/25: R39/23/25: R39/24: R39/24/25: R39/25: R39/26: R39/26/27: R39/26/27/28: R39/26/28: R39/27: R39/27/28: R39/28: R40*/20: R40*/20/21: R40*/20/21/22: R40*/20/22: R40*/22: R40*/21: R40*/21/22:

Irritating to eyes, respiratory system and skin Irritating to eyes and skin Irritating to respiratory system and skin Toxic: danger of very serious irreversible effects through inhalation Toxic: danger of very serious irreversible effects through inhalation and in contact with skin Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed Toxic: danger of very serious irreversible effects through inhalation and if swallowed Toxic: danger of very serious irreversible effects in contact with skin Toxic: danger of very serious irreversible effects in contact with skin and if swallowed Toxic: danger of very serious irreversible effects if swallowed Very Toxic: danger of very serious irreversible effects through inhalation Very Toxic: danger of very serious irreversible effects through inhalation and in contact with skin Very Toxic: danger of very serious irreversible effects through inhalation, in contact with skin and if swallowed Very Toxic: danger of very serious irreversible effects through inhalation and if swallowed Very Toxic: danger of very serious irreversible effects in contact with skin Very Toxic: danger of very serious irreversible effects in contact with skin and if swallowed Very Toxic: danger of very serious irreversible effects if swallowed Harmful: possible risk of irreversible effects through inhalation Harmful: possible risk of irreversible effects through inhalation and in contact with skin Harmful: possible risk of irreversible effects through inhalation, in contact with skin and if swallowed Harmful: possible risk of irreversible effects through inhalation and if swallowed Harmful: possible risk of irreversible effects if swallowed Harmful: possible risk of irreversible effects in contact with skin Harmful: possible risk of irreversible effects in contact with skin and if swallowed

* (See potential phrase change for R40.) R42/43: R48/20: R48/20/21: R48/20/21/22: R48/20/22: R48/21: R48/21/22: R48/22: R48/23: R48/23/24: R48/23/24/25: R48/23/25: R48/24: R48/24/25: R48/25: R50/53: R51/53: R52/53:

May cause sensitisation by inhalation and skin contact Harmful: danger of serious damage to health by prolonged exposure through inhalation Harmful: danger of serious damage to health by prolonged exposure through inhalation and in contact with skin Harmful: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed Harmful: danger of serious damage to health by prolonged exposure in contact with skin Harmful: danger of serious damage to health by prolonged exposure in contact with skin and if swallowed Harmful: danger of serious damage to health by prolonged exposure if swallowed Toxic: danger of serious damage to health by prolonged exposure through inhalation Toxic: danger of serious damage to health by prolonged exposure through inhalation and in contact with skin Toxic: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed Toxic: danger of serious damage to health by prolonged exposure through inhalation and if swallowed Toxic: danger of serious damage to health by prolonged exposure in contact with skin Toxic: danger of serious damage to health by prolonged exposure in contact with skin and if swallowed Toxic: danger of serious damage to health by prolonged exposure if swallowed Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment Toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment


SECTION 2. GLOSSARY1 (As relates to the 7th Directive for Notification of New Substances in the European Community.)

A. GENERAL EINECS European Inventory of Existing Commercial Substances. This inventory contains the definitive list of all substances deemed to be on the Community market on 18 September 1981. It is a closed list searchable by CAS number and chemical name. ELINCS European List of New Chemical Substances. The list contains the EEC number, notification number, trade name, classification, and in some cases the IUPAC name. Notification Document, with the requisite information, presented to the competent authority of a Member State. Placing on the market The making available of a substance to third parties. Incorporation into the community customs territory shall be deemed to be placing on the market. Polymer A substance consisting of molecules characterized by the sequence of one or more types of monomer units and comprising a simple weight majority of molecules containing at least three monomer units which are covalently bound to at least one other monomer unit or other reactant and consists of less than a simple weight majority of molecules of the same molecular weight. Such molecules must be distributed over a range of molecular weights wherein differences in the molecular weight are primarily attributable to differences in the number of monomer units. In the context of this definition a “monomer unit” means the reacted form of a monomer in a polymer. Preparations Mixtures or solutions composed of two or more substances. Process-oriented research and development The further development of a substance in the course of which pilot plant of production trials are used to test the fields of application of the substance. Scientific research and development Scientific experimentation, analysis or chemical research carried out under controlled conditions; it includes the determination of intrinsic properties, performance, and efficacy, as well as scientific investigation related to product development. Substances Chemical elements and their compounds in the natural state or obtained by any production process, including any additive necessary to preserve the stability of the products and any impurity deriving from the process used, but excluding any solvent which may be separated without affecting the stability of the substance or changing its compositions.

B. TOXICOLOGICAL Carcinogenic substances Substances or preparations which, if they are inhaled or ingested or if they penetrate the skin, may induce cancer or increase its incidence. Corrosive substances Substances or preparations which may, on contact with living tissues, destroy them. Environmental toxic substances Substances and preparations which are dangerous for the environment: substances and preparations which, were they to enter the environment, would or may present an immediate or delayed danger for one or more components of the environment. Harmful substances Substances and preparations that may cause death or acute or chronic damage to health when inhaled, swallowed, or absorbed via the skin. Irritant substances Noncorrosive substances and preparations which, through immediate, prolonged, or repeated contact with the skin or mucus membrane, may cause inflammation.


Mutagenic substances Substances and preparations which, if they are inhaled or ingested or if they penetrate the skin, may induce heritable genetic defects or increase their incidence. Reprotoxic substances Substances and preparations which, if they are inhaled or ingested or if they penetrate the skin, may produce, or increase the incidence of, nonheritable adverse effects in the progeny and/or an impairment of male or female reproductive functions or capacity. Sensitizing substances Substances and preparations which, if they are inhaled or if they penetrate the skin, are capable of eliciting a reaction of hypersensitization such that on further exposure to the substance or preparation, characteristic adverse effects are produced. Toxic substances Substances and preparations which in low quantities cause death or acute or chronic damage to health when inhaled, swallowed, or absorbed via the skin. Very toxic substances Substances and preparations which in very low quantities cause death or acute or chronic damage to health when inhaled, swallowed, or absorbed via the skin.

REFERENCES 1. Council Directive 92/32/EEC, April 30, 1993, amending for the seventh time Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances, Official Journal of the European Community, 35, No. L-154, pp. 1–29, 1992. 2. Brooker, P.C., personal communication, Huntingdon Research Centre, Huntingdon, U.K., 1993. 3. Knight, D.J., personal communication, Safepharm Laboratories, Derby, U.K., 1993.


ADDITIONAL RELATED INFORMATION TABLE 31.9 OECD Screening Information Data Set (SIDS) Studies • Physical Chemistry ‚ Melting point ‚ Boiling point ‚ Vapor pressure ‚ Octanol/water partition coefficient • Environmental fate ‚ Photodegradation (estimation) ‚ Hydrolysis-stability in water (estimation) ‚ Transport/distribution (fugacity model) ‚ Inherent biodegradation • Ecotoxicology ‚ Acute toxicity to fish ‚ Acute toxicity to Daphnia ‚ Algal growth inhibition • Genetic Toxicology ‚ Bacterial mutation assay (Ames) ‚ In vitro chromosomal aberration or ‚ In vivo chromosomal aberration • Mammalian toxicology ‚ Acute oral toxicity ‚ Acute dermal toxicity ‚ 28-day oral toxicity ‚ Reproduction/developmental toxicity screening or Combined repeat dose toxicity/reproduction Note: For more information on SIDS program visit www.oecd.org/ehs/sidsman.htm.


32

Regulatory Toxicology: Notification of New Substances in Canada, Korea, Australia, and China Henry C. Fogle, B.S., M.S.

CONTENTS Section Section Section Section Section

1. 2. 3. 4. 5.

Canada Korea Australia Peoples Republic of China Worldwide Internet Addresses Relating to Chemical Products Table 32.1 Worldwide Internet Addresses Relating to Chemical Products

SECTION 1: CANADA The importation or manufacture of chemical substances in Canada is controlled by New Substance Notification Regulations, which implement requirements outlined in Section 26 of the Canadian Environmental Protection Act (CEPA). The information that must be included in the notification and timing are explained in the New Substance Notification Regulations, Part I (Chemicals), Part II (Polymers), and Part III (Administrative and Testing Requirements). The amount of information required and the government review periods are dependent on the type of substance, the quantity to be imported or manufactured, specific use, etc. Parts I, II, and III of these regulations were published in the Canada Gazette, Part II on April 6, 1994 and have been in effect since July 1, 1994. A modification to these regulations was published in Part II of the Canada Gazette on December 28, 1994. Copies of CEPA and the notification regulations with guidelines and related notification forms may be obtained from Environment Canada at: New Substances Division Commercial Chemicals Evaluation Branch Environment Canada 14th Floor Place Vincent Massey Ottawa, Ontario, Canada KIA OH3 Telephone: (800) 567-1999 (toll-free within Canada) (819) 953-7156 (outside Canada)


Most of the definitions, classifications, and procedures used in the United States under the Toxic Substances Control Act (TSCA) have been adopted by Canada. However, unlike TSCA in the United States, Canada has a “menu” of required toxicological test data similar to the European Union (see Chapter 31, Table 31.6). A “new substance” is one that is not on the Canadian Domestic Substances List (DSL). There are quantity triggers for the amount of information that must be included in a new substance notification (NSN), starting from the basic Schedule I (720 kg/calendar year), through Schedule II (71,000 kg/calendar year), to Schedule III (710,000 kg/calendar year). The amount of toxicological and physicochemical data required is directly related to the quantity to be commercialized in Canada. There are a number of categories where “reduced information packages” are allowed. Details regarding toxicological and physicochemical test data required for different categories and different volume levels (there are three different schedules based on quantity and ten other schedules based on use and nature of the substance) are described in Guidelines for Notification and Testing of New Substances: Chemical and Polymers. These guidelines, as well as copies of CEPA and the DSL, may be obtained from Environment Canada at 1-800-567-1999 or, if calling from outside of Canada, 819-953-7156. The test conditions and procedures used to generate the required test data must be consistent with the Organisation for Economic Cooperation and Development (OECD) Guidelines for Testing of Chemicals. The laboratory practices used to develop test data included in new substance notifications must be consistent with the Principles of Good Laboratory Practice (GLP) set out by the OECD. Some deviations from these GLPs and published OECD testing guidelines might be acceptable but they should be discussed with Environment Canada before inclusion in the NSN.

SECTION 2: KOREA Chemical control in Korea is covered by legislation and regulations under the administration and control of two ministries: the Ministry of the Environment (MOE) and the Ministry of Labor (MOL). The Toxic Chemicals Control Law (TCCL) is administered by the MOE and the Industrial Safety and Health Law (ISHL) is administered by the MOL. In Korea, there are three types of notifications requiring differing amounts of data: 1. A “Full Notification” 2. A “Simplified or Reduced Notification” for chemical substances listed on the inventories of at least two other countries (before 1991) and certain polymers (requires acute toxicity and an Ames test) 3. A “Polymer Notification” for substances meeting the definition of a polymer by the OECD, the European Union, or the United States (TSCA). The Korean Chemical Control Law (the TCCL) requires that a notification be approved prior to the importation or manufacture of a new chemical substance for commercial purposes in Korea. A “new” substance is defined as a chemical substance that does not appear on the Korean Existing Core Inventory (KECI) which was published in 1992. (In 1996, about 5000 substances on the MOL inventory were added to KECI.) If a product is composed of a mixture of two or more substances, each component must either be exempt from notification or notified prior to import/manufacture and commercialization in Korea. Although MOE must evaluate and approve notifications related to substances to be produced or shipped into Korea for the first time, “existing chemicals” that are on KECI must be “certified” before entry through customs will be permitted (certification is provided by the Korean Toxic Chemicals Management Association, KTCMA, on behalf of MOE). A notification must also be approved by MOL, but efforts are being made to integrate the two systems. Notification is accomplished through the use of a specific “Notification of Manufacture/Import of Chemical Substances” Copyright © 2002 by Taylor & Francis

form. Test data regarding the toxicity of the notified substance must be included in the notification for evaluation by MOE. Such data must include acute toxicity (e.g., oral, dermal, and inhalation toxicity as tested on mammalia, i.e., rats or mice). Inhalation toxicity data are required for volatile liquids or gases. Data on mutagenicity must include a reverse-mutation assay, a chromosomal aberration assay, and, if either of these tests are positive, in vivo nonbacteriological tests (e.g., micronucleus assays in mouse bone marrow) must be performed. Data on biological degradability may also be required. Additional details can be obtained from the MOE, the Toxic Substance Division, 1-JoongAng-dong, KwaChun-shi, Kyungki-do, Korea 427-760; telephone (02) 504-9288; fax (02) 504-9280. Information relating to the MOL can be obtained from the Work Environment Division, 1-JoongAng-dong, Kwa-Chun-shi, Kyungki-do, Korea 427-760; telephone (02) 500-5635; fax (02) 503-4545. The address for the Korean Toxic Chemicals Management Association (KTCMA) is 5th Floor, Jungwon Bldg., 1556-8, Seocho-dong, Secho-ku, Seoul, Korea; telephone (02) 587-698217; fax (02) 587-6988.

SECTION 3. AUSTRALIA The Australian Industrial Chemicals (Notification and Assessment) Act 1989 provides for a national scheme for the notification and assessment of industrial chemicals to protect people and the environment from potential and harmful effects prior to import, manufacture, or use of a chemical substance for commercial purposes. The scheme, known as the National Industrial Chemicals Notification and Assessment Scheme (NICNAS), began operating in July 1990 and is administered by the National Occupational Health and Safety Commission (Worksafe Australia), which also performs the primary toxicological assessment and the occupational health and safety assessments. The Department of Arts, Sport, the Environment, Tourism and Territories undertakes the environmental hazard assessment, while the Department of Community Services and Health carries out the public health assessment. Under this Australian law, a new chemical substance is one that is not listed on the Australian Inventory of Chemical Substances (AICS). If a chemical substance is listed on AICS, a notification and assessment is not required. Five years after a new substance is issued an assessment certificate by the Director of NICNAS, the identity of that substance will be listed on AICS. The information required for a full notification essentially corresponds to the OECD minimum premarketing set of data (MPD), and is detailed in Parts A, B, and C of the schedule to the Act. OECD toxicological testing guidelines are recommended, but equivalent methods are accepted. Tests must be performed in compliance with GLP, and Australian Codes of Practice on GLP are to be in harmony with OECD GLP principles. The Australian notification scheme has varying data requirements for different classes of notifiable industrial chemicals. In general, more data are required if the quantity is greater, the chemical is not site-limited, and the class of the chemical is more likely to be hazardous. The exact requirements for data also vary depending on exposure potential, potential uses, and the characteristics of the chemical. A full package of data, similar to the premarketing data set of the OECD, is required for a standard notification, which includes information on mammalian toxicity, ecotoxicity and biodegradability/bioaccumulation (see Chapter 31, Table 31.6). A chemical substance cannot be introduced into Australia until it has been assessed and a certificate issued. There is a statutory period of 90 days allowed for government assessment of new chemical notifications. A secondary notification is required if any one of a number of circumstances change after the initial assessment. These include a significant new use or change of use that is likely to increase the risk of adverse effects to health or the environment, a significant increase in production, and new information on the hazardous properties of the chemical. Special conditions for secondary notification can also be set at the time of the initial assessment. Copyright © 2002 by Taylor & Francis

Sources of information on NICNAS are as follows: • AICS, Vols. 1 and 2, 1992 • Chemical Gazette, Commonwealth of Australia Gazette, published monthly • Annual Reports, The Operation of the Industrial Chemicals (Notification and Assessment) Act of 1989 • Handbook for Notifiers, National Industrial Chemicals Notification and Assessment Scheme (NICNAS), Worksafe Australia, GPO Box 58, Sydney, NSW 2001. It contains AICS on microfiche and the legislation, as well as detailed guidance regarding NICNAS

SECTION 4. PEOPLES REPUBLIC OF CHINA The Peoples Republic of China (PRC) has regulations governing the importation (registration) of chemical “products” (as opposed to chemical substances). Such products to be registered can be a single chemical substance or a mixture of several chemical substances. It is the “product” that must be registered, not the chemical substance (unless the product happens to be a single specific chemical substance). The product must be registered under the product name that appears on the container label and the Material Safety Data Sheet (MSDS). If a number of products are very similar in characteristics and content, that number of products can be registered as a “group” or category of products. The regulations relating to this registration requirement is entitled “Regulations for Environmental Management of the First Import of Chemical and the Import and Export of Toxic Chemicals,” May 1994. (The PRC is currently in the process of modifying these regulations and in the future, plans to implement a new law to be called the New Chemical Environmental Pollution Control Law.) Currently, any “product” that is a chemical substance or that contains a chemical substance must be registered with the State Environmental Protection Administration (SEPA) prior to shipment into the PRC for commercial purposes. There are exemptions to this registration requirement, which may apply (similar to notification exemptions in other countries). Upon approval of the “product” registration application, SEPA provides the importer with a “Registration Certificate.” There are registration fees associated with different types of chemical products that have different degrees of toxicity. Currently, the product registration does not require new toxicological testing. Typically, a good MSDS explaining the hazards related to the products and proper handling and disposal is acceptable along with the completed form. Currently, the PRC is also receiving nominations for an inventory of “chemical substances” that have been or are currently being shipped into the PRC. At this time, placement of such chemical substances on this inventory is voluntary and is not a regulatory requirement. This opportunity to place a chemical substance on the PRC inventory may change in 2001 or 2002. This inventory is not currently linked to the “product” regulation process (but may be when the law is modified). Copies of forms and instructions needed to register chemical products to be shipped into the PRC can be obtained from: Director The Chemical Registration Center of the State Environmental Production Administration (CRC-SEPA) Beiyuan, Beijin 100012, P.R. China Tel: +86-10-8491-5286 Fax: +86-10-8491-3897 http://www.crc-sepa.org.cn (contains English versions of forms)


SECTION 5: WORLDWIDE INTERNET ADDRESSES RELATING TO CHEMICAL PRODUCTS TABLE 32.1 Worldwide Internet Addresses Relating to Chemical Products Country Australia

Peoples Republic of China (PRC)

Hong Kong Indonesia Japan

Korea

Malaysia New Zealand Philippines Singapore Taiwan Thailand The European Union

Canada

Description WorkSafe Australia: WorkSafe Online Chemicals in Australia National Industrial Chemicals Notifications and Assessment Scheme China — Laws and Regulations of the PRC China Environmental Protection Agency The Chemical Registration Center Environmental Protection Department BAPEDAL Environment Ministry Ministry of Labor Environmental Agency Ministry of the Environment Ministry of Labor Ministry of Environment Malaysian Ministries Ministry for Science and Technology Department of Labor Ministry of the Environment Environmental Management Bureau Ministry of the Environment Ministry of Labor Environmental Protection Administration Ministry of Industry MOSTE Ministry for Science European Governments on the WWW The European Directory Full text of the last 20 days’ publication of the EU Official Journal CEPA Publications Environment Canada–Commercial Chemicals Web site Health Canada Chemical Sustances on Canada’s Domestic Substances List and Nondomestic Substances List DSL Catagorization and Screening Program Canada Gazette Web site


Address http://www.allette.com.au/worksafe/home.htm http://www.worksafe.gov.au http://jimi.vianet.net.au/∼acted/reg_home.htm http://www.nicnas.gov.au http://www.qis.net/chinalaw/ http://www.ihei.com/ http://www.crc-sepa.org.cn./home.english.htm http://www.info.gov.hk/epd http://www.bapedal.go.id http://www.mol.go.jp/english/index.htm http://www.mx.eic.or.jp.eanet/ http://www.yahoo.com/Regional/Countries/Japan http://www.moenv.go.kr/english/index.htm http://www.molab.go.kr/English/English.html http://www.moenv.go.kr/english/index.html http://www.me.go.kr/english/ http://www.smpke.jpm.my/ http://mastic.gov.my/ http://www.dol.govt.nz/ http://www.mfe.govt.nz/ http://www.psdn.org.ph/emb http://www.gov.sg.env/ http://www.gov.sg.mol/ http://www.epa.gov.tw/english/ http://www.tisi.go.th/moi/index.html http://www.nectec.or.th/bureaux/moste/moste.html/ http://www..lrz-muenchen.de/~a2c0133/222/govt.eur http://www.ukshops.co.uk:8000/thedoor.html http://europa.eu.int/eur-lex/en/oj/index.html http://www.doe.ca/tandi/cepa/etitles.html http://www.ec.gc.ca/ccebl/eng/psap/html http://www.hwc.ca/links/english.html http://www.2.ec.gc.ca/ccebl/cas_e.html

http://www.ec.gc.ca/cceb1/ese/eng/dslprog.htm http://www.ec.gc.ca/cceb1/ese/fre/dslprog.htm www.canada.gc.ca/gazette/hompar/_e.html

33

Miscellaneous Information of Toxicological Significance Mannfred A. Hollinger, Ph.D. and Michael J. Derelanko, Ph.D., D.A.B.T, F.A.T.S.

CONTENTS Section 1. LD50 Values Table 33.1 LD50 Values of Common Xenobiotics Table 33.2 Radiation LD50 Values for Different Species Section 2. Tables of Comparative Anatomical, Physiological, and Biochemical Data Table 33.3 Comparison of Physiological Parameters for Different Body Organs Table 33.4 Comparison of the Blood Flow/Perfusion and Oxygen Consumption of Liver, Lung, Intestine, and Kidney of the Rat In Vivo and in Organ Perfusion Table 33.5 Comparison of Respiratory Volume and Alveolar Ventilation in Relation to Animal Weight Table 33.6 Comparison of Dosage by Weight and Surface Area Table 33.7 Relationship Between Body Weight and Body Surface Area in a Number of Vertebrates Table 33.8 Comparison of Bile Flow and Hepatic Blood Flow Rates in Various Species Table 33.9 Comparison of the pH Value of Contents of Different Parts of the Alimentary Tract in Various Species Table 33.10 Comparison of Enzyme Activity in the Small Intestine of Several Species Table 33.11 Comparison of pH Values in Some Human Body Compartments Table 33.12 Comparison of the Size of the Absorptive Surface of the Various Parts of the Gastrointestinal Tract Table 33.13 Comparison of Physiological Characteristics of Experimental Animals and Humans Table 33.14 Comparison of Certain Physiological Values of Experimental Animals and Humans Table 33.15 Comparison of Biochemical Components in Urine of Normal Experimental Animals and Humans Table 33.16 Comparison of Some Biochemical/Physiological/Morphological Differences of Potential Toxicological Significance Between Rats and Humans Table 33.17 Permittivity and Conductivity of Biological Tissues Section 3. Mathematics, Symbols, Physical Constants, Conversions, and Statistics A. Symbols


Table 33.18 B. Conversions Table 33.19 Table 33.20 Table 33.21

Greek Alphabet

Base Units of SI Representative Derived Units Prefixes and Symbols for Decimal Multiples and Submultiples Table 33.22 Conversion of Human Hematological Values from Traditional Units into SI Units Table 33.23 Conversion of Laboratory Values from Traditional Units into SI Units Table 33.24 Calculation of Milliequivalent Weight, Creatinine Clearance, and Surface Area of Children Table 33.25 Table for Predicting Human Half-Life of Xenobiotics from Rat Half-Life Table 33.26 Table for Predicting Human Volume of Distribution from Rat Volume of Distribution Table 33.27 Approximate Metric and Apothecary Weight Equivalents Table 33.28 Conversion Factors — Metric to English Table 33.29 Conversion Factors — English to Metric Table 33.30 Conversion Factors — General Table 33.31 Temperature Factors Table 33.32 Temperature Conversions Table 33.33 Table of Equivalents Table 33.34 Overview of Units Used to Express the Concentration of a Substance Table 33.35 Conversions of Nucleic Acids and Proteins Table 33.36 Conversion of Radioactivity Units from mCi and μCi to MBq Table 33.37 Conversion Formula for Concentration of Solutions C. Physical Constants Table 33.38 General Physical Constants Table 33.39 Summary of the 1986 Recommended Values of the Fundamental Physical Constants Table 33.40 Standard Atomic Weights D. Statistics Figure 33.1 Decision Tree for Selecting Hypothesis-Testing Procedures Table 33.41 Transformation of Percentages into Logits Table 33.42 Transformation of Percentages into Probits Table 33.43 Areas under the Standard Normal Curve Table 33.44 Poisson Distribution Table 33.45 t-Distribution Table 33.46 χ2 Distribution Table 33.47 Variance Ratio General Statistical References E. Kinetics Table 33.48 Table of Equations and their Uses Section 4. Calculations, Preparation, and Properties of Various Types of Substances Commonly Used in Toxicology A. Molarity, Molality, Normality, Osmolarity Calculations B. Solution Calculations C. pH Calculations D. Glossary of Terms Associated with Solutions Copyright © 2002 by Taylor & Francis

Section 5.

Section 6.

Section 7.

Section 8. Section 9. Section 10.

Section 11.

References (Subsections A.→D.) Table 33.49 Strengths of Concentrated Solutions of Acids and Bases Table 33.50 Physiological Solutions Table 33.51 Composition of Typical Organ Perfusion Medium Table 33.52 Properties of Carrier Gases for Gas Chromatography Table 33.53 Solvents for Ultraviolet Spectrophotometry Table 33.54 13C Chemical Shifts of Useful NMR Solvents Table 33.55 Important Peaks in the Mass Spectra of Common Solvents Table 33.56 Solvents for Liquid Chromatography National Standards Table 33.57 Threshold Limit Values for Airborne Contaminants Table 33.58 U.S. National Primary Drinking Water Standards Design and Performance of Toxicology Studies A. Acceptable Toxicology Study Requirements Table 33.59 Minimum Requirements for an Acceptable Toxicology Study References B. Estimated Costs and Test Sample Requirements for Standard Toxicology Tests Table 33.60 Mammalian Toxicology Tests — Cost and Material Requirements Table 33.61 Genetic Toxicology Tests — Cost and Material Requirements Table 33.62 Aquatic/Ecotoxicology Tests — Cost and Material Requirements C. Contract Laboratories Table 33.63 Directory of Contract Toxicology Laboratories References Organizations and Agencies Table 33.64 Organizations and Agencies Associated with Toxicology and/or Toxicological Issues Internet/Web Sites Table 33.65 Useful Internet Sites Acronyms Table 33.66 Frequently Encountered Acronyms Academic Programs in Toxicology Table 33.67 Academic Programs in Toxicology: United States and Canada References Miscellaneous A. Radioactivity Table 33.68 Decay Constants and Modes for Selected Radionuclides B. Ionization Table 33.69 pKa Values of Common Drugs Table 33.70 Approximate pH Values of Biological Materials and Foods C. Chemical Functional Groups Table 33.71 Chemical Functional Groups Table 33.72 Properties of Common Amino Acids D. Material Safety Data Sheets Table 33.73 Information Disclosed on a Material Data Sheet (MSDS) References


SECTION 1. LD50 VALUES TABLE 33.1 LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Acetaldehyde Rabbit Rat Acetaminophen Rat Acetanilide Mouse Rat Acetone cyanohydrin Rabbit Acetylsalicylic acid Mouse Rat Guinea pig Rabbit Dog Acetylcholine Mouse Rat Rabbit Cat Aconitine Mouse Albuterol sulfate Mouse Rat Alfentanil HCl Mouse Rat Guinea pig Dog Allobarbital Rat Alloxan Mouse Allopurinol Rat Allylamine Mouse Rat Allyl chloride Rat α-Prodine Mouse Rat Guinea pig

IV

IP

300

IM

SC

PO

1,200 640

1,900

500

338

820 800

800

17


495 500

20 22 0.3

>125

1,100 1,500 1,200 1,800 3,000 170 250

3,000 2,500

10 6.9

20 >2,000 >2,000

73 45 75 75 110 200

50

350 4,500 57 106 700

54 18

73 22

98 23

90

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rabbit Alprazolam Rat Altretamine Mouse Rat Amidephrine Mouse Amiloride (base) Mouse Rat Aminocaproic Mouse Rat Aminoglutethimide Rat Dog Aminohippurate sodium Mouse, female Aminophenazone Mouse Rat Rabbit Dog Aminophylline Mouse Rabbit Aminopyrine Mouse Rat Amitriptyline Mouse Rat Rabbit Dog Ammonium fluorosilicate Guinea pig Amobarbital Rat Rabbit Dog Amphetamine Mouse Rat Rabbit Aniline Rat


IV

IP

IM

SC

PO

18.5 331–2,171 437 1,050 1,990

>6,000 56 36–85

3 g/kg 3.2 g/kg

12 g/kg 16.4 g/kg

156 >100

1,800 >100

7.22 g/kg 184 110

350

1,850 1,380 160 150 540

150 184

350

1,850 1,700

328 1,290

289 530 446 200

248 27 10 9.9 10

76 72 72

150 128 75 75

115

25

120 125

160 575 125

160

22 60.5 85 440

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Anileridine (base) Mouse Rat o-Anisidine Mouse Rat Rabbit p-Anisidine Mouse Rat Rabbit Antazoline Rat Antipyrine Mouse Rat Apomorphine Dog Aprobarbital Rat Arsenic pentoxide Mouse Rat Arsenic trioxide Mouse Rat Asparaginase Mouse Rabbit Atracurium besylate Mouse, male Mouse, female Rat, male Atrazine Rat Atropine Mouse Rat Guinea pig Auranofin Mouse Rat Azacyclonol Mouse Azapetine Mouse Rabbit Dog Azathioprine Mouse


IV

IP

25

53 45

IM

SC

PO

100 163

128 175 1,300 1,400 2,900 1,400 2,000 870

1.1 mmol/kg 1,000

1,800 1,800

80 100 50–100 8 30–60 13–30 500K I.U./kg 22K I.U./kg 1.9 2 1.3

283 1.2 g/kg

90

250 280 400

900

400 750 1,100 310 265

177

220

27 28 50

210

600

350

650

725

460

2,500

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Aziridine Rat Guinea pig Barbital Mouse Barium chloride Mouse Rat Beclomethasone dipropionate Mouse Rat Bemegride Mouse Rat Guinea pig Rabbit Benactyzine Mouse Rat Benztropine Mouse Rat Betaxolol Mouse Rat Bethanechol chloride Mouse Bethanidine Mouse Biperiden Mouse Rat Dog Bisacodyl Mouse Rat bis(2-chloroethyl)ether Rat Bitolterol mesylate Mouse Rat Bretylium Mouse Bromoacetic acid Mouse

IV

IP

IM

SC

PO 400 15

14


760

600

500 178

>1 g/kg >1 g/kg 20 16.3 26.5 25

45 23.5

43 30.5

100

100–130 100–130

250

350

103 353

94

25

350–920 860–1,050

1,510 12

150

260

56

520 545 750 340 17,500 4,320

75–150 6,575 5,650 20

49

72

400 100

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Bromomethane Rat 1-Bromopropane Rat Buformin Mouse Rat Bulbocapnine Mouse Bupivacaine HCl Mouse Bupropion HCl Mouse, male Mouse, female Rat, male Rat, female Buspirone Mouse Rat Dog Monkey Busulfan Mouse 1,4-Butynediol Rat Guinea pig Butyronitrile Mouse Rat Cadmium chloride Rat Cadmium cycanide Rat Cadmium fluoride Guinea pig Cadmium nitrate Mouse Cadmium oxide Rat Caffeine Mouse Rat Dog Calcium chloride Rat Capreomycin sulfate Mouse Captan Rat


IV

IP

IM

SC

PO

214 4,000 380 320 195 6–8

38–54 544 636 607 482 655 196 586 356 120 104 130

50 140–220 88 16 150 100 72–296 100 105 175

245

250

500

1,200 200

4,000 514 9,000–1,500

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Carbachol Mouse Rat Carbamazepine Mouse Rat Rabbit Guinea pig Carbenicillin Mouse Rat Dog Rat Rabbit Carbromal Rabbit Dog Cefoxitin sodium Mouse, female Rat Rabbit Ceftazidime Rabbit Cefuroxime sodium Mouse Centchroman Mouse Chloral hydrate Mouse Rat Chloralose Mouse Chlorambucil Mouse Chlordiazepoxide Mouse Rat Rabbit Dog Chlorisondamine Mouse Rat Chloroacetic acid Mouse Rat Chloroacetonitrile Mouse Rat p-Chloroaniline Rat


IV

IP

0.3 0.1

IM

SC

PO

3 4

15 40

350

1,100–3,570 3,850–4,025 1,500–2,680 920 3,600 2,000 >500 1,320

450 124

500–700 450 8 g/kg >10 g/kg >1 g/kg >2 g/kg >10 g/kg 400 890 620

500

200 123 95 165 36

24 28

268

530 800

720 2,000 590 1,000

401

165 76 139 220 310

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal 1-Chloro-2,4-dinitrobenzene Rat 2-Chloroethanol Rat Chloromethyl methyl ether Rat p-Chloronitrobenzene Rat Chloroprocaine HCl Mouse Chlorothiazide Mouse Rat Dog Chlorpheniramine Mouse Chlorpromazine Mouse Rat Rabbit Dog Chlorprothixene (2%) Mouse Chlorprothixene (5% suspension) Mouse Chlorprothixene (injectable) Mouse Cimetidine Mouse Rat Clemastine Mouse Rat Dog Clomethiazole Mouse Clomipamine Mouse Rat Clonazepam Mouse Rat Clonidine HCl Mouse Rat


IV

IP

IM

SC

PO

1,070 58–95

817 810 97

950

1,120

8,510 10,000 >1,000

1,386 1,000

162 26 29 235 228

92 74

300 542

319 493

350

220

>125 140 110

2–5,000 6,000 730 3,550 175

220

800 630 1,450 >2,000 >2,000 206 465

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Clotrimazole Mouse Rat Rabbit Cocaethylene Mouse Cocaine Mouse Rat Rabbit Dog Codeine Mouse Rat Rabbit Colchicine Mouse Rat Cat Coniine Rabbit Guinea pig Cortisone acetate Mouse female m-Cresol Rat o-Cresol Rat p-Cresol Rat Crotonaldehyde Mouse Cyanamide Rat Cyclobarbital Rat Rabbit Dog Cyclobenzaphine HCl Mouse Rat Cycloserine Mouse Cyclosporine Mouse Rat Rabbit Cyproheptadine Mouse Rat


IV

IP

IM

SC

PO

700–1,000 700–1,000 700–1,000 60 75–100 17.5 17 22

95 70

68 55 60

130 102

183 332 32

1.75 1.7 0.25

3.5

3.1 4

250

395 542

56 150 1,405 2,020 1,350 1,800 240 125 205 450 250 338 425 5,290 148 104 46 23

2,329 1,480 >1,000 55 52

107

125 295

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Dactinomycin Rat Decametthonium Mouse Rabbit Desipramine Mouse, male Rat, female Desmetryn Rat Dexamethasone Mouse, female Dexamethasone sodium phosphate Mouse, female Dextroamphetamine Mouse Rat Dextromoramide Mouse o-Dianisidine Rat 2,4-Diaminophenol Rat Diazepam Mouse Rat Rabbit Dog N-(2-Chloroethyl)dibenzylamine Mouse Dibozane Mouse Rabbit 3,4-Dichloroaniline Mouse Rat Dichloroisoproterenol Mouse 2,2′-dichloro-4,4′methylenedianiline Mouse 1,1-Dichloro-1-nitroethane Rat Dichlorphenamide Mouse Rat


IV

IP

IM

SC

PO

0.46 0.75 0.2 290 320 1,390 6.5 g/kg

794 14.3

72.2

84 200

37 80 220 1,920 240

220

970 1,200

8.8 1,000

800 260 43 740 648 48

132

880

410 1,710 2,600

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Dicyclomine HCl Mouse Dicumarol Mouse Rat Guinea pig N,N-Diethylaniline Rat Diethylcarbamazine Mouse Rat Diethylene glycol diacrylate Rat Rabbit Diethylpropion HCl Mouse Rat Dog Diflunisal Mouse, female Rat, female Digitoxin Mouse Rat Guinea pig Digoxin Guinea pig Dihydroergotamine Mouse Rat Rabbit Cat Diisopropyl fluorophosphate Mouse Rat Rabbit Cat Dog Monkey Diltiazem HCl Mouse Rat Dog Dimethindene Rat Guinea pig Dog

IV

IP

IM

SC

PO

625 64 52 59

350

233 542

782 550 395

770 180


600 250 225 500 826 22.2 16.4

32.7 23.8 >100

0.355 118 110 25 68 6

1.8 0.34 1.6 3.4 0.25

3.7 3 1

36.8 6 9.8

3

60 38

415–740 560–810 >50

26.8

618.2 888

45

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal N,N-Dimethylaniline Rat Dimethylnitrosamine Rat Dimethyl phenylpiperazinium Mouse Rat Dimethyl sulfate Rat Dimethyl sulfoxide Mouse Dog 2,4-Dinitrophenol Rat Rabbit Dog 2,4-Dinitrotoluene Rat Diphenhydramine Mouse Rat Guinea pig Rabbit Dog Disopyramide phosphate Mouse Rat Dyclonine Mouse Rat Diphenylamine Rat Doxapram HCl Mouse Rat Cat Dog Doxazosin Mouse Rat Doxepine Mouse Rat Doxycycline Mouse Dropempine Mouse Rat


IV

IP

IM

SC

PO

1,410 26

40

27.5

365 2,000 440

14,700 >10,000 25 30

20

22

30 200 25 268

31 42

84 82 75

127 475

164 500

10 24

700 580

31

90 176 3,300

75 75 40–80 40–80 >1,000 >1,000 20 16

165 400

175

1,600

125

420 370

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Econazole Mouse Rat Guinea pig Dog Edrophonium Mouse Rabbit Dog Enalaprilat Mouse, female Rat Ephedrine Mouse Rat Epinephrine Mouse Rat 2,3-Epoxy-1-propanol Mouse Rat Rabbit 2,3-Epoxypropyl acrylate Rat Rabbit Ergometrine Mouse Ergotamine Mouse Rat Rabbit Cat Ethacrynic acid Mouse Ethacrynate sodium Mouse Ethanol Mouse Rat Guinea pig Rabbit Ethinamate Rat Dog Ethosuximide Mouse Rat N-Ethylaniline Mouse

IV

IP

IM

SC

PO

462 668 272 >160 9 28.5 15

37

130

3,740–5,890

600

2,000 2,000 1,550 650

0.5 0.98

4 3.5

1.47 5

50

431 420 1980

214 400


144 52 62 3.55 11 627 175 1,953

7,260 5,000 5,560

8,285

9,488 13,600 9,500 331 314 1,530 1,820 500

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Rabbit Ethyl biscoumacetate Mouse Rat Rabbit Ethyl chloroacetate Rabbit Ethyl chloroformate Mouse Rat Rabbit Ethylene dibromide Rat Rabbit Ethylene oxide Mouse Rat Ethyl methacrylate Rat Ethylmorphine Mouse Ethylnorepinephrine Mouse Etilefrine Mouse Etrentinate Rat Mouse Fentenyl citrate Rat Mouse Cat Dog Monkey Floxuridine Mouse Rat Rabbit Dog Fluoroacetic acid Mouse Rat Guinea pig Rabbit Cat Dog Monkey Fluorouracil Mouse

IV

IP

IM

SC

PO 290

4,700 880 880 1,100 230 15 270 7,120 300 300


365 330 16g/kg 200 117 1,600 >4,000 >4,000

>4,000 >4,000

3 11.2 1 14 0.03 880 670 94 157 10 0.4 0.35 0.25 0.2 0.06 4 340

5

16 2.5

8 2.5

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Rabbit Dog Flurazepam Mouse Rat Flurbiprofen Mouse Rat Fominoben Mouse Rat Fosinopril Rat 2-Furaldehyde Rat Furosemide Rat Rabbit Dog Gadopentetate dimeglumine Mouse Rat Gallamine Mouse Rat Rabbit Dog Glucagon Mouse Glutethimide Mouse Guanethidine Mouse Rat Haloperidol Mouse Rat Harmaline Mouse HC Blue No. 2 Rat Hemicholinium Mouse Rat Heparin Mouse Hexachlorophene Rat


IV

IP

IM

SC

PO

165 27 32 870 1,230 200 400

750 160

100 100

2,000 2,000 2,600 127 4,600 800 2,000

5–12.5mmol/kg 10–15 mmol/kg 4.3 5.5 0.65 0.8

9.6 2.5

17.4 25 3

425 100

300 350 22 23

1,000

13 19

54

144

120 1,250–5,000 0.064 0.45 1,780 7.5

30

70

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Hexamethonium Mouse Hexobarbital Mouse Rat Rabbit Hexocyclium Mouse Histamine Guinea pig Homatropine Mouse Rat Guinea pig Hydralazine Mouse Rat Hydrazine Rat Hydrochlorothiazide Mouse Rat Rabbit Dog Hydrocordone Mouse Hydrocortisone Mouse, female Hydromorphone Mouse Hydroquinone Rat Hydroxyzine Rat Hyoscyamine-sulfate Rat Ibuprofen Mouse Rat Imipramine Mouse Rat Rabbit Iodoacetic acid Mouse Rat Iodomethane Rat Iohexol Mouse


IV

IP

21

42

IM

SC

PO

484

340 280

468 468

80 10.5

55

360

600

60 82 120

650 800

1,400 1,200 1,000

0.18

83 180 60 884

578 234

1,470 1,270

3,080 6,190

461 250 9 1,740 88

84 700

45

1,000 375 320

35 22 18

115 79

1,300

800 1,600

189 250

400 625

83 60 76 24.2 g iodine/kg

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Ipratropium bromide Mouse Rat Dog Iproniazid Mouse Rat Rabbit Isocarboxazid Mouse Rat Isoflurophate Mouse Isoniazid Mouse Rat Rabbit Dog Isoprenaline Mouse Isoproterenol Mouse Guinea pig Dog Isosorbide dinitrate Rat Ketamine Mouse Rat Labetalol Mouse Rat Lactulose Mouse Rat Lauremine oxide (0.3%) Rat Levallorphan Mouse Rat Levocarnitine Mouse Levorphanol Mouse Rat Rabbit


IV

IP

IM

SC

PO

15 g iodine/kg

1,001–2,010 1,667–4,000 400–1,300 725

690

683

750

150 110 199

968 383 150 173 280 37

153 398 94 50

132

140

160 533 151

142 650

2,221 128

300 0.32

450 270

50 1,100 65 62

360.6

680 490

50–60 50–60

600 >2 g/kg 48.8 ml/kg >30 ml/kg

>20g/kg 184 185

949 949 19.2 g/kg

41.5 20

73

187 110

285 150

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Lidocaine HCl Mouse Rat Lignocaine Mouse Lisinopril Mouse Rat Loperamide Rat Lysergide Mouse Rat Rabbit Malononitrile Mouse Mebendazole Mouse Rat Guinea pig Rabbit Dog Cat Mebutamate Mouse Rat Mecamylamine (base) Mouse Rat Guinea pig Mechlorethamine HCl Mouse Rat Meclofenoxate Mouse Meclozine Mouse Menadiol sodium diphosphate Mouse Rat Mepenzolate Mouse Rat Meperidine Mouse Rat Rabbit


IV

IP

31.5

35

IM

SC

PO

400

457 459

122

520 >20 g/kg >20 g/kg

5.92 54

46 16

3 18.6 1,280 1,280 1,280 1,280 >640 >640 460 410

21

39 54 52

550 1,160

93 145 127

92 171 144

2 1.6 330

1,750 1,600

500 400

6,172 5,250

9.8 21.8

900 1,100

50 34 30

150 93

195 200

178 170 500

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Mephenesin Mouse Rat Rabbit Mephenoxalone Mouse Mephentermine Mouse Mephenytoin Mouse Guinea pig Rabbit Cat Mepivacaine Mouse Meprobamate Mouse Rat Rabbit Mercaptopurine Mouse Mercury (II) bromide Mouse Rat Mercury (II) chloride Rat Mercury (II) cyanide Mouse Rat Mercury (II) nitrate Rat Mercury (II) oxide Mouse Rat Mescaline Mouse Rat Metaraminol Mouse Rat Methacholine-Cl Mouse Rat Methacrylonitrile Rat Methadone Mouse Rat Guinea pig Monkey

IV

IP

186

471

IM

SC

PO

990 625

125 3,820 110 560 215 430 190 23–35

280 710

980 1,600

350 260 250

35 1

100

1 33 26 26 22 18

315


500 370 99 240 1,100 750 0.25 ml/kg 17 10

38 23

33 12 54 15

93.7 95

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Methamphetamine Mouse Methaqualone Rat Metharbital Mouse Methotrexate Mouse Methoxamine Mouse N-Methylaniline Rabbit Methylatropine Mouse 2-Methylazindine Rat Methyl chloroformate Mouse Rabbit Rat Methyldopa Mouse Rat Rabbit Methyldopate HCl Mouse Methylergonovine Mouse Rat Rabbit Methyl isocyanate Mouse Rat Rabbit 1-Methyl-3-nitro-lnitrosoguanidine Rat Methyloxirane Rat Methylpentynol Mouse Rat Guinea pig Methylphenidate Mouse Rat Metyrapone Rat Miconazole nitrate Mouse

IV

IP

10

15

IM

SC

PO

232

100

300

500

500 4.5 92 280

7

250 19

1,750 7,120 60 1,900

406 647

5,300 7,490 713

321 85 23 2.6

187 93 4.5 120 69 90

0.22 ml/kg


90 520–1,140 525 300–900 534 450 48

470

680 367 521 578

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Guinea pig Dog Minoxidil Mouse Rat Morphine Mouse Rat Guinea pig Rabbit Muscarine Mouse Nalorphine Mouse Naloxone Mouse Rat Naltrexone Mouse Rat Guinea pig Naphazoline Mouse Rat Rabbit Naproxen Mouse Rat Hamster Dog Neostigmine Mouse Rat Rabbit Nialamide Mouse Rat Nicotine Mouse Rat Rabbit Dog Nicotinic acid Rat Nifedipine Mouse Rat Nikethamide Mouse


IV

IP

IM

SC

PO >640 276 >160

51 49

1,000–1,300

275 237

500 500

2,500 1,300–2,000 500–700 266–572 391 600

500

745 905

0.23 190

590

0.4

670

150 109

565

1,100 1,450 1,490 170 385 0.8 1,234 543 4,110 >1,000 0.36 0.16

0.62

0.31 0.42 0.31

0.8 0.37

742

14.4

1,000 1,700

7.1 33.5

3.3 >24

9.4 5 7,000 16 4

490 1,020 174

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Rabbit Nitrazepam Mouse Rat m-Nitroaniline Rat ø-Nitroaniline Rat p-Nitroaniline Rat Nitrofurazone Mouse Rat 2-Nitro-p-anisidine Rat 2-Nitronaphthalene Rat 2-Nitropropane Rat o-Nitrotoluene Rat p-Nitrotoluene Mouse Rat 5-Nitro-o-toluidine Rat Nitroprusside Mouse Rat Rabbit Dog Nizatidine Mouse Rat Norephedrine Rabbit Norepinephrine Rat Noscapine Rat Obidoxime Mouse Rat Rabbit Octreotide acetate Mouse Rat Orciprenaline Mouse


IV

IP

191

300 225

IM

SC

PO

470

1,800 2,000 535 1,600 750

30

747 590 14,100 4,400 720 891 1,231 2,144 574

8.4 11.2 2.8 5 232 301 75 29

132 800

100 100 100

200 200 200

2,240

72 18 4,800

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Osmium tetroxide Mouse Ouabain Mouse Rat Guinea pig Cat Oxazepam Mouse Oxilorphan Mouse Oxotremorine Mouse Papaverine Mouse Rat Paracetamol Rat Paraldehyde Mouse Guinea pig Rabbit Cat Dog Paraquat (mmol/kg) Mouse Rat Guinea pig Cat Pargyline Mouse Rat Cat Dog Monkey Pentachlorophenol Rat Pentazocine-HCl Mouse Pentobarbital Mouse Rat Guinea pig Rabbit Cat Pentolinium Mouse Pentylenetetrazol Mouse

IV

IP

IM

SC

PO

162 20 97 0.26 0.11 >1,500

7,500

32

315 5

33.1

750 63

420

2,500 745 3,700 1,650

1,230 450 450 500

3,500 0.12 0.12 0.018

0.66 0.57–0.94 0.19 0.22

370 142 200

680 300 175

150 96


3,570 80

130 75 50

124

107

280 118

70

45

275 100

29

36

51

92

512 101

162

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Perphenazine Mouse Phalloidine Mouse Phenacetin Mouse Rat Phenazone Rat Phenelzine Mouse p-Phenetidine Mouse Rat Pheniprazine Mouse Rat Phenobarbital Mouse Rat Rabbit Cat Phenoxybenzamine Mouse Rat Guinea pig Phentolamine Mouse Rat Phentolamine mesylate Mouse Rat Phenylbutazone Mouse Rat Rabbit m-Phenylenediamine Rat p-Phenylenediamine Rat Phenylephedrine Mouse Rat Phenylhydrazine Mouse Rat Rabbit Guinea pig


IV

IP 70

IM

SC

PO

100

70 1.9 1,030–2,000 1,650 1,800 157

150

156 530 580

60.6 44.5

122

95 45.3

73 34.1

340 190

230 200

325 660

185 175 1,535 2,500 500

75

275

1,000 1,250

1,000 1,250 123 150

336 215

417.5 650–1,000 146 650 98

21

1,000

70 92

120 350–1,120 175 188 80 80

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Phenyl oxirane Rat Rabbit Phenytoin Mouse Rat Rabbit Phloroglucinol Rat Physostigmine Mouse Phytonadione (1%) Mouse Phytonadione Mouse 4-Picoline Rat Rabbit Picrotoxin Mouse Rat Pilocarpine Mouse Pimozide Mouse Rat Dog Pipradrol Mouse Rat Rabbit Potassium bromate Rat Potassium cyanide Mouse Potassium fluorosilicate Guinea pig Pralidoxime Mouse Rat Guinea pig Rabbit Praziquantel Mouse Rat Prednisolone phosphate disodium Mouse, female

IV

IP

IM

SC

PO

4,290 930–1,060 200 280

490

125 3,180

4,850

5,200

1

0.54

3

52 ml/kg >25 g/kg

>25 g/kg 1,290 14.8

270


3

7.2 6.5

7

14.8

500 >5, 100 >5,100 40 94

240 240

30 15 50–200

365 180

200–400 6

16

500 90 96

155

180 150 168

4,100

95 2,500 2,500

1,190

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Prenylamine Mouse Rat Primidone Mouse Rat Proadifen Mouse Rat Probenecid Mouse Rat Rabbit Dog Probucol Mouse Rat Procaine Mouse Rat Guinea pig Rabbit Dog Prochlorperazine Mouse Rat Prodilidine Mouse Rat Promazine Mouse Rat Rabbit Promethazine Mouse Rat Guinea pig Rabbit Prontalol Mouse Rat Propanidid Mouse Rat Dog Rabbit Propantheline Rat Propargyl alcohol Mouse


IV

IP

IM

SC

PO

200 1,000 600–800 1,500–2,000 60

117.5 163

458

230 394

538 2,140 1,156 611

1,666 1,604

304 270 >5 g/kg >5 g/kg 45 50 51 57 62.4

230 250

92

125

630 1,600

91 74 38 17–29 21

800 2,100

500

350

750 1,800

194 188

318 253

113 233

28–75 20–45 42.5 19

150

50 50

124

216 250

216–485 343–650 125 750 225

375–575 480

900 900

90 80 80 75 298

370 50

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Guinea pig Propiverine Mouse Rat Rabbit Propofol Mouse Propoxyphene HCl Mouse Rat Propoxyphene napsylate Mouse Rat Rabbit Dog Propranolol Mouse Rat Propyl chloroformate Mouse Protoveratrine Mouse Rat Rabbit Cat Protriptyline Mouse Pseudoephedrine Mouse Rat Rabbit Psilocin Mouse Rat Psilocybine Mouse Rat Pyrethrin Rat Pyrilamine Mouse Rat Guinea pig Quinidine Mouse Rat Cat


IV

IP

IM

SC

PO 55 60

113 29 13

490 2,100 620

53 204 131

282 230

915 647 >183 >183 27

114

380 533 650

0.05

0.4 0.6 0.11 0.5

5

0.05

37

192

269 726 2,206 1,177

202

74 75 285 280 1,500 30

102

150 150 70

24.4 69 23.1 21.6

190

200

235

594 1,000

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Ramipril Mouse Rat Ranitidine HCl Mouse Rat Reserpine Mouse Rat Resorcinol Rat Guinea pig Rifampin Mouse Rat Rabbit Ritodrine Mouse Rabbit Rotenone Rat Salicylamide Mouse Rat Rabbit Scopolamine Mouse Secobarbital Cat Selenium Rat Selenium disulfide Rat Selenium monosulfide Mouse Selenium tetrachloride Guinea pig Semicarbazide Mouse Serotonin Mouse Rat Sodium bromide Mouse Rat Sodium fluoride Rat Sodium tetradecyl sulfate Mouse


IV

IP

IM

SC

PO

10–11 g/kg 10–11 g/kg 77 83 70

390–500

18 370 370 885 1,720 2,120 540 64 100–300 313

1,400 1,200 3,000

600 153.5

590 50 6,700 138 370 19

125.6

23.3

160 30

868 117

125.5

176

5,020

7,000 3,500

750

200

90

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rat Solanine Rat Rabbit Sheep Sotalol Mouse Rat Rabbit Dog g-Strophanthine Guinea pig Stearyl Heptanoate Rat Strychnine Mouse Rat Succinylcholine Mouse Rabbit Sulfacetamide Mouse Sulfadimethoxine Rat Rabbit Sulfafurazole Rat Rabbit Sulfaguanidine Mouse Sulfamethoxazole Mouse Sufentanil citrate Mouse Rat Guinea pig Dog Sulfamethoxazole Mouse Rat Rabbit Sulfanilamide Mouse Rat Dog Sulfasalazine Mouse Sulfisoxazole Mouse Rat


IV

IP

IM

SC

PO

72–128 75 20–23

590 225

670 680

2,600 3,450 1,000

330 0.26 >5g/kg 0.98 0.09–1.4 0.75 1

4

0.85 1.2

16.2 125

16.5 g/kg >4,000 >2,000 >10,000 >2,000 15,000 3,200 17 10.5 12.5 10.1–19.5 2,300 3,000 >2,000 3,700–4,300 3,900–10,000 2,000 >12 g/kg 5,700 >10,000

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rabbit Syrosingopine Rat Talinolol Mouse Rat Terbutaline Mouse Rat Terconazole Rat, male Rat, female Dog, male Dog, female Terfenadine Mouse Rat Rat newborn Tetrabenazine Mouse Tetracaine Mouse Guinea pig Dog 1,1,2,2-Tetrabromoethane Mouse Rat Rabbit Guinea pig 1,1,2,2-Tetrachloroethane Rat 2,3,4,5-Tetrachlorophenol Mouse Rat Guinea pig 2,3,4,6-Tetrachlorophenol Mouse Rabbit Tetraethylammonium Mouse Rat Rabbit Dog Tetrahydrocannabinol Mouse

IV

IP

IM

SC

PO >2,000

50 600–1,450 1,180–2,580 3,000 18,000 1,741 849 1,280 640 >5,000 >5,000 438 150

400

6.6 15.6 4.3

269 1,100 400 400

5,250

800

400 140 250

109 250


29 63 72 55

56 115

510

655

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Tetryzoline Mouse Thebaine Rat Theophylline Mouse Thioglycolic acid Rat Rabbit Thioguanine Rat, male Rat, female Thiram Rat Rabbit Thiopental Mouse Rat Guinea pig Rabbit Dog Timolol maleate Mouse, female Rat, female Tocinide HCl Mouse Rat Guinea pig Tolazoline Mouse o-Tolidine Rat Toluene-2,4-diisocyanate Rat o-Toluidine Rat Rabbit P-Toluidine Mouse Rat Rabbit Toxaphene Rat Tranylcypromine Mouse Trazodone Mouse Rat

IV

IP

IM

SC

PO

39 13.9 350 114 848 823 740 865 210 112 67.5 55 40 55

200 120 57.5

350

600 150 1,190 900 800 1,000 230 500 404

5,800 670 3,250 794 656 890


40–120 37

38

96

610 486

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Rabbit Triamterene Mouse Triazolam Mouse Rat Trichloroacetonitrile Rat Rabbit Trichloronitromethane Rat Trichlorotrinitrobenzene Rat Trichothecene Rat Guinea pig Trifluperidol Mouse Rat Trifluoperazine Mouse Rat Dog Trimethadione Mouse Rat Rabbit Trimipramine Mouse Tripelennamine Mouse Rat Guinea pig Triphenyltin acetate Rat Rabbit Triphenyltin hydroxide Mouse Rat Tubocurarine Mouse Rat Rabbit

IV

IP

IM

SC

PO 560 300–380 >1,000 >5,000 250

900


250

30 g/kg 0.75 1.3 988 113 36

442 740

60 2,000

1,800

2,200 2,200

1,500

1,500 250

17 13

70

75 225 30.2

210 570 155 140 30

245 46 0.14 0.25 0.35

TABLE 33.1 (Continued) LD50 Values of Common Xenobiotics (mg/kg unless stated otherwise) Dermal Ursodiol Mouse Rat Vancomycine HCl Mouse Rat Veratradine Mouse Rat Vidarabine Mouse Rat Vinyl chloride Rat Vinyl cyclohexene diepoxide Rat Rabbit Warfarin Mouse Rat Guinea pig Rabbit Dog 2,4-Xylenol Mouse Rat 2,5-Xylenol Mouse 2,6-Xylenol Mouse Rat Zinc phosphide Mouse Rat Rabbit Cat Zoxazolamine Mouse Rat Dog

IV

IP

IM

SC

PO

>7,500 >5,000 400 319 0.42

1.35 3.5 >5,020 >5,020 500

2,130 620 165 186

374 323 182 800 250

150 250 1,040

3,200 383 920

980 296 40 2.7–40.5 40 250

2,000–5,000

376 102

825 376

117

Data taken from Drug Dosage in Laboratory Animals: A Handbook, R.E. Borchard, C.D. Barnas, and L.G. Eltherington, Eds., CRC Press, Boca Raton, FL, 1992; Chemical Safety Data Sheets, Vols. 4a and 4b, Royal Society of Chemistry, Indispensable Publications Ltd., Bugbrooke, Northamptonshire, U.K., 1991; Physician Desk Reference, 47 ed., Medical Economics Data, Montvale, NJ, 1993; Toxicological Analysis, R. Klaus Müller, Ed., Ullstein Mosby GmbH & Co., KG, Berlin, 1992.


0370_frame_C33(1) Page 1270 Thursday, July 12, 2001 12:38 PM

TABLE 33.2 Radiation LD50 Values for Different Species LD50 (Midline Absorbed Dose, Rads)

Species Sheep Burro Swine Goat Dog Man

,155 ,155 , 195 ,230 , 265 , 270(?) , 243(?) ,255(?) ,840 ,900 , 900 ,900 1,059 1,100–1,200

Rabbit Mouse Rat Hamster Gerbil Wild mice Desert mice Praomys formusus Praomys longimembris Guinea pig Monkey Marmoset

1,300 1,520 , 255(?) ,398 ,200

From CRC Handbook of Radiobiology, Kedar N. Prasad, 1984. With permission.

SECTION 2. TABLES OF COMPARATIVE ANATOMICAL, PHYSIOLOGICAL, AND BIOCHEMICAL DATA TABLE 33.3 Comparison of Physiological Parameters for Different Body Organs

Organ Adrenal glands Blood Bone Brain Fat Heart Kidneys Liver Portal Arterial Lungs Muscle Skin Thyroid gland Total body

Weight (kg)

Percent of Body Volume

Percent Water

5.4 10 1.5 10 0.3 0.3 1.5

0.03 7 16 2 10 0.5 0.4 2.3

83 22 75 10 79 83 68

1.0 30 5 0.03

0.7 42 18 0.03 100

79 76 72 60

Blood Flow (ml/min) 25 5,000 250 700 200 200 1,100 1,350 1,050 300 5,000 750 300 50 5,000

Plasma Flow (ml/min)

Blood Flow (ml/kg)

Blood Flow Fraction

15 150 420 120 120 660 810 630 180 3,000 450 180 30 3,000

780 0.05 250 1,200 1,500

900 250

0.25

0.19

Source: Modified from Illing, Xenobiotic Metabolism and Disposition: The Design of Studies on Novel Compounds, CRC Press, Boca Raton, FL, 1989. Data are for hypothetical 70-kg human.



TABLE 33.4 Comparison of the Blood Flow/Perfusion and Oxygen Consumption of Liver, Lung, Intestine, and Kidney of the Rat In Vivo and in Organ Perfusiona Parameter (unit) In vivo Blood flow (ml min–1) Blood pressure S/D (torr) pO2-arterial (torr) pO2-venous (torr) O2-consumption (µl min–1) In perfusion Perfusion flow (ml min–1) Perfusion pressure (torr) pO2-arterial (torr) pO2-venous (torr) max. O2-supplyb (µl min–1)

Liver

Lung

Intestine

Kidney

13–20 150/100 95 40 500–800

55–70 25/10 40 100 From air

5–8 150/100 95 50 40–160

4–6 150/100 95 70 100–200

30–50 100–120 600 200 380–630

50 10–20 600 ? ?

6 100–120 400 180 120c

20–35 100–120 600 400 120–220

a

These values are indications of the most common values measured for the various organs in a rat of 250 to 300 g. The figures provided for the kidney apply to a single kidney. The values measured in organ perfusions may differ greatly, depending on the set-up, method of gassing, etc. S = systolic; D = diastolic. b Calculated from pO arterial, pO -venous, and perfusion flow. 2 2 c With 20% FC-43 emulsion in KRB; other figures apply to KRB buffer without erythrocytes or oxygen carrier. (KRB = Krebs-Ringer buffer.) From Toxicology: Principles and Applications, CRC Press, Boca Raton, FL, 1996. With permission.

TABLE 33.5 Comparison of Respiratory Volume and Alveolar Ventilation in Relation to Animal Weight

Species

Weight (g)

Respiratory Volume per Minute (cm3)

Respiratory Volume per Minute (cm3) Weight (g)

Mouse Cotton rat Hamster White rat Guinea pig Rabbit Monkey Human

19.8 76.8 91.6 112.8 466.0 2,069.0 2,682.0 68,500.0

24.5 39.6 60.9 72.9 155.6 800.0 863.5 8,732.0

1.24 0.52 0.67 0.65 0.33 0.39 0.32 0.13

Alveolar Ventilation (l/hr) Weight (g) 0.067

0.070

0.005

Source: Modified from Calabrese, Principles of Animal Extrapolation, Lewis Publishers, Chelsea, MI, 1991.



TABLE 33.6 Comparison of Dosage by Weight and Surface Area Species

Weight (g)

Dosage (mg/kg)

Dose (mg/animal)

Surface Area (cm2)

Dosage (mg/cm2)

Mouse Rat Guinea pig Rabbit Cat Monkey Dog Human

20 200 400 1,500 2,000 4,000 12,000 70,000

100 100 100 100 100 100 100 100

2 20 40 150 200 400 1,200 7,000

46 325 565 1,270 1,380 2,980 5,770 18,000

0.043 0.061 0.071 0.118 0.145 0.134 0.207 0.388

Source: Casarett and Doull’s Toxicology, 4th ed., M. O. Amdur, J. Doull, and C. D. Klassen, Eds., Pergamon Press, New York, 1991. With permission.

TABLE 33.7 Relationship Between Body Weight and Body Surface Area in a Number of Vertebrates Species Mouse Rat Guinea pig Rabbit Cat Monkey Dog Man

Weight (g)

Surface Area (cm2)

20 200 400 1,500 2,000 4,000 12,000 70,000

46 325 565 1,270 1,380 2,980 5,770 18,000

From Toxicology: Principles and Applications, CRC Press, Boca Raton, FL, 1996. With permission.

TABLE 33.8 Comparison of Bile Flow and Hepatic Blood Flow Rates in Various Species

Species Rat Guinea pig Rabbit Cat Dog Hen Sheep Monkey

Liver Weight as Percentage of Total Body Weight

Hepatic Blood Flow Rate (ml Blood per 100 g Liver/min)

Bile Flow Rate (ml Bile per kg Body Weight/Day)

3.36 3.86 3.2 3.59 2.94 1.53 2.97 2.09

79 — 74 35–48 82 — — —

28.6–47.1 228 118 14 12 14 12 28

From Calabrese, Principles of Animal Extrapolation, Lewis Publisher, Chelsea, MI, 1991. With permission.



TABLE 33.9 Comparison of the pH Value of Contents of Different Parts of the Alimentary Tract in Various Species pH Value (Median for Different Animals) of Contents of Species (No. Examined) Monkey (3) Dog (3) Cat (6) Ox (3) Sheep (3) Horse (3) Pig (20) Rabbit (11) Guinea Pig (6) Rat (7) Mouse (3) Hamster (3) Gerbil (3) Fowl (6) Duck (3) a

Stomacha

Small Intestine Portion

A

P

1

3

5

7

Cecum

Colon

Fecesa

4.8 5.5 5.0 6.0 6.4 5.4 4.3 1.9 4.5 5.0 4.5 6.9 5.5 4.9 5.0

2.8 3.4 4.2 2.4 3.0 3.3 2.2 1.9 4.1 3.8 3.1 2.9 3.8 4.2 4.5

5.6 6.2 6.2 6.7 5.7 6.7 6.0 6.0 7.6 6.5 — 6.1 6.7 5.8 6.1

5.8 6.2 6.7 7.0 6.6 7.0 6.2 6.8 7.7 6.7 — 6.6 7.0 6.2 6.5

6.0 6.6 7.0 7.3 7.7 7.3 6.9 7.5 8.1 6.8 — 6.8 7.8 7.0 7.4

6.0 7.5 7.6 7.9 8.0 7.9 7.5 8.0 8.2 7.1 — 7.1 8.2 7.8 8.0

5.0 6.4 6.0 7.0 7.3 7.0 6.3 6.6 7.0 6.8 — 7.1 7.0 7.0 —

5.1 6.5 6.2 7.4 7.8 7.4 6.8 7.2 6.7 6.6 — — 7.4 — —

5.5 6.2 7.0 7.5 8.0 7.5 7.1 7.2 6.7 6.9 — — 7.5 7.6 7.3

A = Anterior portion of stomach; P = posterior portion; feces = contents of posterior rectum.

From Calabrese, Principles of Animal Extrapolation, Lewis Publisher, Chelsea, MI, 1991. With permission.

TABLE 33.10 Comparison of Enzyme Activity in the Small Intestine of Several Species Enzymatic Activity in the Small Intestine as a Percentage of That in the Liver Species

Ethylmorphine N-Demethylase

Biphenyl Hydroxylase

Aniline Hydroxylase

AHH

Cytochrome c Reductase

Cytochrome P-450

Rabbit Guinea pig Cat Mouse Hamster

18.6 23.3 NDa ND ND

14.1 16.4 9.3 9.0 6.8

20.4 19.8 ND ND ND

30.0 37.4 4.6 6.0 5.7

75.7 78.7 42.0 79.6 60.7

34.6 12.4 ND 4.0 13.0

a

ND = not detectable.




TABLE 33.11 Comparison of pH Values in Some Human Body Compartments Blood Oral cavity Stomach (at rest) Duodenum Jejunum Ileum Colon Rectum Cerebral fluid Vagina Urine Sweat Milk

7.35–7.45 6.2–7.2 1.0–3.0 4.8–8.2 6.3–7.3 7.6 7.8–8.0 7.8 7.3–7.4 3.4–4.2 4.8–7.5 4.0–6.8 6.6–7.0


TABLE 33.12 Comparison of the Size of the Absorptive Surface of the Various Parts of the Gastrointestinal Tract Oral cavity Stomach Small intestine Large intestine Rectum

0.02 0.1–0.2 100 0.5–1.0 0.04–0.07



Cardiac Function Body Wt. (kg)

Surface Area (cm2)

0.1–0.5

0.03–0.06

Rabbit Monkey

1–4 2–4

0.23 0.31

Dog

5–31

0.39–0.78

Man

54–94

1.65–1.83

Pig

100–250

2.9–3.2

Ox

500–800

4.2–8.0

Horse

650–800

5.8–8.0

Species Rat

a

Energy Metabolisma

Heart

Heart

Stroke

Cardiac

Cardiac

Wt. (g/100g)

Rate (beats/min)

Vol (ml/beat)

Output (l/min)

Index (l/m2/m)

Arterial Blood Pressure (mm Hg) Systolic

Diastolic

cal/kg/day

cal/m2/day

120–140 (B) 47 49 (B) 34–39 (B) 23–26 (B) 14–17 (B) 15 (B) 25 (R)

760–905 (B) 810 675

0.24–0.58

250–400

1.3–2.0

0.015–0.079

1.6

88–184

58–145

0.19–0.36 0.34–0.39

123–330 165–240

1.3–3.8 8.8

0.25–0.75 1.06

1.7 —

95–130 137–188

60–90 112–152

770–800 (B) 790–910 (B) 1,100–1,360 (B) 1635 (B) 2,710–2,770 (R)

0.65–0.96

72–130

14–22

0.65–1.57

2.9

95–136

43–66

0.45–0.65

41–108

62.8

5.6

3.3

92–150

53–90

0.25–0.40

55–86

39–43

5.4

4.8

144–185

98–120

0.31–0.53

40–58

244

146

—

121–166

80–120

0.39–0.94

23–70

852

188

4.4

86–104

43–86

B = basal; R = resting.

Source: Mitruka, B.M., and Rawnsley, H.M., Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals, Masson Publishing, New York, 1977. With permission.



TABLE 33.13 Comparison of Physiological Characteristics of Experimental Animals and Humans


TABLE 33.14 Comparison of Certain Physiological Values of Experimental Animals and Humans

Species Mouse Rat Hamster Guinea pig Rabbit Chicken Cat Dog Monkey Pig Goat Sheep Cattle Horse Man

Body Temperature (°C) 36.5 37.3 36.0 37.9 38.8 41.4 38.6 38.9 38.8 39.3 39.5 38.8 38.6 37.8 36.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.70 1.40 0.50 0.95 0.65 0.25 0.70 0.65 0.80 0.30 0.60 0.80 0.30 0.25 0.35

Whole Blood Volume (ml/ kg body wt) 74.5 58.0 72.0 74.0 69.4 95.5 84.6 92.6 75.0 69.4 71.0 58.0 57.4 72.0 77.8

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17.0 14.0 15.0 7.00 12.0 24.0 14.5 29.5 14.0 11.5 14.0 8.50 5.00 15.0 15.0

Plasma Volume (ml/kg body wt) 48.8 31.3 45.5 38.8 43.5 65.6 47.7 53.8 44.7 41.9 55.5 41.9 38.8 51.5 47.9

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

17.0 12.0 7.50 4.50 9.10 12.5 12.0 20.1 13.0 8.90 13.0 12.0 2.50 12.0 8.70

Plasma pH 7.40 7.35 7.39 7.35 7.32 7.52 7.43 7.42 7.46 7.40 7.41 7.48 7.38 7.42 7.39

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

Plasma CO2 content (mM/l)

CO2 Pressure (mm Hg)

22.5 ± 4.50 24.0 ± 4.70 37.3 ± 2.50 22.0 ± 6.60 22.8 ± 8.60 23.0 ± 2.50 20.4 ± 3.50 21.4 ± 3.90 29.3 ± 3.8 30.2 ± 2.5 25.2 ± 2.8 26.2 ± 5.00 31.0 ± 3.0 28.0 ± 4.00 27.0 ± 2.00

40.0 ± 5.40 42.0 ± 5.70 59.0 ± 5.00 40.0 ± 9.80 40.0 ± 11.5 26.0 ± 4.50 36.0 ± 4.60 38.0 ± 5.50 44.0 ± 4.8 43.0 ± 5.60 50.0 ± 9.40 38.0 ± 8.50 48.0 ± 4.80 47.0 ± 8.50 42.0 ± 5.00

0.06 0.09 0.08 0.09 0.03 0.04 0.03 0.04 0.06 0.08 0.09 0.06 0.05 0.03 0.06

Source: Mitruka, B. M. and Rawnsley, H. M., Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals, Masson Publishing, New York, 1977. With permission.

TABLE 33.15 Comparison of Biochemical Components in Urines of Normal Experimental Animals and Humans Component (mg/kg body wt/day) or Property Volume (ml/kg body wt/day) Specific gravity pH Calcium Chloride Creatinine Magnesium Phosphorous, inorganic Potassium Protein, total Sodium Urea nitrogen (g/kg/day) Uric acid

Volume (ml/kg body wt/day) Specific gravity

pH

Rat

Rabbit

Cat

Dog

Goat

Sheep

150–350 1.040–1.076 7.30–8.50 3.00–9.00 50.0–75.0 24.0–40.0 0.20–1.90 20.0–40.0 50.0–60.0 1.20–6.20 90.4–110. 1.00–1.60 8.00–12.0

20.0–350 1.003–1.036 7.60–8.80 12.1–19.0 190–300 20.0–80.0 0.65–4.20 10.0–60.0 40.0–55.0 0.74–1.86 50.0–70.0 1.20–1.50 4.00–6.00

10.0–30.0 1.020–1.045 6.00–7.00 0.20–0.45 89.0–130 12.0–30.0 1.50–3.20 39.0–62.0 55.0–120 3.10–6.82 — 0.80–4.00 0.20–13.0

20.0–167 1.015–1.050 6.00–7.00 1.00–3.00 5.00–15.0 15.0–80.0 1.70–3.00 20.0–50.0 40.0–100 1.55–4.96 2.00–189. 0.30–0.50 3.1–6.0

7.0–40.0 1.015–1.062 7.5–8.80 1.00–3.40 186–376 10.0–22.0 0.15–1.80 0.5–1.6 250–360 0.74–2.48 140.–347. 0.14–0.47 2.00–5.00

10.0–40.0 1.015–1.045 7.50–8.80 1.00–3.00 — 5.80–14.5 0.10–1.50 0.10–0.50 300–420 0.74–2.17 0.80–2.00 0.11–0.17 2.00–4.00

Swine

Cattle

Horse

Monkey

Man

5.00–30.0 1.010–1.050 Swine

17.0–45.0 1.025–1.045 Cattle

3.0–18.0 1.020–1.050 Horse

70.0–80.0 1.015–1.065 Monkey

8.60–28.6 1.002–1.040 Man

6.25–7.55

7.60–8.40

7.80–8.30

5.50–7.40

4.80–7.80



TABLE 33.15 (Continued) Comparison of Biochemical Components in Urines of Normal Experimental Animals and Humans Calcium Chloride Creatinine Magnesium Phosphorous, inorganic Potassium Protein, total Sodium Urea nitrogen (g/kg/day) Uric acid

— — 20.0–90.0 — — — 0.33–1.49 — 0.28–0.58 1.00–2.00

0.10–3.60 10.0–140. 15.0–30.0 2.00–7.00 0.01–6.20 240–320. 0.25–2.99 2.00–40.0 0.05–0.06 1.00–4.00

— 81.0–120 — — 0.05–2.00 — 0.62–0.99 — 0.20–0.80 1.00–2.00

10.0–20.0 80.0–120. 20.0–60.0 3.20–7.10 9.00–20.6 160–245. 0.87–2.48 — 0.20–0.70 1.00–2.00

0.60–8.30 40.0–180. 15.0–30.0 0.42–2.40 10.0–15.0 16.0–56.0 0.81–1.86 25.0–94.0 0.20–0.50 0.80–3.00

Source: Mitruka, B. M. and Rawnsley, H. M., Clinical Biochemical and Hematological Reference Values in Normal Experimental Animals, Masson Publishing, New York, 1977. With permission.

TABLE 33.16 Comparison of Some Biochemical/Physiological/Morphological Differences of Potential Toxicological Significance Between Rats and Humans Rat A. Skin characteristics 1. Stratum corneum 2. Dermal vasculature 3. Sweat glands

4. Hair follicles 5. Dermal absorption based on the above characteristics B. Respiratory parameters 1. Histamine content (µg/g) 2. Exogenous histamine catabolism (%) 3. Histamine release (%) µg/g Compound 48/80 Cotton dust 4. Lung morphometry a. Branching angles b. Symmetry c. Diameter ratio of daughter branches at bifurcation d. Number of diversions of tracheobronchial tree 5. Mucus flow patterns 6. Bronchial glands 7. Position of lung to ground


Missing from general body surface; eccrine sweat glands located in foot pads to moisten frictional surface Densely haired with up to 4000 hairs/cm2 Considerably more efficient absorber than humans for a wide variety of organic compounds

Human

Much thicker than rat Much thicker than rat Numerous coiled tubular sweat glands (100–600/m2) Much fewer hairs, with 40–70 hairs/cm2 on skin of the trunk and limbs

15.8 44.2

27.7 29.2

17.1 0.0

43.2 16.1

Decreases with increasing depth in the lung Less than humans Greater than humans

Increases with increasing depth in the lung

More variable than humans 13.5 mm/min Absence Horizontal

15 mm/min Numerous Vertical


TABLE 33.16 (Continued) Comparison of Some Biochemical/Physiological/Morphological Differences of Potential Toxicological Significance Between Rats and Humans Rat 8. Breathing C. Gut flora location

D. Estimated β-glucuronidase activity Proximal small intestine Distal small intestine E. Plasma protein binding

F. Biliary excretion

G. Metabolism 1. Conjugations Sulfate Glucuronidation Acetylation

Obligate nose breathers Numerous flora in stomach and proximal small intestine Numerous flora in distal small intestine, large intestine, rectum, and feces Very high (304.0 units) Very high (1,341.0 units) Generally not as extensive a binder as the human; a number of leading researchers feel that it is not possible to adequately predict the extent of human binding with the rat model The rat is perhaps the most efficient biliary excreter, whereas limited evidence suggests that the human is not an efficient excreter of intermediately weighted compounds.

Less active than human More active than human Effective

Deacetylation

Displays a relatively low ability

Amino acid with carboxylic acid substrates Benzoic acid

80–100% conjugation with glycine

Phenylacetic acid 2. Rodanese activity (liver) 3. Epoxide hydrase 4. Red blood cell enzymes that prevent oxidant stress (in values relative to humans, which are given as 1) Glutathione peroxidase Glutathione reductase Catalase Glucose-6-phosphate dehydrogenase Superoxide dismutase Methemoglobin reductase 5. Comparison of rat versus human for 23 substances according to qualitative and quantitative similarity of metabolic pathway


Human

Little or no flora in stomach and proximal small intestine Similar to rat

0.02 units 0.09 units

Humans display both effective and slow acetylator phenotypes Human data inadequate to assess 100% conjugation with glycine

Strongly favors glutamine conjugation

Strongly favors glycine conjugation Considerably more active than humans Less active in humans

10.2 0.2 0.2 2.4 1.7 2.4 Good predictor 4 of 23; Invalid predictor 8 of 23

1 1 1 1 1 1


TABLE 33.16 (Continued) Comparison of Some Biochemical/Physiological/Morphological Differences of Potential Toxicological Significance Between Rats and Humans Rat 6. Concentration of urine

H. Dermatotoxicity 1. Ocular 2. Skin

3. Allergic hypersensitivity

I. DNA repair

J. Teratogenicity

K. High-risk animal models 1. Respiratory a. Asthma

b. Bronchitis


Typical laboratory rat has the ability to concentrate its urine approximately 2 times as much as that of humans, as indicated by urine/plasma ratios; the desert rat will concentrate its urine 4–5 times more than humans Practical reasons preclude its widespread use as a predictive model (e.g., not sufficiently docile); the rabbit is the model of choice for historical reasons and practical considerations: docility, large skin surface, large nonpigmented eyes The rat is not a good model, because it does not produce anaphylactic antibodies in response to the diversity of allergens, which humans do; the guinea pig model is favored in such studies for qualitative predictions. In absolute terms, less efficient than humans in excision repair; when adjusted for the influence of life span, little difference between these species is found; more efficient than humans in postreplicative repair. Prolonged dependence of the rat (up to the 20–25th somite) on the inverted yolk sac placenta during organogenesis as compared with higher mammals, especially humans (5th somite), thereby making the rodent generally much more susceptible to teratogens than humans.

Rats are not considered appropriate; with the exception of humans, dogs are the only animal that develop a defined hypersensitivity disease related to aeroallergens. Rats are not considered an appropriate model, because of its version of chronic respiratory disease that involves bronchitis displays, excessive inflammation, and involvement of the pulmonary parenchyma.

Human


TABLE 33.16 (Continued) Comparison of Some Biochemical/Physiological/Morphological Differences of Potential Toxicological Significance Between Rats and Humans Rat 2. Cardiovascular a. Atherosclerosis

Human

The rat is generally not considered a very effective model, because it is very resistant to developing this disease. Numerous predictive rat strains exist; the rat is the animal of choice.

b. Hypertension

From Principles of Animal Extrapolation, Calabrese, E. J., Ed., Lewis Publishers, Chelsea, MI, 1991. With permission.

TABLE 33.17 Permittivity and Conductivity of Biological Tissues : Relative permittivity : Conductivity in S/m (siemens per meter) All values refer to 37C. Frequencies are those commonly used for therapy.

Material Artery Blood Bone With marrow In Hank’s solution Bowell (plus contents) Brain White matter Grey matter Fat Kidney Liver Lung Inflated Deflated Muscle Ocular tissues Choroid Cornea Iris Lens cortex Lens nucleus Retina Skin Spleen Tumor tissues Hemangiopericytoma

433 MHz

915 MHz

ε

σ

ε

σ

ε

σ

ε

σ

ε

σ

— —

— 155

— 1.16

— 110

— 1.19

— 66

— 1.27

— 62

1.41

43 60

1.85 2.04

44–55

11 28 73

0.03 0.021 —

9 24 49

0.04 0.024 —

5.2 — —

0.11 — —

4.9 — —

0.15 — —

4.8 — —

0.21 —

182 310 38 402 288

0.27 0.40 0.21 0.72 0.49

123 186 22 229 182

0.33 0.45 0.21 0.83 0.58

48 57 15 60 47

0.63 0.83 0.26 1.22 0.89

41 50 15 55 46

0.77 1.0 0.35 1.41 1.06

35.5 43 12 50 44

1.04 1.43 0.82 2.63 1.79

73–78

42 94 152

0.11 0.29 0.74

29 57 112

0.13 0.32 0.76

— 35 57

— 0.71 1.12

— 33 55.4

0.78 1.45

— — 49.6

— — 2.56

78 75 77 65 — 89 60–76 76–81

240 132 240 175 50.5 464 120 269

0.97 1.55 0.90 0.53 0.13 0.90 0.25 0.86

144 100 150 107 48.5 250 98 170

1.0 1.57 0.95 0.58 0.15 1.0 0.40 0.93

60 55 59 55 31.5 61 47 —

1.32 1.73 1.18 0.80 0.29 1.50 0.84 —

55 51.5 55 52 30.8 57 45 —

1.40 1.90 1.18 0.97 0.50 1.55 0.97 —

52 49 52 48 26 56 44 —

2.30 2.50 2.10 1.75 1.40 2.50 1.85 —

136

1.10

106

1.14

57

1.37

55.4

1.61

50

2.86

Wt % Water

60–82 68–73 82–85 5–20 78–79 73–77 80–83


13.56 MHz

27.12 MHz

2450 MHz


TABLE 33.17 (Continued) Permittivity and Conductivity of Biological Tissues : Relative permittivity : Conductivity in S/m (siemens per meter) All values refer to 37C. Frequencies are those commonly used for therapy. Wt % Water

Material Intestinal leiomysarcoma Splenic hematoma Rat hepatoma D23 Canine fibrosarcoma Mouse KHT tumor

13.56 MHz

27.12 MHz

ε

σ

ε

σ

309 297 305 48 30 —

0.83 0.64 0.74 0.18 0.65 —

183 243 178 29 19 135

0.91 0.74 0.87 0.19 0.66 —

433 MHz

915 MHz

ε

ε

σ

2450 MHz

σ

62 1.23 60 1.49 54 0.93 52 1.06 — — — — (Before hyperthermia) (After 43C hyperthermia) 61 1.12 60 1.61

ε

σ

54 49 —

2.63 1.89 —

54

2.56

Note: The dielectric properties of the same tissue types from different animals have been found to be similar. The data given in this table should be considered as representing average values, and the likely spread of the electrical properties for each tissue type can be estimated from the range of tissue water contents. These data have been derived from Pethig, R., IEEE Trans. Elec. Insul. El-19, 453, 1984, from which full references may be obtained. The electrical properties of other biological materials at other frequencies have been tabulated by Geddes, L. A. and Baker, L. E., Med. Biol. Eng., 5, 271, 1967 and by Stuchly, M. A. and Stuchly, S. S., J. Microwave Power 15, 19, 1980. From Handbook of Chemistry and Physics, Lide, D. R., Ed., CRC Press, Boca Raton, FL, 1990. With permission.

SECTION 3. MATHEMATICS, SYMBOLS, PHYSICAL CONSTANTS, CONVERSIONS, AND STATISTICS A. SYMBOLS TABLE 33.18 Greek Alphabet Greek Letter

Greek Name

Α Β Γ ∆ Ε Ζ Η Θ Ι Κ Λ Μ

Alpha Beta Gamma Delta Epsilon Zeta Eta Theta Iota Kappa Lambda Mu

α β γ δ ε ζ η θ ι κ λ µ

See end of section for source.


English Equivalent a b g d e˘ z e th i k l m

Greek Letter

Greek Name

Ν Ξ Ο Π Ρ Σ Τ Υ Φ Χ Ψ Ω

Nu Xi Omicron Pi Rho Sigma Tau Upsilon Phi Chi Psi Omega

ν ξ ο π ρ σ τ υ φ χ ψ ω

e

ϕ

English Equivalent n x o p r s t u ph ch ps o


B. CONVERSIONS The Système international d’unités (International System of Units) or SI is a modernized version of the metric system. The primary goal of the conversion to SI units is to revise the present confused measurement system and to improve test-result communications. The SI has 7 basic units from which other units are derived:

TABLE 33.19 Base Units of SI Physical Quantity Length Mass Time Amount of substance Thermodynamic temperature Electric current Luminous intensity

Base Unit meter kilogram second mole kelvin ampere candela

SI Symbol m kg s mol K A cd


Combinations of these base units can express any property, although for simplicity, special names are given to some of these derived units.

TABLE 33.20 Representative Derived Units Derived Unit

Name and Symbol

Derivation from Base Units

Area Volume Force Pressure Work, energy Mass density Frequency Temperature Concentration Mass Substance Molality Density

square meter cubic meter newton (N) pascal (Pa) joule (J) kilogram per cubic meter hertz (Hz) degree Celsius (C)

m2 m3 kg · m · s–2 kg · m1 · s–2 (N/m2) kg · m2 · s–2 (N · m) kg/m3 s–1 C = K – 273.15

kilogram/liter mole/liter mole/kilogram kilogram/liter

kg/l mol/l mol/kg kg/l


Prefixes to the base unit are used in this system to form decimal multiples and submultiples. The preferred multiples and submultiples listed below change the quantity by increments of 103 or 10–3. The exceptions to these recommended factors are indicated by the asterisk.



TABLE 33.21 Prefixes and Symbols for Decimal Multiples and Submultiples Factor 18

10 1015 1012 109 106 103 102* 101* 10–1* 10–2* 10–3 10–6 10–9 10–12 10–15 10–18

Prefix

Symbol

exa beta tera giga mega kilo hecto deka deci centi milli micro nano pico femto atto

E P T G M k h da d c m µ n p f a


To convert xenobiotic concentrations to or from SI units: Conversion factor (CF) = 1000/mol. wt.; conversion to SI units: g/ml CF = mol/l; conversion from SI units: mol/l CF = g/ml.

TABLE 33.22 Conversion of Human Hematological Values from Traditional Units into SI Units Constituent Clotting time Prothrombin time Hematocrit (erythrocytes, volume fraction) Hemoglobin Leukocyte count (leukocytes, number concentration) Erythrocyte count (erythrocytes, number concentration) Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH) (Erc-Hemoglobin, amount of substance) Mean corpuscular hemoglobin concentration (MCHC) (Erc-Hemoglobin, substance concentration) Erythrocyte sedimentation rate Platelet count (Blood platelets, number concentration) Reticulocyte count (Erc-Reticulocytes, number fraction)

Traditional Units

SI Units

Minutes Seconds % g/100 ml per mm3 million per mm3 µ3 pg

0.06 1.0 0.01 0.6205 106 106 1.0 0.06205

ks arb. unit 1 mmol/l 109/1 1012/1 fl fmol

%

0.6205

mmol/l

mm/hour mm3 % red cells

1.0 106 0.01

arb.unit 109/1 1

Source: Young, D. S., N. Engl. J. Med., 292, 795, 1975. With permission.


Multiplication Factor


TABLE 33.23 Conversion of Laboratory Values from Traditional Units into SI Units Constituent

Multiplication Factor

SI Units

mm Hg Sigma Bodansky mg/100 ml g/100 ml

1.0 43.06 17.10 17.10 0.2495 1.0 1.0 0.01667 88.40 0.05551 0.01667 0.02586 0.50 0.1333 1.0 0.133 278.4 0.08967 0.3229 10

arb · unit µmol/l µmol/l µmol/l mmol/l mmol/l mmol/l µmol S–1/l µmol/l mmol/l µmol S–1/l mmol/l mmol/l kPa 1 kPa nmol S–1/l nmol S–1/l nmol/l g/l

% total % total % total % total % total m Eq/l m Eq/l Karmen mg/100 ml mg/100 ml

0.01 0.01 0.01 0.01 0.01 1.0 1.0 0.008051 0.3569 0.65948

1 1 1 1 1 mmol/l mmol/l mol S–1/1 mmol/l mmol/l

Traditional Units

Amylase Bilirubin (direct) conjugated total Calcium Carbon dioxide Chloride Creatine phosphokinase (CPK) Creatinine Glucose Lactic dehydrogenase Cholesterol Magnesium PCO 2 pH PO2 Phosphatase, acid Phosphatase, alkaline Phosphorus, inorganic Protein, total Protein, electrophoreses Albumin Globulin, α1 α2 β γ Potassium Sodium Transaminase (SGOT) (aminotransferase) Urea nitrogen Uric acid

Units/l mg/100 ml mg/100 ml mg/100 ml mg/100 ml mEq/1 mEq/1 mU/ml mg/100 ml mg/100 ml mU/ml mg/100 ml mEq/l mm Hg

Source: Young, D. S., N. Engl. J. Med., 292, 795, 1975. With permission.

TABLE 33.24 Calculation of Milliequivalent Weight, Creatinine Clearance, and Surface Area of Children To calculate milliequivalent weight: mEq = mEq =

Gram molecular weight / valence 1000

mg eq wt

Equivalent weight or eq wt =

Chloride Sodium Calcium

Gram molecular weight Valence

35.5 mg = 1 mEq 23 mg = 1 mEq 20 mg = 1 mEq


Commonly Used mEq Weights Magnesium Potassium

12 mg = 1 mEq 39 mg = 1 mEq


TABLE 33.24 (Continued) Calculation of Milliequivalent Weight, Creatinine Clearance, and Surface Area of Children To calculate creatinine clearance (Ccr) from serum creatinine: Male: Ccr =

Weight (kg) × (140 - age) Female: Ccr = 0.85 × calculation for males 72 × serum creatinine (mg / dl)

To calculate ideal body weight (kg): Male = 50 kg + 2.3 kg (each inch > 5ft) Female = 45.5 kg + 2.3 kg (each inch > 5ft) To approximate surface area (ml) of children from weight (kg): Surface Area (m2)

Weight Range (kg) 1–5 6–10 11–20 21–40

(0.05 (0.04 (0.03 (0.02

kg) kg) kg) kg)

+ + + +

0.05 0.10 0.20 0.40

To calculate body surface area (BSA) in adults and children: 1) Dubois method: SA (cm2) wt (kg)0.425 ht (cm)0.725 71.84 SA (m2) = K × 3 wt 2 ( kg) (common K value 0.1 for toddlers, 0.103 for neonates) 2) Simplified method: BSA( m 2 ) =

ht(cm) × wt( kg) 3, 600

TABLE 33.25 Table for Predicting Human Half-Life of Xenobiotics from Rat Half-Life Rat Half-life (Hours)

Lower 95%

90%

0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0.019 0.034 0.048 0.062 0.074 0.087 0.099 0.110 0.122 0.13 0.24 0.34 0.43 0.51 0.60 0.68

0.025 0.045 0.064 0.081 0.098 0.114 0.130 0.146 0.161 0.18 0.31 0.44 0.56 0.68 0.79 0.89


80%

Human Half-Life Estimate (Hours)

Upper 80%

90%

95%

0.034 0.062 0.087 0.111 0.134 0.156 0.178 0.199 0.220 0.24 0.43 0.60 0.77 0.92 1.07 1.22

0.106 0.189 0.264 0.335 0.404 0.469 0.533 0.596 0.657 0.72 1.27 1.78 2.26 2.72 3.17 3.60

0.327 0.574 0.799 1.011 1.213 1.408 1.598 1.782 1.963 2.14 3.78 5.28 6.70 8.05 9.36 10.63

0.451 0.790 1.098 1.387 1.664 1.930 2.189 2.441 2.687 2.93 5.17 7.21 9.14 10.99 12.77 14.51

0.598 1.045 1.450 1.830 2.193 2.543 2.882 3.213 3.536 3.85 6.79 9.47 12.00 14.42 16.76 19.04


TABLE 33.25 (Continued) Table for Predicting Human Half-Life of Xenobiotics from Rat Half-Life Rat Half-life (Hours)

Lower 95%

90%

0.8 0.9 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90 100 200 300 400 500 600 700 800 900 1000 1100

0.76 0.84 0.92 1.63 2.27 2.88 3.46 4.02 4.56 5.08 5.60 6.1 10.7 14.9 18.8 22.6 26.2 29.6 33.0 36.3 39.5 68.9 95.2 119.8 143.2 165.5 187.1 208.1 228.5 248.4 267.9

1.00 1.10 1.20 2.13 2.98 3.78 4.54 5.28 5.99 6.68 7.36 8.0 14.1 19.7 24.9 29.8 34.6 39.2 43.6 48.0 52.3 91.4 126.7 159.7 191.0 221.1 250.1 278.3 305.8 332.7 359.0

80%

Human Half-Life Estimate (Hours)

Upper 80%

90%

95%

1.36 1.50 1.64 2.91 4.07 5.16 6.20 7.21 8.19 9.14 10.06 11.0 19.4 27.0 34.1 41.0 47.5 53.9 60.1 66.1 72.0 126.5 175.7 221.7 265.6 307.7 348.5 388.1 426.8 464.6 501.7

4.02 4.44 4.84 8.60 12.04 15.28 18.39 21.39 24.31 27.15 29.94 32.7 58.1 81.3 103.2 124.1 144.4 164.1 183.3 202.1 220.6 391.9 548.6 696.4 837.9 974.7 1107.6 1237.3 1364.3 1488.9 1611.3

11.88 13.09 14.29 25.40 35.59 45.24 54.49 63.46 72.18 80.71 89.07 97.3 174.0 244.6 311.6 376.1 438.6 499.5 559.2 617.7 675.2 1214.2 1712.7 2186.8 2643.8 3087.5 3520.5 3944.6 4361.2 4771.1 5175.1

16.20 17.86 19.49 34.66 48.58 61.76 74.42 86.69 98.64 110.32 121.77 133.0 238.3 335.6 427.9 516.9 603.3 687.5 770.0 851.1 930.8 1679.5 2374.3 3036.7 3676.1 4297.9 4905.5 5501.3 6086.9 6663.7 7232.7

21.26 23.44 25.57 45.47 63.76 81.09 97.74 113.88 129.61 144.99 160.08 174.9 313.9 442.4 564.7 682.7 797.2 909.1 1018.7 1126.4 1232.4 2230.5 3159.3 4046.6 4904.4 5739.6 6556.7 7358.6 8147.4 8925.0 9692.5

Note: The following examples indicate how this table is used. For a xenobiotic with a rat halflife of 0.8 h, the prediction or best guess of the human half-life is 4.02 h. The table indicates that the actual half-life would fall between 1.0 and 16.2 h with a confidence of 90%. Values falling between those indicated in the table can be linearly interpolated, for example, a rat half-life of 2.7 h gives a human half-life of 11.01 h. From Bachmann, K. M., Pardoe, D., and White, D., Scaling basic toxicokinetic parameters from rat to man, Environ. Health Perspect., 104, 400–407, 1996. With permission.



TABLE 33.26 Table for Predicting Human Volume of Distribution from Rat Volume of Distribution Lower

Upper

Rat volume (l/kg)

95%

90%

80%

Human Volume Estimate (l/kg)

80%

90%

95%

0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 2 3 4 5 6 7 8 9 10 20 30 40 50 60 70 80 90

0.002 0.004 0.006 0.008 0.010 0.012 0.013 0.015 0.017 0.019 0.035 0.051 0.066 0.081 0.096 0.111 0.125 0.139 0.15 0.29 0.41 0.53 0.65 0.77 0.88 0.99 1.11 1.21 2.25 3.22 4.15 5.06 5.94 6.80 7.65 8.48

0.003 0.005 0.008 0.010 0.013 0.015 0.017 0.019 0.022 0.024 0.045 0.065 0.085 0.104 0.123 0.142 0.160 0.178 0.20 0.37 0.53 0.69 0.84 0.99 1.14 1.28 1.42 1.56 2.90 4.16 5.37 6.54 7.69 8.81 9.91 11.00

0.004 0.007 0.011 0.014 0.017 0.020 0.023 0.026 0.029 0.032 0.060 0.087 0.113 0.139 0.164 0.189 0.213 0.237 0.26 0.49 0.70 0.91 1.18 1.32 1.51 1.71 1.90 2.08 3.88 5.57 7.20 8.78 10.33 11.84 13.34 14.81

0.011 0.020 0.029 0.038 0.046 0.055 0.063 0.071 0.079 0.087 0.164 0.236 0.307 0.376 0.443 0.510 0.575 0.640 0.70 1.32 1.91 2.48 3.03 3.58 4.11 4.64 5.17 5.69 10.66 15.40 19.99 24.48 28.88 33.22 37.49 41.72

0.031 0.057 0.082 0.105 0.128 0.151 0.174 0.196 0.217 0.239 0.445 0.641 0.831 1.016 1.198 1.377 1.554 1.729 1.90 3.57 5.16 6.71 8.23 9.72 11.19 12.65 14.09 15.52 29.32 42.60 55.54 68.24 80.77 93.14 105.39 117.54

0.041 0.076 0.109 0.141 0.172 0.202 0.232 0.261 0.290 0.319 0.593 0.853 1.105 1.352 1.593 1.832 2.067 2.299 2.53 4.75 6.87 8.93 10.96 12.95 14.92 16.86 18.79 20.70 39.20 57.04 74.46 91.59 108.50 125.22 141.79 158.23

0.054 0.099 0.141 0.182 0.222 0.261 0.299 0.337 0.374 0.411 0.762 1.096 1.419 1.735 2.045 2.350 2.652 2.950 3.25 6.09 8.82 11.47 14.08 16.64 19.17 21.68 24.17 26.63 50.54 73.63 96.23 118.48 140.45 162.21 183.79 205.22

Note: See note of previous table for examples of how this table is used. From Bachmann, K. M., Pardoe, D., and White, D., Scaling basic toxicokinetic parameters from rat to man, Environ. Health Perspect., 104, 400–407, 1996. With permission.



TABLE 33.27 Approximate Metric and Apothecary Weight Equivalents Metric 1 gram (g) 0.6 g (600 mg) 0.5 g (500 mg) 0.3 g (300 mg) 0.2 g (200 mg) 0. 1 g (100 mg) 0.06 g (60 mg)

Apothecary = = = = = = =

15 grains 10 grains 712 grains 5 grains 3 grains 112 grains 1 grain

Metric

Apothecary

0.05g (50 mg) 0.03 g (30 mg) 0.015 g (15 mg) 0.001 g (1 mg) 0.6 mg 0.5 mg 0.4 mg

= 3/4 grain = 1/2 grain = 1/4 grain = 1/80 grain = 1/100 grain = 1/120 grain = 1/150 grain

Approximate household, apothecary, and metric volume equivalents Household 1 1 2 1 1 1 1

Apothecary

teaspoon (t or tsp) tablespoon (T or tbs) tablespoons measuring cupful pint (pt) quart (qt) gallon (gal)

= = = = = = =

1 fluidram (f3) 1 fluidounce (f ) 2 3 1 fluidounce 8 fluidounces 16 fluidounces 32 fluidounces 128 fluidounces

Metric = 4 or 5 mla = 15 ml = 30 ml = 240 ml = 473 ml = 946 ml = 3,785 ml

a

1 ml = 1 cubic centimeter (cc); however, ml is the preferred measurement term today. See end of section for source.

TABLE 33.28 Conversion Factors — Metric to English To Obtain

Multiply

By

Inches Feet Yards Miles Ounces Pounds Gallons (U.S. Liquid) Fluid ounces Square inches Square feet Square yards Cubic inches Cubic feet Cubic yards

Centimeters Meters Meters Kilometers Grams Kilograms Liters Milliliters (cc) Square centimeters Square meters Square meters Milliliters (cc) Cubic meters Cubic meters

0.3937007874 3.280839895 1.093613298 0.6213711922 3.527396195 10–2 2.204622622 0.2641720524 3.381402270 10–2 0.1550003100 10.76391042 1.195990046 6.102374409 10–2 35.31466672 1.307950619




TABLE 33.29 Conversion Factors — English to Metrica To Obtain

Multiply

By

Microns Centimeters Meters Meters Kilometers Grains Kilograms Liters Millimeters (cc) Square centimeters Square meters Square meters Milliliters (cc) Cubic meters Cubic meters

Mils Inches Feet Yards Miles Ounces Pounds Gallons (U.S. Liquid) Fluid ounces Square inches Square feet Square yards Cubic inches Cubic feet Cubic yards

25.4 2.54 0.3048 0.9144 1.609344 28.34952313 0.45359237 3.785411784 29.57352956 6.4516 0.09290304 0.83612736 16.387064 2.831684659 10–2 0.764554858

a

Boldface numbers are exact; others are given to ten significant figures where so indicated by the multiplier factor. See end of section for source.

TABLE 33.30 Conversion Factors — Generala To Obtain

Multiply

By

Atmospheres Atmospheres Atmospheres Pascal Pascal Pascal BTU BTU Cubic feet Degree (angle) Ergs Feet Feet of water @ 40C Foot-pounds Foot-pounds Foot-pounds per min Horsepower Inches of mercury @ 0C Joules Joules Kilowatts Kilowatts

Feet of water @ 4C Inches of mercury @ 0C Pounds per square inch Millibar Millimeter mercury 0C Pound per square inch Foot-pounds Joules Cords Radians Foot-pounds Miles Atmospheres Horsepower-hours Kilowatt-hours Horsepower Foot-pounds per sec Pounds per square inch BTU Foot-pounds BTU per min Foot-pounds per min

2.950 10–2 3.342 10–2 6.804 10–2 1 102 1.33 102 6.894 103 1.285 10–3 9.480 10–4 128 57.2958 1.356 107 5,280 33.90 1.98 106 2.655 106 3.3 104 1.818 10–3 2.036 1.054.8 1.35582 1.758 10–2 2.26 10–5



TABLE 33.30 (Continued) Conversion Factors — Generala To Obtain

Multiply

Kilowatts Knots Miles Nautical miles Radians Square feet Watts

By

Horsepower Miles per hour Feet Miles Degrees Acres BTU per min

0.745712 0.86897624 1.894 10–4 0.86897624 1.745 10–2 43,560 17.5796

a Boldface numbers are exact; others are given to ten significant figures where so indicated by the multiplier factor. See end of section for source.

See www.onlineconversion.com for other conversions.

TABLE 33.31 Temperature Factorsa F Fahrenheit temperature C Celsius temperature Fahrenheit temperature

From Celsius

Fahrenheit

= = = = =

9/5 (C) + 32 1.8 (temperature in kelvins) – 459.67 5/9 [(F) – 32)] temperature in kelvins – 273.15 1.8 (Celsius temperature) + 32

Conversion of Temperatures To Fahrenheit Kelvin Rankine Celsius Kelvin

Kelvin Rankine

a

Rankine Celsius Rankine Farenheit Kelvin

tF = (tc 1.8) + 32 TK = tc + 273.15 TR = (tc+ 273.15) 18 TY =

t F − 32 1.8

t F − 32 + 273.15 1.8 TR = tf + 459.67 tc = TK – 273.15 TR = TK 1.8 tF = TR – 459.67 Tk =

TK =

tR 1.8

Boldface numbers are exact; others are given to ten significant figures where so indicated by the multiplier factor. See end of section for source.



TABLE 33.32 Temperature Conversions F

C

F

C

F

C

F

C

F

C

–10 –5 0 +5 10 15 20 25 30 32

–23.3 –20.5 –17.8 –15.0 –12.2 –9.4 –6.6 –3.9 –1.1 0

35 40 45 50 55 60 65 70 75 80

+1.6 4.4 7.2 10.0 12.8 15.5 18.3 21.1 23.9 26.6

85 90 95 100 105 110 115 120 125 130

29.4 32.2 35.0 37.8 40.5 43.3 46.1 48.9 51.6 54.4

135 140 145 150 155 160 165 170 175 180

57.2 60.0 62.8 65.5 68.3 71.1 73.9 76.6 79.4 82.2

185 190 195 200 205 210 212

85.0 87.8 90.5 93.3 96.1 98.9 100

TABLE 33.33 Table of Equivalents Kg g mg µg 1 lb oz acre hectare

= = = = = = = = =

1,000 g, 1 million mg, 2.2 lbs 1,000 mg, 1 million µg, approx. 0.035 oz 1,000 µg, 1 million ng 1,000 ng approx. 1 quart, approx. 33 oz 16 oz, 454.5 g, 0.45 kg 28.4 g 4,047 m2 2.5 acres

When referring to the concentration of a chemical in food or other medium: mg/kg = ppm, µg/g mg/l = ppm = 0.0001% µg/kg = ppb, ng/g ng/kg = ppt ppm = mg/kg, g/g ppb = µg/kg, ng/g ppt = ng/kg See end of section for source.

HOW MANY MOLECULES? To calculate the number of molecules in any quantity of a chemical, one needs to know the weight of a mole of the chemical and the number of molecules in a mole. A mole of any chemical is its molecular weight expressed in grams. Avogadro’s number, 6 1023, is the number of molecules in a mole. The number of molecules in 10 µg of benzpyrene is calculated as follows: the molecular weight of benzpyrene is 252; therefore, a mole would weigh 252 g. 252 g is equal to 2.52 108 µg. The number of molecules in a µg is obtained by dividing the number of molecules in a mole by the number of µg in a mole. For benzpyrene, 6 1023 divided by 2.52 108 equals 2.4 1015. The number of molecules in 10 µg benzpyrene would be 10 times as much, or 2.4 1016.



TABLE 33.34 Overview of Units Used to Express the Concentration of a Substance Compartment Air (gases)

Water

Soil

Units µg m–3 µmol m–3 µl m–3 µ1–1 µg 1–1 mg 1–1 µmol 1–1 µg kg–1 mg kg–1 µg g–1 µmol g–1

Abbreviation

Conversiona 1 µg m–3 is V/A µ1 m–3 1 µmol m–3 is V µ1 m–3

ppbv ppmv ppb ppm µM ppb ppm ppm

1 µmol 1–1 is A µg 1–1

1 µmol g–1 is A µg g–1

a A = molecular weight of the substance; V = molar volume at current pressure and temperature.


TABLE 33.35 Conversions of Nucleic Acids and Proteins I.

II.

III.

IV.

V.

Weight conversions 1 mg = 10–6 g 1 ng = 10–9 g 1 pg = 10–12 g 1 fg = 10–15 g Spectrophotometric conversions 1A260 unit of double-stranded DNA = 50 µg/ml 1A260 unit of single-stranded DNA = 33 µg/ml 1A260 unit of single-stranded RNA = 40 µg/ml DNA molar conversions 1 µg of 1,000 bp DNA = 1.52 pmol (3.03 pmol of ends) 1 µg of pBR322 DNA = 0.36 pmol DNA 1 pmol of 1,000 bp DNA = 0.66 µg 1 pmol of pBR322 DNA = 2.8 µg Protein molar conversions 100 pmol of 100,000 MW protein = 10 µg 100 pmol of 50,000 MW protein = 5 µg 100 pmol of 10,000 MW protein = 1 µg Protein/DNA conversions 1 kb of DNA = 333 amino acids of coding capacity = 3.7 104 MW 10,000 MW protein = 270 bp DNA 30,000 MW protein = 810 bp DNA 50,000 MW protein = 1.35 kb DNA 100,000 MW protein = 2.7 kb DNA



TABLE 33.36 Conversion of Radioactivity Units from mCi and µCi to MBq mCi

MBq

mCl

200 150 100 90 80 70 60 50 40 30 20

7,400 5,550 3,700 3,330 2,960 2,590 2,220 1,850 1,480 1,110 740

10 9 8 7 6 5 4 3 2 1

MBq 370 333 296 259 222 185 148 111 74.0 37.0

µCi

MBq

Ci

MBq

1,000 900 800 700 600 500 400 300 200 100 90

37.0 33.3 29.6 25.9 22.2 18.5 14.8 11.1 7.4 3.7 3.33

80 70 60 50 40 30 20 10 5 2 1

2.96 2.59 2.22 1.85 1.48 1.11 0.74 0.37 0.185 0.074 0.037


TABLE 33.37 Conversion Formula for Concentration of Solutions A = Weight per cent of solute B = Molecular weight of solvent E = Molecular weight of solute F = Grams of solute per liter of solution Concentration of Solute SOUGHT

Concentration of Solute — GIVEN A

N

G

M

F

—

100 N × E N × E + (1 − N )B

100G × E 1, 000 + G × E

M×E 10 R

F 10 R

A E A 100 − A + E B

—

B×G B × G + 1, 000

B× M M(B − E ) + 1, 000 R

B× F F(B − E ) + 1, 000 R × E

1, 000 A E(100 − A)

1, 000 N B− N×B

—

1, 000 M 1, 000 R − (M × E)

1, 000 F E(1, 000 R − F )

10R × A E

1, 000 R × N N × E + (1 − N )B

1, 000 R × G 1, 000 + E × G

—

F E

10AR

1, 000 R × N × E N × E + (1 − N )B

1, 000 R × G × E 1, 000 + G × ES

A N

G M F

G = Molality M = Molarity N = Mole fraction R = Density of solution grams per cc

M×E

—

Source: Handbook of Chemistry and Physics, Lide, D. R., Ed., CRC Press, Boca Raton, FL, 1990. With permission.



C. PHYSICAL CONSTANTS TABLE 33.38 General Physical Constants Equatorial radius of the earth = 6378.388 km = 3963.34 miles (statute). Polar radius of the earth = 6356.912 km = 3949.99 miles (statute). 1 degree of latitude at 40 = 69 miles. 1 international nautical mile = 1.15078 miles (statute) = 1852 m = 6076.115 ft. Mean density of the earth = 5.522 g/cm3 = 344.7 lb/ft3 Constant of gravitation (6.673 ± 0.003) 10–8 cm3 gm–1s–2. Acceleration due to gravity at sea level, latitude 45 = 980.6194 cm/s2 = 32.1726 ft/s2. Length of seconds pendulum at sea level, latitude 45 = 99.3575 cm = 39.1171 in. 1 knot (international) = 101.269 ft/min = 1.6878 ft/s = 1.1508 miles (statute)/h. 1 micron = 10–4 cm. 1 ångstrom = 10–5 cm. Mass of hydrogen atom = (1.67339 ± 0.0031) 10–24 g. Density of mercury at 0C = 13.5955 g/ml. Density of water at 3.98C = 1.000000 g/ml. Density, maximum, of water, at 3.98C = 0.999973 g/cm3. Density of dry air at 0C. 760 mm = 1.2929 g/l. Velocity of sound in dry air at 0C = 331.36 m/s – 1087.1 ft/s. Velocity of light in vacuum = (2.997925 ± 0.000002) 1010 cm/s. Heat of fusion of water 0C = 79.71 cal/g. Heat of vaporization of water 100C = 539.55 cal/g. Electrochemical equivalent of silver = 0.001118 g/s international amp. Absolute wavelength of red cadmium light in air at 15C. 760 mm pressure = 6438.4696 Å. Wavelength of orange-red line of krypton 86 = 6057.802 Å. Source: Handbook of Electrical Engineering, Dorf, R. C., Ed., CRC Press, Boca Raton, FL, 1993. With permission.

TABLE 33.39 Summary of the 1986 Recommended Values of the Fundamental Physical Constants

Quantity

Symbol

Speed of light in vacuum Permeability of vacuum

c µo

Permittivity of vacuum

εo

Newtonian constant of gravitation Planck constant h/2 Elementary charge Magnetic flux quantum. h/2e Electron mass Proton mass Proton-electron mass ratio Fine-structure constant, µ0ce2/2h


G h h e o me mp mp /me α

Value 299,792,458 4 10–7 = 12.566 370614… 1/ oc2 = 8.854 187 817… 6.672 59(85) 6.626 0755(40) 1.054 572 66(63) 1.602 177 33(49) 2.067 834 61(61) 9.109 3897(54) 1.672 6231(10) 1836.152701(37) 7.297 353 08(33)

Units –1

Relative Uncertainty (ppm)

ms N A–2 10–7 N A–2

(exact)

10–12 F m–1 10–11 m–3 kg–1 s–2 10–3 J s 10–3J s 10–19 C 10–15 Wb 10–31 kg 10–27 kg

(exact) 128 0.60 0.60 0.30 0.30 0.59 0.59 0.020 0.045

10–3

(exact)


TABLE 33.39 (Continued) Summary of the 1986 Recommended Values of the Fundamental Physical Constants Relative Uncertainty (ppm)

Quantity

Symbol

Value

Inverse fine-structure constant Rydberg constant, mec2/2h Avogadro constant Faraday constant, NAe Molar gas constant Boltzmann constant, R/NA Stefan-Boltzmann constant, (r9/60)k9/h7c7 Non-SI units used with SI Electronvolt. (e/C)J = (e)J (Unified) atomic mass unit. 1 u = mu = 1/12m(12C)

α Rx NAL F R k σ

137 035 9895(61) 10 973 731.534(13) 6.022 1367(36) 96 485.309(29) 8.314 510(70) 1.380 658(12) 5.670 51(19)

m–1 1023mol–1 C mol–1 J mol–1 K–1 10–23 J K–1 10–8 W m–2 K–4

eV u

1.602 17733(40) 1.660 5402(10)

10–19 J 10–27 kg

–1

Units

0.045 0.0012 0.59 0.30 8.4 8.5 34 0.30 0.59

Note: An abbreviated list of the fundamental constants of physics and chemistry based on a least-squares adjustment with 17 degrees of freedom. The digits in parentheses are the one-standard-deviation uncertainty in the last digits of the given value. Since the uncertainties of many entries are correlated, the full covariance matrix must be used in evaluating the uncertainties of quantities computed from them. Source: Handbook of Electrical Engineering, Dorf, R. C., Ed., CRC Press, Boca Raton, FL, 1993. With permission.

TABLE 33.40 Standard Atomic Weights (1987) (Scaled to Ar (12C) = 12) The atomic weights of many elements are not invariant but depend on the origin and treatment of the material. The footnotes to this table elaborate the types of variation to be expected for individual elements. The values of Ar (E) and uncertainty Ur (E) given here apply to elements as they exist naturally on earth. Name Actinium* Aluminium Americium* Antimony (Stibium) Argon Arsenic Astatine* Barium Berkelium* Beryllium Bismuth Boron Bromine Cadmium Caesium Calcium


Symbol

No.

Ac Al Am Sb Ar As At Ba Bk Be Bi B Br Cd Cs Ca

89 13 95 51 18 33 85 56 97 4 83 5 35 48 55 20

Atomic Weight

Footnotes A

26.981539(5) A 121.75(3) 39.948(1) 74.92159(2)

g

r A

137.327(7) A 9.012182(3) 208.98037(3) 10.811(5) 79.904(l) 112.411(8) 132.90543(5) 40.078(4)

g g g

m

r


TABLE 33.40 (Continued) Standard Atomic Weights (1987) (Scaled to Ar (12C) = 12) The atomic weights of many elements are not invariant but depend on the origin and treatment of the material. The footnotes to this table elaborate the types of variation to be expected for individual elements. The values of Ar (E) and uncertainty Ur (E) given here apply to elements as they exist naturally on earth. Name Californium* Carbon Cerium Chlorine Chromium Cobalt Copper Curium* Dysprosium Einsteinium* Erbium Europium Fermium* Fluorine Francium* Gadolinium Gallium Germanium Gold Hafnium Helium Holmium Hydrogen Indium Iodine Iridium Iron Krypton Lanthanum Lawrencium* Lead Lithium Lutetium Magnesium Manganese Mendelevium* Mercury Molybdenum Neodymium Neon Neptunium* Nickel Niobium


Symbol

No.

Cf C Ce Cl Cr Co Cu Cm Dy Es Er Eu Fm F Fr Gd Ga Ge Au Hf He Ho H In I Ir Fe Kr La Lr Pb Li Lu Mg Mn Md Hg Mo Nd Ne Np Ni Nb

98 6 58 17 24 27 29 96 66 99 68 63 100 9 87 64 31 32 79 72 2 67 1 49 53 77 26 36 57 103 82 3 71 12 25 101 80 42 60 10 93 28 41

Atomic Weight

Footnotes A

12.011(1) 140.115 (4) 35.4527(9) 51.9961(6) 58.93320(1) 63.546(3)

r g

r A

162.50(3)

g

167.26(3) 151.965(9)

g g

A

A 18.9984032(9) A 157.25(3) 69.723(1) 72.61(2) 196.96654(3) 178.49(2) 4.002602(2) 164.93032(3) 1.00794(7) 114.82(1) 126.90447(3) 192.22(3) 55.847(3) 83.80(l) 138.9055(2)

g

g

r

g

m

g g

m

r

A 207.2(1) 6.941(2) 174.967(1) 24.3050(6) 54.93805(l)

g g g

m

r r

A 200.59(3) 95.94(l) 144.24(3) 20.1797(6)

g g

m A

58.69(1) 92.90638(2)


TABLE 33.40 (Continued) Standard Atomic Weights (1987) (Scaled to Ar (12C) = 12) The atomic weights of many elements are not invariant but depend on the origin and treatment of the material. The footnotes to this table elaborate the types of variation to be expected for individual elements. The values of Ar (E) and uncertainty Ur (E) given here apply to elements as they exist naturally on earth. Name Nitrogen Nobelium* Osmium Oxygen Palladium Phosphorus Platinum Plutonium* Polonium* Potassium (Kalium) Praseodymium Promethium* Protactinium* Radium* Radon* Rhenium Rhodium Rubidium Ruthenium Samarium Scandium Selenium Silicon Silver Sodium (Natrium) Strontium Sulfur Tantalum Technetium* Tellurium Terbium Thallium Thorium* Thulium Tin Titanium Tungsten (Wolfram) Unnilquadium Unnilpentium Unnihexium Unnilseptium Uranium* Vanadium


Symbol

No.

Atomic Weight

Footnotes

N No Os O Pd P Pt Pu Po K Pr Pm Pa Ra Rn Re Rh Rb Ru Sm Sc Se Si Ag Na Sr S Ta Tc Te Tb Tl Th Tm Sn Ti W Unq Unp Unh Uns U V

7 102 76 8 46 15 78 94 84 19 59 61 91 88 86 75 45 37 44 62 21 34 14 47 11 38 16 73 43 52 65 81 90 69 50 22 74 104 105 106 107 92 23

14.00674(7)

g

190.2(l) 15.9994(3) 105.42(1) 30.973762(4) 195.08(3)

g g g

r A r

A A 39.0983(l) 140.90765(3) A A A A 186.207(l) 102.90550(3) 85.4678(3) 101.07(2) 150.36(3) 44.955910(9) 78.96(3) 28.0855(3) 107.8682(2) 22.989768(6) 87.62(l) 32.066(6) 180.9479(l)

g g g

r g g

r r A

127.60(3) 158.92534(3) 204.3833(2) 232.0381(1) 168.93421(3) 118.710(7) 47.88(3) 183.85(3)

238.0289(1) 50.9415(1)

g

g

Z

g

g

m

A A A A Z


TABLE 33.40 (Continued) Standard Atomic Weights (1987) (Scaled to Ar (12C) = 12) The atomic weights of many elements are not invariant but depend on the origin and treatment of the material. The footnotes to this table elaborate the types of variation to be expected for individual elements. The values of Ar (E) and uncertainty Ur (E) given here apply to elements as they exist naturally on earth. Name Xenon Ytterbium Yttrium Zinc Zirconium

Symbol

No.

Atomic Weight

Xe Yb Y Zn Zr

54 70 39 30 40

131.29(2) 173.04(3) 88.90585(2) 65.39(2) 91.224(2)

Footnotes g g

m

g

g = Geological specimens are known in which the element has an isotopic composition outside the limits for normal material. The difference between the atomic weight of the element in such specimens and that given in the table may exceed the implied uncertainty. m = Modified isotopic compositions may be found in commercially available material because it has been subjected to an undisclosed or inadvertent isotopic separation. Substantial deviations in atomic weight of the element from that given in the table can occur. r = Range in isotopic composition of normal terrestrial material prevents a more precise Ar(E) being given: the tabulated Ar(E) value should be applicable to any normal material. A = Radioactive element that lacks a characteristic terrestrial isotopic composition. Z = An element, without stable nuclide(s), exhibiting a range of characteristic terrestrial compositions of long-lived radionuclide(s) such that a meaningful atomic weight can be given. * Element has no stable nuclides. See end of section for source.

D. STATISTICS Statistical analysis is an integral part of a toxicology study. Advances in computer technology have allowed for more sophisticated statistical analyses to be conducted more easily and quickly on data generated in toxicology studies than would ever have been thought possible just a few decades ago. Figure 33.1 presents a decision tree for choosing the proper statistical tests for analysis of a variety of data. Although other equally valid tests may be used in place of those indicated, the scheme that is shown is a good representation of the statistical approach commonly used for assessing toxicological data. Refer to the referenced texts for a detailed discussion of the various statistical tests and their uses. The reader should be aware of several important limitations associated with the use of statistics in toxicology: 1) statistics cannot make “poor” data “better”; 2) statistical significance may not imply biological significance; 3) an effect that may have biological significance may not be statistically significant; 4) the lack of statistical significance does not prove safety. The importance and relevance of any effect observed in a study must be assessed within the limitations imposed by the study design and the species being studied.


Yes

Assumed Parametric Data

No

Continuous data such as body weights, blood-cell counts, etc. Comparison of three or more groups

Comparison of two groups

Bartlett's homogeneity of variance

Not Sig. (-)

Sig. (+)

(Homogeneous)(Heterogeneous)

SCATTER* GRAM

Not Normal (+)

Normal (-)

Not Sig. (-)

Comparison of two groups or if the variance in one or more groups=0 (no variation within group)

F

Analysis of Variance

Sig. (+)

Test

Not Sig. (-)

N1=N2

If answer to either is no

Data not significant. No more tests necessary.

Comparison of three or more groups all with some variation within group

Sig. (+)

(Homogen) Intracomparison only of groups vs. controls? Group sizes approximately equal?

Non-Parametric Data Includes such data as percentage values, ranks, etc.

(Heterogen) N1=N2

Student's t-test df=(N2 + N2) -2

Wilcoxon Rank Sum Test (2 groups)

KruskalWallis Non-parametric Anova Sig. (+)

Categorical (Quantal) Data Frequency data such as mortalities, pathology findings, etc. 3 or more groups

Fisher's Sig. (+) R x C Chi Exact Square Test

Not Sig. (-)

If answer to both is yes

Duncan's Multiple Range Test

Dunnett's

Student's t-test df=N1 -1

Cochran t-test

Distribution Free Multiple Comparisons

Data not significant No more tests necessary

Not Sig. (-)

* If plot does not clearly demonstrate lack of normality exact tests may be employed. - If continuous data, Kolmogorov Smirnov test. - If discontinuous data, Chi-Square Goodness-of-Fit test may be used.

FIGURE 33.1 Decision tree for selecting hypothesis-testing procedures. Dunn’s summed rank test is generally performed after the Kruskal-Wallis ANOVA for multiple comparisons of nonparametric data. For trend analysis of parametric data, Jonckheere test for monotonic trend can be used. From Gad, S. and Weil, C.S., in Statistics and Experimental Design for Toxicologists, Telford Press, Caldwell, NJ, 1986. With permission.



Visually examine data Do they appear normally distributed?


TABLE 33.41 Transformation of Percentages into Logits Percentage

0

1

2

3

4

5

6

7

8

9

50 60 70 80 90 99

0 0.41 0.85 1.38 2.20 4.60

0.04 0.45 0.90 1.45 2.31 4.70

0.08 0.49 0.94 1.52 2.44 4.82

0.12 0.53 0.99 1.59 2.59 4.95

0.16 0.58 1.05 1.66 2.75 5.11

0.20 0.62 1.10 1.73 2.94 5.29

0.24 0.66 1.15 1.82 3.18 5.52

0.28 0.71 1.21 1.90 3.48 5.81

0.32 0.75 1.27 1.99 3.89 6.21

0.36 0.80 1.32 2.09 4.60 6.91


TABLE 33.42 Transformation of Percentages into Probits Percentage

0

1

2

3

4

5

6

7

8

9

0 10 20 30 40 50 60 70 80 90 99

[–] 3.72 4.16 4.48 4.75 5.00 5.25 5.52 5.84 6.28 7.33

2.67 3.77 4.19 4.50 4.77 5.03 5.28 5.55 5.88 6.34 7.37

2.95 3.82 4.23 4.53 4.80 5.05 5.31 5.58 5.92 6.41 7.41

3.12 3.87 4.26 4.56 4.82 5.08 5.33 5.61 5.95 6.48 7.46

3.25 3.92 4.29 4.59 4.85 5.10 5.36 5.64 5.99 6.55 7.51

3.36 3.96 4.33 4.61 4.87 5.13 5.39 5.67 6.04 6.64 7.58

3.45 4.01 4.36 4.64 4.90 5.15 5.41 5.71 6.08 6.75 7.65

3.52 4.05 4.39 4.67 4.92 5.18 5.44 5.74 6.13 6.88 7.75

3.59 4.08 4.42 4.69 4.95 5.20 5.47 5.77 6.18 7.05 7.88

3.66 4.12 4.45 4.72 4.97 5.23 5.50 5.81 6.23 7.33 8.07




TABLE 33.43 Areas under the Standard Normal Curve

0

_ z

z

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0

0.0000 0.0398 0.0793 0.1179 0.1554 0.1915 0.2257 0.2580 0.2881 0.3159 0.3413 0.3643 0.3849 0.4032 0.4192 0.4332 0.4452 0.4554 0.4641 0.4713 0.4772 0.4821 0.4861 0.4893 0.4918 0.4938 0.4953 0.4965 0.4974 0.4981 0.4987

0.0040 0.0438 0.0832 0.1217 0.1591 0.1950 0.2291 0.2611 0.2910 0.3186 0.3438 0.3665 0.3869 0.4049 0.4207 0.4345 0.4463 0.4564 0.4649 0.4719 0.4778 0.4826 0.4864 0.4896 0.4920 0.4940 0.4955 0.4966 0.4975 0.4982 0.4987

0.0080 0.0478 0.0871 0.1255 0.1628 0.1985 0.2324 0.2642 0.2939 0.3212 0.3461 0.3686 0.3888 0.4066 0.4222 0.4357 0.4474 0.4573 0.4656 0.4726 0.4783 0.4830 0.4868 0.4898 0.4922 0.4941 0.4956 0.4967 0.4976 0.4982 0.4987

0.0120 0.0517 0.0910 0.1293 0.1664 0.2019 0.2357 0.2673 0.2967 0.3238 0.3485 0.3708 0.3907 0.4082 0.4236 0.4370 0.4484 0.4582 0.4664 0.4732 0.4788 0.4834 0.4871 0.4901 0.4925 0.4943 0.4957 0.4968 0.4977 0.4983 0.4988

0.0160 0.0557 0.0948 0.1331 0.1700 0.2054 0.2389 0.2704 0.2995 0.3264 0.3508 0.3729 0.3925 0.4099 0.4251 0.4382 0.4495 0.4591 0.4671 -0.4738 0.4793 0.4838 0.4875 0.4904 0.4927 0.4945 0.4959 0.4969 0.4977 0.4984 0.4988

0.0199 0.0596 0.0987 0.1368 0.1736 0.2088 0.2422 0.2734 0.3023 0.3289 0.3531 0.3749 0.3944 0.4115 0.4265 0.4394 0.4505 0.4599 0.4678 0.4744 0.4798 0.4842 0.4878 0.4906 0.4929 0.4946 0.4960 0.4970 0.4978 0.4984 0.4989

0.0239 0.0636 0.1026 0.1406 0.1772 0.2123 0.2454 0.2764 0.3051 0.3315 0.3554 0.3770 0.3962 0.4131 0.4279 0.4406 0.4515 0.4608 0.4686 0.4750 0.4803 0.4846 0.4881 0.4909 0.4931 0.4948 0.4961 0.4971 0.4979 0.4985 0.4989

0.0279 0.0675 0.1064 0.1443 0.1808 0.2157 0.2486 0.2794 0.3078 0.3340 0.3577 0.3790 0.3980 0.4147 0.4292 0.4418 0.4525 0.4616 0.4693 0.4756 0.4808 0.4850 0.4884 0.4911 0.4932 0.4949 0.4962 0.4972 0.4979 0.4985 0.4989

0.0319 0.0714 0.1103 0.1480 0.1844 0.2190 0.2517 0.2823 0.3106 0.3365 0.3599 0.3810 0.3997 0.4162 0.4306 0.4429 0.4535 0.4625 0.4699 0.4761 0.4812 0.4854 0.4887 0.4913 0.4934 0.4951 0.4963 0.4973 0.4980 0.4986 0.4990

0.0359 0.0753 0.1141 0.1517 0.1879 0.2224 0.2549 0.2852 0.3133 0.3389 0.3621 0.3830 0.4015 0.4177 0.4319 0.4441 0.4545 0.4633 0.4706 0.4767 0.4817 0.4857 0.4890 0.4916 0.4936 0.4952 0.4964 0.4974 0.4981 0.4986 0.4990



Each number in this table represents the probability of obtaining at least X successes, or the area under the histogram to the right of and including the rectangle whose center is at X. m X=0 X=1 X=2 X=3 X=4 X=5 X=6 X=7 X=8 X =9 X =10 X =11 X =12 X =13 X = 14 .10 .20 .30 .40 .50 .60 .70 .80 .90 1.00 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0

1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000

.095 .181 .259 .330 .393 .451 .503 .551 .593 .632 .667 .699 .727 .753 .777 .798 .817 .835 .850 .865 .889 .909 .926 .939 .950 .959 .967 .973 .978 .982 .985 .988 .990 .992 .993

.005 .018 .037 .062 .090 .122 .156 .191 .228 .264 .301 .337 .373 .408 .442 .475 .507 .537 .566 .594 .645 .692 .733 .769 .801 .829 .853 .874 .893 .908 .922 .934 .944 .952 .960



.001 .004 .008 .014 .023 .034 .047 .063 .080 .100 .120 .143 .167 .191 .217 .243 .269 .296 .323 .377 .430 .482 .531 .577 .620 .660 .697 .731 .762 .790 .815 .837 .857 .875

.001 .002 .003 .006 .009 .013 .019 .026 .034 .043 .054 .066 .079 .093 .109 .125 .143 .181 .221 .264 .308 .353 .397 .442 .485 .527 .567 .605 .641 .674 .706 .735

.001 .001 .002 .004 .005 .008 .011 .014 .019 .024 .030 .036 .044 .053 .072 .096 .123 .152 .185 .219 .256 .294 .332 .371 .410 .449 .487 .524 .560

.001 .001 .002 .002 .003 .004 .006 .008 .010 .013 .017 .025 .036 .049 .065 .084 .105 .129 .156 .184 .215 .247 .280 .314 .349 .384

x _ .001 .001 .001 .002 .003 .003 .005 .007 .012 .017 .024 .034 .045 .058 .073 .091 .111 .133 .156 .182 .209 .238

.001 .001 .001 .002 .003 .005 .008 .012 .017 .023 .031 .040 .051 .064 .079 .095 .113 .133

.001 .001 .002 .004 .006 .008 .012 .016 .021 .028 .036 .045 .056 .068

.001 .001 .002 .003 .004 .006 .008 .011 .015 .020 .025 .032

.001 .001 .002 .003 .004 .006 .008 .010 .014

.001 .001 .002 .003 .004 .005

.001 .001 .001 .002

.001


TABLE 33.44 Poisson Distribution


TABLE 33.45 t-Distribution

-t_

_t

0

Deg. freedom. f

90% (P = .1)

95% (P = .05)

99% (P = .01)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 inf.

6.314 2.920 2.353 2.132 2.015 1.943 1.895 1.860 1.833 1.812 1.796 1.782 1.771 1.761 1.753 1.746 1.740 1.734 1.729 1.725 1.721 1.717 1.714 1.711 1.708 1.706 1.703 1.701 1.699 1.645

12.706 4.303 3.182 2.776 2.571 2.447 2.365 2.306 2.262 2.228 2.201 2.179 2.160 2.145 2.131 2.120 2.110 2.101 2.093 2.086 2.080 2.074 2.069 2.064 2.060 2.056 2.052 2.048 2.045 1.960

63.657 9.925 5.841 4.604 4.032 3.707 3.499 3.355 3.250 3.169 3.106 3.055 3.012 2.977 2.947 2.921 2.898 2.878 2.861 2.845 2.831 2.819 2.807 2.797 2.787 2.779 2.771 2.763 2.756 2.576




TABLE 33.46 χ2 Distribution

x2

0 v

0.05

0.025

0.01

0.005

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

3.841 5.991 7.815 9.488 11.070 12.592 14.067 15.507 16.919 18.307 19.675 21.026 22.362 23.685 24.996 26.296 27.587 28.869 30.144 31.410 32.671 33.924 35.172 36.415 37.652 38.885 40.113 41.337 42.557 43.773

5.024 7.378 9.348 11.143 12.832 14.449 16.013 17.535 19.023 20.483 21.920 23.337 24.736 26.119 27.488 28.845 30.191 31.526 32.852 34.170 35.479 36.781 38.076 39.364 40.646 41.923 43.194 44.461 45.722 46.979

6.635 9.210 11.345 13.277 15.086 16.812 18.475 20.090 21.666 23.209 24.725 26.217 27.688 29.141 30.578 32.000 33.409 34.805 36.191 37.566 38.932 40.289 41.638 42.980 44.314 45.642 46.963 48.278 49.588 50.892

7.879 10.597 12.838 14.860 16.750 18.548 20.278 21.955 23.589 25.188 26.757 28.300 29.819 31.319 32.801 34.267 35.718 37.156 38.582 39.997 41.401 42.796 44.181 45.558 46.928 48.290 49.645 50.993 52.336 53.672




TABLE 33.47 Variance Ratio F (95%) n1 n2

1

2

3

4

5

6

8

12

24

∞

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 40 60 120 ∞

161.4 18.51 10.13 7.71 6.61 5.99 5.59 5.32 5.12 4.96 4.84 4.75 4.67 4.60 4.54 4.49 4.45 4.41 4.38 4.35 4.32 4.30 4.28 4.26 4.24 4.22 4.21 4.20 4.18 4.17 4.08 4.00 3.92 3.84

199.5 19.00 9.55 6.94 5.79 5.14 4.74 4.46 4.26 4.10 3.98 3.88 3.80 3.74 3.68 3.63 3.59 3.55 3.52 3.49 3.47 3.44 3.42 3.40 3.38 3.37 3.35 3.34 3.33 3.32 3.23 3.15 3.07 2.99

215.7 19.16 9.28 6.59 5.41 4.76 4.35 4.07 3.86 3.71 3.59 3.49 3.41 3.34 3.29 3.24 3.20 3.16 3.13 3.10 3.07 3.05 3.03 3.01 2.99 2.98 2.96 2.95 2.93 2.92 2.84 2.76 2.68 2.60

224.6 19.25 9.12 6.39 5.19 4.53 4.12 3.84 3.63 3.48 3.36 3.26 3.18 3.11 3.06 3.01 2.96 2.93 2.90 2.87 2.84 2.82 2.80 2.78 2.76 2.74 2.73 2.71 2.70 2.69 2.61 2.52 2.45 2.37

230.2 19.30 9.01 6.26 5.05 4.39 3.97 3.69 3.48 3.33 3.20 3.11 3.02 2.96 2.90 2.85 2.81 2.77 2.74 2.71 2.68 2.66 2.64 2.62 2.60 2.59 2.57 2.56 2.54 2.53 2.45 2.37 2.29 2.21

234.0 19.33 8.94 6.16 4.95 4.28 3.87 3.58 3.37 3.22 3.09 3.00 2.92 2.85 2.79 2.74 2.70 2.66 2.63 2.60 2.57 2.55 2.53 2.51 2.49 2.47 2.46 2.44 2.43 2.42 2.34 2.25 2.17 2.10

238.9 19.37 8.84 6.04 4.82 4.15 3.73 3.44 3.23 3.07 2.95 2.85 2.77 2.70 2.64 2.59 2.55 2.51 2.48 2.45 2.42 2.40 2.38 2.36 2.34 2.32 2.30 2.29 2.28 2.27 2.18 2.10 2.02 1.94

243.9 19.41 8.74 5.91 4.68 4.00 3.57 3.28 3.07 2.91 2.79 2.69 2.60 2.53 2.48 2.42 2.38 2.34 2.31 2.28 2.25 2.23 2.20 2.18 2.16 2.15 2.13 2.12 2.10 2.09 2.00 1.92 1.83 1.75

249.0 19.45 8.64 5.77 4.53 3.84 3.41 3.12 2.90 2.74 2.61 2.50 2.42 2.35 2.29 2.24 2.19 2.15 2.11 2.08 2.05 2.03 2.00 1.98 1.96 1.95 1.93 1.91 1.90 1.89 1.79 1.70 1.61 1.52

254.3 19.50 8.53 5.63 4.36 3.67 3.23 2.93 2.71 2.54 2.40 2.30 2.21 2.13 2.07 2.01 1.96 1.92 1.88 1.84 1.81 1.78 1.76 1.73 1.71 1.69 1.67 1.65 1.64 1.62 1.51 1.39 1.25 1.00

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

4,052 98.50 34.12 21.20 16.26 13.74 12.25 11.26 10.56 10.04 9.65 9.33 9.07 8.86 8.68 8.53 8.40

4,999 99.00 30.82 18.00 13.27 10.92 9.55 8.65 8.02 7.56 7.20 6.93 6.70 6.51 6.36 6.23 6.11

5,403 99.17 29.46 16.69 12.06 9.78 8.45 7.59 6.99 6.55 6.22 5.95 5.74 5.56 5.42 5.29 5.18

5,625 99.25 28.71 15.98 11.39 9.15 7.85 7.01 6.42 5.99 5.67 5.41 5.20 5.03 4.89 4.77 4.67

5,859 99.33 27.91 15.21 10.67 8.47 7.19 6.37 5.80 5.39 5.07 4.82 4.62 4.46 4.32 4.20 4.10

5,982 99.37 27.49 14.80 10.29 8.10 6.84 6.03 5.47 5.06 4.74 4.50 4.30 4.14 4.00 3.89 3.79

6,106 99.42 27.05 14.37 9.89 7.72 6.47 5.67 5.11 4.71 4.40 4.16 3.96 3.80 3.67 3.55 3.45

6,234 99.46 26.60 13.93 9.47 7.31 6.07 5.28 4.73 4.33 4.02 3.78 3.59 3.43 3.29 3.18 3.08

6,366 99.50 26.12 13.46 9.02 6.88 5.65 4.86 4.31 3.91 3.60 3.36 3.16 3.00 2.87 2.75 2.65


F (99%) 5,764 99.30 28.24 15.52 10.97 8.75 7.46 6.63 6.06 5.64 5.32 5.06 4.86 4.69 4.56 4.44 4.34


TABLE 33.47 (Continued) Variance Ratio F (99%) n1 n2

1

2

3

4

5

6

8

12

24

∞

18 19 20 21 22 23 24 25 26 27 28 29 30 40 60 120 ∞

8.28 8.18 8.10 8.02 7.94 7.88 7.82 7.77 7.72 7.68 7.64 7.60 7.56 7.31 7.08 6.85 6.64

6.01 5.93 5.85 5.78 5.72 5.66 5.61 5.57 5.53 5.49 5.45 5.42 5.39 5.18 4.98 4.79 4.60

5.09 5.01 4.94 4.87 4.82 4.76 4.72 4.68 4.64 4.60 4.57 4.54 4.51 4.31 4.13 3.95 3.78

4.58 4.50 4.43 4.37 4.31 4.26 4.22 4.18 4.14 4.11 4.07 4.04 4.02 3.83 3.65 3.48 3.32

4.25 4.17 4.10 4.04 3.99 3.94 3.90 3.86 3.82 3.78 3.75 3.73 3.70 3.51 3.34 3.17 3.02

4.01 3.94 3.87 3.81 3.76 3.71 3.67 3.63 3.59 3.56 3.53 3.50 3.47 3.29 3.12 2.96 2.80

3.71 3.63 3.56 3.51 3.45 3.41 3.36 3.32 3.29 3.26 3.23 3.20 3.17 2.99 2.82 2.66 2.51

3.37 3.30 3.23 3.17 3.12 3.07 3.03 2.99 2.96 2.93 2.90 2.87 2.84 2.66 2.50 2.34 2.18

3.00 2.92 2.86 2.80 2.75 2.70 2.66 2.62 2.58 2.55 2.52 2.49 2.47 2.29 2.12 1.95 1.79

2.57 2.49 2.42 2.36 2.31 2.26 2.21 2.17 2.13 2.10 2.06 2.03 2.01 1.80 1.60 1.38 1.00


GENERAL STATISTICAL REFERENCES Adler, H. L. and Roessler, E. B., Introduction to Probability and Statistics, 6th ed., H. Freeman, New York, 1977. Gad, S. and Weil, C. S., Statistics and Experimental Design for Toxicologists, Telford Press, NJ, 1986. Hollander, M. and Wolfe, D. A., Nonparametric Statistical Methods, John Wiley & Sons, New York, 1973. Snedecor, G. W. and Cochran, W. G., Statistical Methods, 6th ed., Iowa State University Press, Ames, IA, 1967. Tallarida, R. J. and Murray, R. B., Manual of Pharmacologic Calculations with Computer Programs, 2nd ed., Springer-Verlag, New York, 1987.

E. KINETICS TABLE 33.48 Table of Equations and their Uses k=

0.693 t1/2

(1)

Equation 1 is useful for determining the value of the rate constant when the half-time is known or for determining the half-time when the rate constant is known. Rate constants and half-times are measured in dynamic binding experiments. The ratio of the dissociation rate constant to the association rate constant is an alternative way to determine the equilibrium dissociation constant. This relationship is only valid for first order processes or pseudo first order processes where all but one component is constant over time. Plotting the log of the concentration of the substance of interest vs. time will give a straight line if the process is first order. ∆G 0 = − RT ln KD


(2)


TABLE 33.48 (Continued) Table of Equations and their Uses Equation 2 is useful for determining the free energy difference between reactants and products at equilibrium when the equilibrium constant for the reaction is known. Alternatively, if the energy difference between reactants and products at equilibrium is known, Equation 2 can be used to determine the equilibrium constant. Calorimetric measurements can be used to measure ∆G0 values associated with binding interactions. BL =

BLmax [ L] K D + [ L]

(3)

The Langmuir binding isotherm1 describes the characteristics of binding of molecules to other molecules or surfaces. We are concerned here with the binding of ligands to receptors. The useful parameters are the maximum binding capacity of the receptors (Bmax) and their affinity for binding the ligand (KD). Nonlinear fitting to Equation is the best way of evaluating these parameters. Y=

[ L] K D + [ L]

(4)

An alternative means of expressing the Langmuir binding isotherm is in terms of fractional receptor occupancy (Y). This has the effect of confining the dependent variable to a scale from 0 to 1. This allows direct graphic comparisons (Y vs. log [L]) of the affinity of the interactions of different receptor-ligand systems. The ordinate scale is the same for each receptorligand interaction regardless of the magnitude of Bmax.The only difference is the position of the curve, right or left, which is a measure of the affinity. 1 1 KD 1 = + BL [ L] BL max BL max

(5)

The double-reciprocal or Lineweaver-Burk equation is the most widely used and understood of the linear transformations of Equation 3. For this reason it is useful as a means of displaying data for graphic presentation. With the wide availability of computer programs for nonlinear fitting, Equation 5 is now of only limited value for evaluating binding parameters. B BL B = − L + L max KD KD [ L]

(6)

The Rosenthal-Scatchard equation is particularly sensitive to the presence of heterogeneity of binding and for the presence of multiple binding sites for the same ligand on a single receptor. Either of these phenomena have the potential to produce nonlinearity. Multiple binding sites for the same ligand on a single receptor will produce convex curvature while binding site heterogeneity can produce convex, concave, or no curvature. The value of this transformation of Equation is in graphic analysis for nonlinearity. BL = −

BL K + BL max [ L] D

(7)

The Eadie-Hofstee equation is essentially the same equation as the Rosenthal-Scatchard equation. The major difference between the two equations is that the axes are reversed. Equation 7 is useful for the same analyses as the Rosenthal-Scatchard equation and it has the additional advantage of providing the values of the binding parameters directly rather than as reciprocals or in combinations. KD [ L] [ L] =− + BL BL max BL max

(8)

The Woolf equation is the least used of the linear transformations of the Langmuir binding isotherm. It is the best one for minimizing the effect of weighting errors. However, since nonlinear fitting to the Langmuir binding isotherm, as a means of evaluating binding parameters, is now widely available this feature of the equation is of diminished importance.



TABLE 33.48 (Continued) Table of Equations and their Uses  [ R]  pL = pK D + log   [ RL] 

(9)

Equation 9 is the ligand binding equivalent of the Henderson Hasselbalch pH equation. This equation can be used like the Henderson-Hasselbalch equation, for example, to calculate the fractional receptor occupancy for any given ligand concentration when the KD is known. EL α = KD Emax +1 [ L]

(10)

Equation 10 is a rearrangement that incorporates terms for relative intrinsic activity due to the phenomenon of partial agonism. When the maximum physiological effect for a receptor system is known, Equation 10 can be employed to evaluate the relative intrinsic activity (α) of any given agonist. log

 Y  = n log[ L] − log K D 1− Y 

(11)

The Hill equation is useful for evaluating the presence of interacting binding sites on a receptor (cooperativity). The Hill equation also detects multiple noninteracting binding sites for the same ligand with different affinities on the same receptor. It is incapable of distinguishing between these two mechanisms. A Hill coefficient (n) greater than 1 is an indication of either cooperativity of binding or multiple noninteracting binding sites on the same receptor. The deficiency of the Hill equation is that the KD value is a composite of multiple values and the Hill coefficient determined from experimental data, which is supposed to represent the number of different or interacting binding sites, is rarely a simple integer value. B BL B = − L + L max KD KD [ L] n

(12)

Equation 12 is a form of the Scatchard equation that takes into consideration the possibility of multiple binding sites for the same ligand on the receptor. The n in this equation is the same as the Hill coefficient. When n is greater than 1 the Scatchard plot will exhibit pronounced convex curvature. This equation has the same deficiency as the Hill equation. log

K    Y  = log[ L] − log K D2 − log1 + D  1− Y   [ L] 

(13)

Equation 13 is a modification of the Hill equation that takes into consideration the individual affinity constants for a receptor with two binding sites for the ligand where only the RLL complex is capable of producing a physiological effect. When these sites interact, or when they have different affinities, they will produce a downward curving Hill plot. When such behavior is noted, this equation can be used to evaluate the relative contributions of the two binding sites to the overall binding and effect. log

K D2    Y  = 2 log[ L] − log( K D1 K D2) + log1 + k 1− Y  [ L]  

(14)

Equation 14 is a modified version of the Hill equation that takes into consideration the individual affinity constants for a receptor with two binding sites when both RL and RLL are capable of producing a physiological effect. This mechanism will produce Hill plots with an upward curvature. This equation, like Equation 13, is useful for evaluation of the relative contributions of the two binding sites to the overal binding and effect. BL =


BL max [ L]  [I]  K D 1 + + [ L] K I  

(15)


TABLE 33.48 (Continued) Table of Equations and their Uses Equation 15 is a modified version of the Langmuir binding isotherm which takes into consideration the contribution of a competitive inhibitor. This equation, in combination with Equation 3, is useful for the evaluation of the inhibition constant employing nonlinear fitting methods. 1 1 KD = BL [ L] BL max

 1 [I]  1 + K  + B   I L max

(16)

Equation 16 is a modified version of the double-reciprocal transformation of the Langmuir binding isotherm that takes into consideration the contribution of a competitive inhibitor. This equation is useful for the graphic analysis of competitive inhibition. If the inhibitor changes only the slope, and not the abscissal intercept, this indicates that the inhibition is competitive.  [ L ′]  log − 1 = log[ I ] + pK I  [ L] 

(17)

The Schild equation is useful for calculating the concentration of agonist needed to restore the same level of effect when a known concentration of competitive inhibitor is added. Alternatively, it is useful for calculating the competitive inhibitor concentration when a known increase in agonist concentration is necessary to maintain the same level of effect. EL =

E L max [ L]   [I]  [I]  K D 1 + + [ L ] 1 + K I  K I   

(18)

Equation 18 is a modified version of the Langmuir binding isotherm which takes into consideration the contribution of a simple noncompetitive inhibitor. This equation is useful for the evaluation of the inhibition constant employing nonlinear fitting methods. 1 1 KD = E L [ L] E L max

 1 [I]  1 + K  + E  I  L max

 [I]  1 + K   I 

(19)

Equation 19 is a modified version of the double-reciprocal transformation of the Langmuir binding isotherm that takes into consideration the contribution of a simple noncompetitive inhibitor. This equation is useful for the graphic analysis of simple noncompetitive inhibition. If the inhibitor changes both the slope and the abscissal intercept, this indicates that the inhibition is noncompetitive. EL = E L max

[ L]    [I] [I]  K DI 1 +  + [ L ] 1 +  K I1  K I2   

(20)

Equation 20 is a modified version of the Langmuir binding isotherm which takes into consideration the contribution of a heterotropic-cooperative noncompetitive inhibitor. This equation is useful for the evaluation of the effect of agonist binding on the inhibition constant employing nonlinear fitting methods. 1 1 K D1  1  [I]  [I]  = 1 + + 1 +  E L [ L] E L max  K I1  E L max  K I2 

(21)

Equation 21 is a modified version of the double-reciprocal transformation of the Langmuir binding isotherm that takes into consideration the contribution of a heterotropic-cooperative noncompetitive inhibitor. This equation is useful for the graphic analysis of heterotropic-cooperative noncompetitive inhibition. If the inhibitor changes both the slope and the abscissal intercept, this indicates that the inhibition is noncompetitive. If the intersection of the lines is above or below the ordinate, this indicates that the binding of substrate influences the binding of inhibitor and the binding of inhibitor influences the binding of substrate. When this is true, the mechanism is heterotropic-cooperative noncompetitive inhibition.



TABLE 33.48 (Continued) Table of Equations and their Uses Y=

1  [ I]  K D1 1 +  K I1    [ I]  [ L]  1 +  K I2  

(22) +1

Equation 22 is a modified version of the Langmuir binding isotherm which takes into consideration the contribution of a heterotropic-cooperative noncompetitive inhibitor that reduces the affinity of the receptor for the agonist without preventing the physiological effect. This is allosteric competitive inhibition. This equation is useful by virtue of the fact that it predicts that, at very high inhibitor concentration, there will be a decrease in the BLmax. Thus, allosteric competitive inhibition can be distinguished from simple competitive inhibition by employing very high inhibitor concentrations. Y=

1 K D2  K S1   KS  1+ + 1 + 2    [ L]  [ S]   [ S] 

(23)

Equation 23 is a modified version of the Langmuir binding isotherm which takes into consideration the contribution of a simple noncompetitive stimulator. This equation is useful for the evaluation of the stimulation constant employing nonlinear fitting methods. An alternative way to think about a stimulator is that it can also represent, under some circumstances, a second agonist at the receptor where both agonists must bind before a physiological effect can be produced. 1 1 K D2 = E L [ L] E L max

K S1   1 1 + [ S ]  + E   L max

K S2   1 + [ S ]   

(24)

Equation 24 is a modified version of the double-reciprocal transformation of the Langmuir binding isotherm that takes into consideration the contribution of a simple noncompetitive stimulator. This equation is useful for the graphic analysis of simple noncompetitive stimulation. If the stimulator changes both the slope and the abscissal intercept, this indicates that the stimulation is noncompetitive. EL 1 = E L max KD  [I ]  + 1+ [ L]  K I 

(25)

Equation 25 is a modified version of the Langmuir binding isotherm which takes into consideration the contribution of an uncompetitive inhibitor. This equation is useful for the evaluation of the inhibition constant using nonlinear fitting methods. 1 1 KD 1 = + E L [ L] E L max E L max

 [I]  1 + K   I 

(26)

Equation 26 is a modified version of the double-reciprocal transformation of the Langmuir binding isotherm that takes into consideration the contribution of an uncompetitive inhibitor. This equation is useful for the graphic analysis of uncompetitive inhibition. If the inhibitor changes the abscissal intercept without affecting the slope, this indicates that the inhibition is uncompetitive. Uncompetitive inhibition can, under some circumstances, represent a special case of heterotropic-cooperative noncompetitive inhibition where binding of substrate increases the affinity of binding of the inhibitor from near zero to its final value. V=


Vmax [ S] K M + [ S]

(27)


TABLE 33.48 (Continued) Table of Equations and their Uses The Michaelis-Menten equation for enzyme kinetics is equivalent in form to the Langmuir binding isotherm. A number of special conditions and assumptions are involved in the derivation of the Michaelis-Menten equation which are not necessary for the Langmuir binding isotherm. If these conditions and assumptions are not met, the Michaelis-Menten equation is not valid for the analysis of enyme kinetic data. This equation is useful for the evaluation of the KM and Vmax parameters of enzyme reactions using nonlinear fitting methods. V=

Vmax [ S1 ][ S2 ] K D1 K M2 + K M1 [ S2 ] + K M2 [ S1 ] + [ S1 ][ S2 ]

(28)

Equation 28 is a modified version of the Michaelis-Menten equation for enzyme reactions that require two substrates to add to the enzyme before any product formation can occur. Normally, one substrate is present at saturating concentration. When this is true, the equation simplifies to Equation 27. This equation is useful for analyzing kinetic parameters when it is not feasible to hold one substrate at saturation. V=

Vmax [ S1 ][ S2 ] K M1 [ S2 ] + K M2 [ S1 ] + [ S1 ][ S2 ]

(29)

Equation 29 is a modified version of the Michaelis-Menten equation for enzyme reactions that exhibit a ping-pong mechanism. These enzymes have two substrates and two products. The first substrate combines with the enzyme and is converted to product. This reaction alters the enzyme such that it can then combine with the second substrate and convert it to product. The second reaction converts the enzyme back to its original form. Normally one of the substrates is present at saturating concentration. When this is true, the equation simplifies to Equation 27. This equation is useful for analyzing kinetic parameters when it is not feasible to hold one substrate at saturation. Source: Matthews, J. C., Fundamentals of Receptor, Enzyme, and Transport Kinetics, CRC Press, Boca Raton, FL, 1993. With permission.

MATHEMATICS, SYMBOLS, PHYSICAL CONSTANTS, CONVERSIONS, AND STATISTICS Materials in this section were reprinted from the following sources: Lide, D. R., Ed., CRC Handbook of Chemistry and Physics, 73rd ed., CRC Press, Boca Raton, FL, 1992: International System of Units (SI), conversion constants and multipliers (conversion of temperatures), symbols and terminology for physical and chemical quantities, fundamental physical constants, classification of electromagnetic radiation, electrical resistivity (pure metals, selected alloys), dielectric constants, properties of semiconductors, properties of magnetic alloys, resistance of wires. Beyer, W. H., Ed., CRC Standard Mathematical Tables and Formulae, 29th ed., CRC Press, Boca Raton, FL, 1991: Greek alphabet, conversion constants and multipliers (recommended decimal multiples and submultiples, metric to English, English to metric, general, temperature factors), physical constants, series expansion, integrals, the Fourier transforms, numerical methods, probability, positional notation. Tallarida, R. J., Pocket Book of Integrals and Mathematical Formulas, 2nd ed., CRC Press, Boca Raton, FL, 1992: Elementary algebra and geometry; determinants, matrices, and linear systems of equations; trigonometry; analytic geometry; series; differential calculus; integral calculus; vector analysis; special functions; statistics; tables of probability and statistics; table of derivatives. Pankow, J. F., Aquatic Chemistry Concepts, Chelsea, MI, Lewis Publishers, 1991: Periodic table of the elements. Shackelford, J. and Alexander, W. Eds., CRC Materials Science and Engineering Handbook, CRC Press, Boca Raton, FL, 1992: Electrical resistivity of selected alloy cast irons, resistivity of selected ceramics.



SECTION 4. CALCULATIONS, PREPARATION, AND PROPERTIES OF VARIOUS TYPES OF SUBSTANCES COMMONLY USED IN TOXICOLOGY A. MOLARITY, MOLALITY, NORMALITY, OSMOLARITY CALCULATIONS Number of moles of solute Liter of solution Grams of chemical Where: Number of moles = Molecular weight

1. Molarity (M) =

2. Molality (m) =

Number of moles of solute Kilogram of solution

Number of equivalents of solute Liter of solution Grams of chemical Where: Number of equivalents = Equivalent weight

3. Normality (N) =

Molecular weight n For acids and bases, n = The number of replaceable H+ or OH– ions per molecule Equivalent weight =

4. Normality = n Molarity Where n: = Number of replaceable H+ or OH– ions per molecule. 5. Osmolarity = n Molarity Where n: = Number of dissociable ions per molecule.

B. SOLUTION CALCULATIONS Volume of solute × 100 Volume of solution Weight of solute 7. Weight percent (% w / w) = × 100 Weight of solution 6. Volume percent (% v / v) =

8. Weight / volume percent (% w / v) =

Weight of solute (g) × 100 Volume of solution (ml)

Weight of solute (mg) × 100 100 mL of solution Weight of solute 10. Parts per million (ppm) = × 10 6 Weight of solution 11. Parts per million (for gases) 9. Milligram percent (mg%) =

(mg / m 3 ) (R) Molecular weight Where: R = 24.5 at 25°C. ppm =

12. (volumeC) (concentrationC) = (volumeD) (concentrationD) Where: C = Concentrated solution Where: D = Dilute solution The above relationship is useful in preparing dilute solutions from concentrated solutions. Copyright © 2002 by Taylor & Francis


C.

PH

CALCULATIONS

13. pH = − log [H + ] = log

1 [H + ]

[ A− ] [ HA] Where: HA ⇔ H+ + A– (weak acid) (conjugate base) pKa = –log Ka (equilibrium constant)

14. pH = pKa + log

D. GLOSSARY

OF

TERMS ASSOCIATED

WITH

SOLUTIONS

Dispersion Two-phase system that consists of finely divided particles distributed throughout a bulk substance. Examples include gas:liquid (foam); solid:gas (aerosol); gas:solid (foamed plastic); liquid:gas (fog); liquid:liquid (emulsion); solid:liquid (paint); solid:solid (carbon black in rubber). Emulsion A system containing two or more immiscible liquids in which one is dispersed in the form of very small globules throughout the other. Miscible Capable of being mixed and remaining so after the mixing process ceases. Materials that do not mix at all are said to be immiscible (e.g., oil and water). Mixture A mutual incorporation of two or more substances without chemical union, the physical characteristics of each of the components being retained. The components may or may not be uniformly dispersed and can usually be separated by mechanical means. Mixtures can be broadly grouped into two classes: mechanical mixtures which consist of a mixture of particles or masses distinguishable as such under a microscope or by other methods; physical mixtures consisting of a more intimate mixture of molecules such as with gases and many solutions. Solubility The ability or tendency of one substance to blend uniformly with another. Examples include solid in liquid, liquid in liquid, gas in liquid, gas in gas. Usually, liquids and gases are said to be miscible in other liquids and gases rather than soluble. Soluble Capable of being dissolved. Solution A uniformly dispersed mixture at the molecular or ionic level of one or more substances (the solute) in one or more other substances (the solvent). Suspension The dispersion through a liquid of very small particles (solid, semisolid, or liquid) of a size large enough to be detected by optical means. If the particles are small enough to pass through filter membranes but still large enough to scatter light, they will generally remain dispersed indefinitely and the system is called a colloidal suspension.

REFERENCES (SUBSECTIONS A.→D.) 1. Segel, I. H., Biochemical Calculations, 2nd ed., John Wiley & Sons, New York, 1976. 2. Skoog, D. A. and West, D. M., Fundamentals of Analytical Chemistry, 2nd ed., Holt, Rinehart and Winston, New York, 1969. 3. Sax, N. I. and Lewis, R. J. Sr., Hawley’s Condensed Chemical Dictionary, 11th ed., Van Nostrand Reinhold Company, New York, 1987. 4. Stedman’s Medical Dictionary, 22nd ed., Williams & Wilkins Company, Baltimore, 1972.



TABLE 33.49 Strengths of Concentrated Solutions of Acids and Bases Acid or Base

Specific Gravity % by Weight Weight (g/l) Approximate Molarity

Hydrochloric acid (HCl) Sulfuric acid (H2SO4) Nitric acid (HNO3) Acetic acid (CH3COOH) Ammonium hydroxide (NH4OH) Sodium hydroxide (saturated solution) (NaOH) Potassium hydroxide (saturated solution) (KOH)

1.19 1.84 1.42 1.06 0.880 1.50–1.53 1.55

37.0 96.0 70.0 99.5 29.0 about 50 about 50

440 1,730 990 1,060 250 600–700 800

12.1 18.0 15.7 17.4 15–17 15–18 14.0

TABLE 33.50 Physiological Solutions (g/l) Saline (mammal) Ringer (mammal) Ringer (by Cattell) Ringer (by Dresel) Ringer (by Evans) Ringer (by Genell) Ringer (by Moran) Ringer-Dale (by Stewart) Ringer-Locke (same as Locke’s) Ringer-Locke (by Feldberg) Ringer-Locke (by Gaddum) Ringer-Locke (by Hukovic) Locke’s (by Burn) Krebs-Henseleit Krebs-Henseleit- Ringer Krebs-Henseleit (by Furchgott) Krebs (by Hukovic) Beauvilain’s McEwan’s Tyrode (isolated gut) Feigen’s (isolated heart) a b c

NaCl

KCl

CaCl2

MgCl2

NaHCO3

NaH2PO4

9.00 9.00 9.00 6.00 — 8.00 7.00 9.00 9.00 9.00 9.00 9.00 9.00 6.87 6.90 6.90 6.60 9.00 7.60 8.00 9.00

— 0.42 0.42 0.531 0.42 0.42 0.42 0.42 0.42 0.20 0.42 0.42 0.42 0.40 0.354 0.354 0.350 0.42 0.42 0.20 0.42

— 0.24 0.12 0.35 0.12 0.24 0.24 2.015 .024 0.20 0.06 0.24 0.24 0.28 0.280 0.282 0.280 0.06 0.24 0.20 0.62

— — — — 0.200 0.005 0.200 0.003 — — — — 0.005 — — — — 0.005 — 0.100 —

— 0.50 — 2.10 — 1.00 2.10 0.50 0.15 0.30 0.50 0.50 0.50 2.10 2.10 2.10 2.10 0.50 2.10 1.00 0.60

— — — — — — — — — — — — 0.140 — — — — 0.143 0.050 —

KH2PO4 — 0.100 0.081 — — — — — — — — — — 0.162 0.162 0.162 — — — —

K2SO4 = 22.00. KHCO3 = 3.60. Sucrose = 4.50.

TABLE 33.51 Composition of a Typical Organ Perfusion Medium Component Electrolytes

Buffer Source of energy Oncotic agent Other additions


NaCl KCl MgSO4 CaCl2 NaH2PO4 NaHCO3 Glucose Glutamine Bovine serum albumin Alanine Glutathione

Concentration 115 mM 5.4 mM 2.4 mM 3.0 mM 1.5 mM 25 mM 5 mM 2 mM 60 g 1–1 2 mM 2 mM

MgSO4

Glucose

— 0.147 — — — — — — — — — 0.140 0.294 0.294 0.294 — — — —

— 1.00 1.00 0.90 1.00a,b 0.50 1.80 0.50 1.75 1.00 0.50 2.00 0.50 2.00 — 1.80 2.08 0.50 2.00 c 1.00 1.00


TABLE 33.52 Properties of Carrier Gases for Gas Chromatography

Density (kg/m3)

Thermal Conductivity × 10–2 (W/[m · K])

Hydrogen

0.08988

19.71

Helium

0.17847

Methane

Carrier Gas

Thermal Conductivity Differences δλ (He)

δλ (N2)

δλ (Al )

3.97

16.96

17.81

15.74

—

12.99

13.84

0.71680

3.74

–12.00

0.99

1.84

Oxygen

1.42904

2.85

–12.89

0.10

0.95

Nitrogen

1.25055

2.75

–12.99

—

0.85

Carbon monoxide

1.25040

2.67

–13.07

–0.08

0.77

Ethane

1.35660

2.44

–13.30

–0.31

0.54

Ethene

1.26040

2.30

–13.44

–0.45

0.40

Propane

2.00960

2.03

–13.71

–0.72

0.13

Argon

1.78370

1.90

–13.84

–0.85

—

Carbon dioxide

1.97690

1.83

–13.91

–0.92

–0.07

n-Butane Sulfur hexafluoride

2.51900 6.50 (20°C)

1.82 1.63

–13.92 –14.11

–0.93 –1.12

–0.08 –0.27

Viscosity × 10−3 (Pa ⋅ s)

Heat Capacity (J/[kg ⋅ K])

Relative Molecular Mass

0.876 (20.7°C) 1.086 (129.4°C) 1.381 (299.0°C) 1.941 (20.0°C) 2.281 (100.0°C) 2.672 (200.0°C) 1.087 (20.0°C) 1.331 (100.0°C) 1.605 (200.5°C) 2.018 (19.1°C) 2.568 (127.7°C) 3.017 (227.0°C) 1.781 (27.4°C) 2.191 (127.2°C) 2.559 (226.7°C) 1.753 (21.7°C) 2.183 (126.7°C) 2.548 (227.0°C) 0.901 (17.2°C) 1.143 (100.4°C) 1.409 (200.3°C) 1.008 (20.0°C) 1.257 (100.0°C) 1.541 (200.0°C) 0.795 (17.9°C) 1.009 (100.4°C) 1.253 (199.3°C) 2.217 (20.0°C) 2.695 (100.0°C) 3.223 (200.0°C) 1.480 (20.0°C) 1.861 (99.1°C) 2.221 (182.4°C) 0.840 (14.7°C) 1.450 (21.1°C)

14,112.7

2.016

5,330.6

4.003

2,217.2

16.04

915.3

32.00

1,030.5

28.016

1,030.7

28.01

1,614.0

30.07

—

28.05

—

44.09

523.7

39.94

836.6

44.01

— 647.0

58.12 146.05

Note: Values refer to a pressure of 101 kPa (760 torr). Density values are given at 0°C (120°F). Reprinted from Bruno, T. J. and Svoronos, P. D. N., CRC Handbook of Basic Tables for Chemical Analysis, CRC Press, Boca Raton, FL, 1989, p. 4. With permission.



TABLE 33.53 Solvents for Ultraviolet Spectrophotometry Solvent Acetic acid Acetone Acetonitrile Benzene 2-Butanol n-Butyl acetate Carbon disulfide Carbon tetrachloride 1-Chlorobutane Chloroforma Cyclohexane 1,2-Dichloroethane 1,2-Dimethoxyethane N,N-Dimethylacetamide N,N-Dimethylformamide Dimethylsulfoxide 1,4-Dioxane Diethyl ether Ethanol 2-Ethoxyethanol Ethyl acetate Glycerol n-Hexadecane n-Hexane Methanol 2-Methoxyethanol Methyl cyclohexane Methyl ethyl ketone Methyl isobutyl ketone 2-Methyl- 1 -propanol N-Methyl-2-pyrrolidone Pentane n-Pentyl acetate 1-Propanol 2-Propanol Pyridine Tetrachloroethyleneb Tetrahydrofuran Toluene 1,1,2-Trichloro-1,2,2-trifluoroethane 2,2,4-Trimethylpentane o-Xylene m-Xylene p-Xylene Water a b

Wavelength Cutoff (nm) 260 330 190 280 260 254 380 265 220 245 210 226 240 268 270 265 215 218 210 210 255 207 200 210 210 210 210 330 335 230 285 210 212 210 210 330 290 220 286 231 215 290 290 290

Dielectric Constant (20°C) 6.15 20.7 37.5 2.284 15.8 2.641 2.238 7.39 4.806 2.023 10.19 59 36.7 4.7 2.209 4.335 24.30 6.02 42.5 2.06 1.890 32.63 16.9 2.02 18.5

(25°C)

(25°C)

(25°C)

(25°C) (83°C)

(25°C) (25°C) (25°C) (25°C) (25°C) (25°C) (25°C)

1 32.0 1.844 20.1 18.3 12.3 7.6 2.379 1.936 2.568 2.374 2.270 78.54

(25°C) (25°C) (25°C)

(25°C) (25°C)

(25°C)

Stabilized with ethanol to avoid phosgene formation. Stabilized with thymol (isopropyl meta-cresol).

Reprinted from Bruno, T. J. and Svoronos, P. D. N., CRC Handbook of Basic Tables for Chemical Analysis, CRC Press, Boca Raton, FL, 1989, p. 212. With permission.



TABLE 33.54 13C Chemical Shifts of Useful NMR Solvents Solvent

Formula

Chemical Shift (npm)

Acetone-d6 Acetonitrile-d3 Benzene-d6 Carbon disulfide Carbon tetrachloride Chloroform-d3 Cyclohexane-d12 Dichloromethane-d2 Dimethylformamide-d7 Dimethylsulfoxide-d6 Dioxane-d8 Methanol-d4 Nitromethane-d3 Pyridine-d5 1, 1,2,2-Tetrachloroethane-d2 Tetrahydrofuran-d8 Trichlorofluoromethane Water (heavy)

(CD3)2C=O CD3C≡N C6D6 CS2 CCl4 CDCl3 C6D12 CD2Cl2 (CD3)2NCOD (CD3)2S=O C6D8O2 CD3OD CD3-NO2` C5D5N CDCl2-CDCl2 C4D8O CFC13 D 2O

29.2 (CD3) 204.1 (>C = O) 1.3 (CD3) 117.1 (C ≡ N) 128.4 192.3 96.0 77.05 27.5 53.6 31 (CD3) 36 (CD3) 162.4 (DC = O) 39.6 67.4 49.3 57.3 123.9 (C-3) 135.9 (C-4) 150.2 (C-2) 75.5 25.8 (C-2) 67.9 (C-1) 117.6 —


TABLE 33.55 Important Peaks in the Mass Spectra of Common Solvents Solvents

Formula

Water Methanol Acetonitrile Ethanol Dimetyl ether Acetone Acetic acid Ethylene glycol Furan Tetrahydrofuran n-Pentane Dimethylformamide (DMF) Diethylether Methylacetate Carbon disulfide Benzene Pyridine Dichloromethane Cyclohexane n-Hexane p-Dioxane Tetramethylsilane (TMS)

H 2O CH3OH CH3CN CH3CH2OH CH3OCH3 CH3COCH3 CH3CO2H HOCH2CH2OH C4H4O C4H8O C5H12 HCON(CH3)2 (C2H5)2O CH3CO2CH3 CS2 C6H6 C5H5N CH2Cl2 C6H12 C6H14 C4H8O2 (CH3)4Si


M+ 18(100%) 32 41(100%) 46 46(100%) 58 60 62 68(100%) 72 72 73(100%) 74 74 76(100%) 78(100%) 79(100%) 84 84 86 88(100%) 88

Important Peaks (m/e) 17 31 (100%), 29, 15 40, 39, 38, 28, 15 45, 31 (100%), 27, 15 45,29, 15 43 (100%), 42, 39, 27, 15 45, 43, 18, 15 43, 33, 31 (100%), 29, 18, 15 42, 39, 38, 37, 29, 18 71, 43, 42 (100%), 41, 40, 39, 27, 18, 15 57, 43 (100%), 42, 41, 39, 29, 28, 27, 15 58, 44, 42, 30, 29, 28, 18, 15 59, 45, 41, 31 (100%), 29, 27, 15 59, 43 (100%), 42, 32, 29, 28, 15 64, 44, 38, 32 77, 52, 51, 50, 39, 28 80, 78, 53, 52, 51, 50, 39, 26 86, 51, 49 (100%), 48, 47, 35, 28 69, 56, 55, 43, 42, 41, 39, 27 85, 71, 69, 57, (100%), 43, 42, 41, 39, 29, 28, 27 87, 58, 57, 45, 43, 31, 30, 29, 28 74, 73, 55, 45, 43, 29


TABLE 33.55 (Continued) Important Peaks in the Mass Spectra of Common Solvents Solvents

M+

Important Peaks (m/e)

90 92 118 119 152 (not seen) 164 (not seen)

60, 58, 45 (100%), 31, 29 91 (100%), 65, 51, 39, 28 120, 83, 81 (100%), 47, 35, 28 121, 84, 82 (100%), 48, 47, 35, 28 121, 119, 117 (100%), 84, 82, 58.5, 47, 35, 28 168, 166 (100%), 165, 164, 131, 128, 129, 95, 94, 82, 69, 59, 47, 31, 24

Formula

1,2-Dimethoxy ethane Toluene Chloroform Chloroform-d1 Carbon tetrachloride Tetrachloroethene

(CH3OCH2)2 C6H5CH3 CHCl3 CDCl3 CCl4 CCl2 = CC12


TABLE 33.56 Solvents for Liquid Chromatography

Solvent Acetic acid Acetone Acetonitrile Benzene 1-Butanol 2-Butanol n-Butyl acetate n-Butyl chloride Carbon tetrachloride Chlorobenzene Chloroform Cyclohexane Cyclopentane o-Dichlorobenzene N,N-Dimethylacetamide Dimethylformamide Dimethyl sulfoxide Dioxane 2-Ethoxyethanol Ethyl acetate Ethyl ether Glyme (ethylene glycol dimethyl ether) Heptane Hexadecane Hexane Isobutyl alcohol Methanol 2-Methoxyethanol 2-Methoxyethyl acetate Methylene chloride


Viscosity (mPa . s; 20ºC) 1.31(15) 0.30(25) 0.34(25) 0.65 2.95 4.21 0.73 0.47(15) 0.97 0.80 0.58 0.98 0.44 1.32(25) 2.14 0.92 2.20 1.44(15) 2.05 0.46 0.24 0.46(25) 0.42 3.34 0.31 4.70(15) 0.55 1.72 0.45(15)

UV Cutoff (nm)

330 190 278 215 260 254 220 263 287 245 200 200 295 268 268 286 215 210 256 218 220 200 200 200 200 205 210 254 233

Refractive Index (20ºC) 1.372 1.359 1.344 1.501 1.399 1.397 1.394 1.402 1.460 1.525 1.446 1.426 1.406 1.551 1.438 1.430 1.478 1.422 1.408 1.372 1.352 1.380 1.388 1.434 1.375 1.396 1.328 1.402 1.402 1.424

Normal Boiling Point (ºC) 117.9 56.3 81.6 80.1 117.7 99.6 126.1 78.4 76.8 131.7 61.2 80.7 49.3 180.5 166.1 153.0 189.0 101.3 135.6 77.1 34.6 93.0 98.4 287.0 68.7 107.7 64.7 124.6 144.5 39.8

Dielectric Constant (20ºC) 6.15 20.7(25) 37.5 2.284 17.8 15.8(25)

2.238 2.708 4.806 2.023 1.965 9.93(25) 37.8 36.7 4.7 2.209(25) 6.02(25) 4.335 1.92 1.890 15.8(25) 32.63(25) 16.9 9.08


TABLE 33.56 (Continued) Solvents for Liquid Chromatography

Solvent Methylethylketone Methylisoamylketone Methylisobutylketone N-Methyl-2-pyrrolidone Nonane Pentane Petroleum ether β−Phenethylamine 1-Propanol 2-Propanol Propylene carbonate Pyridine Tetrachloroethylene Tetrahydrofuran Tetramethyl urea Toluene Trichloroethylene 1,2,2-Trichloro- 1,2,2-trifluoroethane 2,2,4-Trimethylpentane Water o-Xylene p-Xylene

Viscosity (mPa . s; 20ºC)

UV Cutoff (nm)

0.42(15)

329 330 334 285 200 200 226 285 210 205

0.54(25) 1.67(25) 0.72 0.24 0.30 2.26 2.86(15) 0.95 0.93(15) 0.55 0.59 0.57 0.71 0.50 1.00 0.81

330 295 212 265 284 273 231 215 10.00 10.4 8.4 7.1 2.8 0.7 7.6 10.3 6.2 8.3 7.2 5.4, 7.4 5.0, 7.4



The following table gives approximate pH values for a number of substances of biological importance. All values are rounded off to the nearest tenth and are based on measurements made at 25°C.

TABLE 33.70 Approximate pH Values of Biological Materials and Foods Blood, plasma, human Spinal fluid, human Blood, whole, dog Saliva, human

7.3–7.5 7.3–7.5 6.9–7.2 6.5–7.5

Biological Materials Gastric contents, human Duodenal contents, human Feces, human Urine, human

1.0–3.0 4.8–8.2 4.6–8.4 4.8–8.4

Milk, human Bile, human

6.6–7.6 6.8–7.0

Apples Apricots Asparagus Bananas Beans Beer Beets Blackberries Bread, white Butter Cabbage Carrots Cheese Cherries Cider Corn Crackers Dates Eggs, fresh white Flour, wheat

2.9–3.3 3.6–4.0 5.4–5.8 4.5–4.7 5.0–6.0 4.0–5.0 4.9–5.5 3.2–3.6 5.0–6.0 6.1–6.4 5.2–5.4 4.9–5.3 4.8–6.4 3.2–4.0 2.9–3.3 6.0–6.5 6.5–8.5 6.2–6.4 7.6–8.0 5.5–6.5

Foods Gooseberries Grapefruit Grapes Hominy (lye) Jams, fruit Jellies, fruit Lemons Limes Maple syrup Milk, cows Olives Oranges Oysters Peaches Pears Peas Pickles, dill Pickles, sour Pimento Plums

2.8–3.0 3.0–3.3 3.5–4.5 6.8–8.0 3.5–4.0 2.8–3.4 2.2–2.4 1.8–2.0 6.5–7.0 6.3–6.6 3.6–3.8 3.0–4.0 6.1–6.6 3.4–3.6 3.6–4.0 5.8–6.4 3.2–3.6 3.0–3.4 4.6–5.2 2.8–3.0

Potatoes Pumpkin Raspberries Rhubarb Salmon Sauerkraut Shrimp Soft drinks Spinach Squash Strawberries Sweet potatoes Tomatoes Tuna Turnips Vinegar Water, drinking Wine

5.6–6.0 4.8–5.2 3.2–3.6 3.1–3.2 6.1–6.3 3.4–3.6 6.8–7.0 2.0–4.0 5.1–5.7 5.0–5.4 3.0–3.5 5.3–5.6 4.0–4.4 5.9–6.1 5.2–5.6 2.4–3.4 6.5–8.0 2.8–3.8

Source: Handbook of Chemistry and Physics, Lide, D. R., Ed., CRC Press, Boca Raton, FL, 1990. With permission.

C. CHEMICAL FUNCTIONAL GROUPS Table 33.71 presents the chemical structure of functional groups frequently encountered in toxicology.

TABLE 33.71 Chemical Functional Groups Acetamido (acetylamino) Acetimido (acetylimino) Acetoacetamido Acetoacetyl Acetonyl Acetonylidene Acetyl Acrylyl


CH3CONH– CH3C(=NH)– CH3COCH2CONH– CH3COCH2CO– CH3COCH2– CH3COCH= CH3CO– CH2=CHCO–


TABLE 33.71 (Continued) Chemical Functional Groups Adipyl (from adipic acid) Alanyl (from alanine) β-alanyl Allophanoyl Allyl (2-propenyl) Allylidene (2-propenylidene) Amidino (aminoiminomethyl) Amino Amyl (pentyl) Anilino (phenylamino) Anisidino Anisyl (from anisic acid) Anthranoyl (2-aminobenzoyl) Arsino Azelaoyl (from azelaic acid) Azido Azino Azo Azoxy Benzal Benzamido (benzylamino) Benzhydryl (diphenylmethyl) Benzimido (benzylimino) Benzoxy (benzoyloxy) Benzoyl Benzyl Benzy1idine Benzyldyne Biphenylyl Biphenylene Butoxy Sec-butoxy Tert-butoxy Butyl Iso-butyl (3-methylpropyl) sec-butyl (1-methylpropyl) Tert-butyl (1,1, dimethylethyl) Butyryl Caproyl (from caproic acid) Capryl (from capric acid) Caprylyl (from caprylic acid) Carbamido Carbamoyl (aminocarbonyl) Carbamyl (aminocarbonyl) Carbazoyl (hydrazinocarbonyl) Carbethoxy Carbobenzoxy Carbonyl Carboxy Cetyl Chloroformyl (chlorocarbonyl)


–OC(CH2)4CO– CH3CH(NH2)CO– HN(CH2)2CO– H2NCONHCO– CH2=CHCH2– CH2 =CHCH= H2NC(=NH)– H2N– CH3(CH2)4– C6H5NH– CH3OC6H4NH– CH3OC6H4CO– 2–H2NC6H4CO– AsH2– –OC(CH2)7CO– N 3– =NN= –N=N– –N(O)N– C6H5CH= C6H5CONH– (C6H5)2CH– C6H5COO– C6H5COO– C6H5CO– C6H5CH2– C6H5CH= C6H5C≡ C6H5C6H5– –C6H4C6H4– C4H9O– C2H5CH(CH3)O– (CH3)3CO– CH3(CH2)3– (CH3)2(CH2)2– C2H5CH(CH3)– (CH3)3C– C3H7CO– CH3(CH2)4CO– CH3(CH2)6CO– CH3(CH2)6CO– H2NCONH– H2NCO– H2NCO– H2NNHCO– C2H5O2C– C6H5CH2O2C– –C=O– HOOC– CH3(CH2)15– ClCO–


TABLE 33.71 (Continued) Chemical Functional Groups Cinnamyl (3-phenyl-2-propenyl) Cinnamoyl Cinnamylidene Cresyl (hydroxymethylphenyl) Crotoxyl Crotyl (2-butenyl) Cyanamido (cyanoamino) Cyanato Cyano Decanedioyl Decanoly Diazo Diazoamino Disilanyl Disiloxanoxy Disulfinyl Dithio Enanthyl Epoxy Ethenyl (vinyl) Ethinyl Ethoxy Ethyl Ethylthio Formamido (formylamino) Formyl Fumaroyl (from fumaric acid) Furfuryl (2-furanylmethyl) Furfurylidene (2-furanylmethylene) Furyl (furanyl) Glutamyl (from glutamic acid) Glutaryl (from glutaric acid) Glycidyl (oxiranylmethyl) Glycinamido Glycolyl (hydroxyacetyl) Glycyl (aminoacetyl) Glyoxylyl (oxoacetyl) Guanidino Guanyl Heptadecanoyl Heptanamido Heptanedioyl Heptanoyl Hexadecanoyl Hexamethylene Hexanedioyl Hippuryl (N-benzoylglycyl) Hydantoyl Hydrazino Hydrazo Hydrocinnamoyl


C6H5CH=CHCH2– C6H5CH=CHCO– C6H5CH=CHCH= HO(CH3)C6H4– CH3CH=CHCO– CH3CH=CHCH2– NCNH– NCO– NC– –OC(CH2)6CO– CH3(CH2)6CO– N 2= –NHN=N– H2SiSiH2– H3SiOSiH20– –S(O)S(O)– –SS– CH3(CH2)5CO– –O– CH2=CH– HC≡C– C2H5O– CH3CH2 C2H5S– HCONH– HCO– –OCCH=CHCO– OC4H3CH2– OC4H3CH= OC4H3– –OC(CH2)2CH(NH2)CO– –OC(CH2)CO– CH2–CHCH2– H2NCH2CONH– HOCH2CO– H2NCH2CO– HCOCO– H2NC(=NH)NH– H2NC(=NH)– CH3(CH2)15CO– CH3(CH2)15CONH– –OC(CH2)5CO– CH3(CH2)5CO– CH3(CH2)4CO– –(CH2)6– –OC(CH2)4CO– C6H5CONHCH2CO– H2NCONHCH2CO– N2NNH– –HNNH– C6H5(CH2)2CO–


TABLE 33.71 (Continued) Chemical Functional Groups Hydroperoxy Hydroxamino Hydroxy Imino Lodoso Isoamyl (isopentyl) Isobutenyl (2-methyl-1-propenyl) Isobutoxy Isobutyl Isobutylidene Isobutyryl Isocyanato Isocyano Isohexyl Isoleucyl (from isoleucine) Isonitroso Isopentyl Isopentylidene Isopropenyl Isopropoxy Isopropyl Isopropylidene Isothiocyanato (isothiocyano) Isovaleryl (from isovaleric acid) Keto (oxo) Lactyl (from lactic acid) Lauroyl (from lauric acid) Leucyl (from leucine) Levulinyl (From levulinic acid) Malonyl (from malonic acid) Mandelyl (from mandelic acid) Mercapto Methacrylyl (from methacrylic acid) Methallyl Methionyl (from methionine) Methoxy Methyl Methylene Methylenedioxy Methylenedisulfonyl Methylol Methylthio Myristyl (from myristic acid) Naphthal Naphthobenzyl Naphthoxy Naphthyl Naphthylidene Neopentyl Nitramino Nitro


HOO– HONH– HO– HN= OI– (CH3)2CH(CH2)2– (CH3)2C=CH– (CH3)2CHCH O– (CH3)2CHCH2– (CH3)2CHCH= (CH3)2CHCO– OCN– CN– (CH3)2CH(CH2)3– C2H3CH(CH3)CH(NH4)CO– HON= (CH3)2CH(CH2)2– (CH3)2CHCH2CH= H2C=C(CH3)– (CH3)2CHO– (CH3)2CH– (CH3)2C= SCN– (CH3)2CHCH2CO– O= CH3CH(OH)CO– CH3(CH2)10CO– (CH3)2CHCH2CH(NH2)CO– CH3CO(CH2)2CO– –OCCH2CO– C6H5CH(OH)CO– HS– CH2=C(CH3)CO– CH2=C(CH3)CH2– CH3SCH2CH2CH(NH2)CO2– CH3O– H3C– H2C= –OCH2O– –O2SCH2SO2– HOCH2– CH2S– CH3(CH2)12CO– (C10H7)CH= (C10H7)CH2– (C10H7)O– (C10H7)– (C10H6)= (CH3)3CCH2– O2NNH– O2N–


TABLE 33.71 (Continued) Chemical Functional Groups Nitrosamino Nitrosimino Nitroso Nonanoyl (from nonanoic acid) Oleyl (from oleic acid) Oxalyl (from oxalic acid) Oxamido Oxo (keto) Palmityl (from palmitic acid) Pelargonyl (from pelargonic acid) Pentamethylene Pentyl Phenacyl Phenacylidene Phenanthryl Phenethyl Phenoxy Phenyl Phenylene Phenylenedioxy Phosphino Phosphinyl Phospho Phosphono Phthalyl (from phthalic acid) Picryl (2,4,6-trinitrophenyl) Pimelyl (from pimelic acid) Piperidino Piperidyl (piperidinyl) Piperonyl Pivalyl (from pivalic acid) Prenyl (3-methyl-2-butenyl) Propargyl (2-propynyl) Propenyl iso-propenyl Propionyl Propoxy Propyl iso-propyl Propylidene Pyridino Pyridyl (pyridinyl) Pyrry1 (pyrrolyl) Salicyl (2-hydroxybenzoyl) Selenyl Seryl (from serine) Siloxy Silyl Silylene Sorbyl (from sorbic acid)


ONNH– ONN= ON– CH3(CH2)7CO– CH3(CH2)7CH=CH(CH2)7CO– –OCCO– H2NCOCONH– O= CH3CH2)14CO– CH3(CH2)7CO– –(CH2)5– CH3(CH2)4– C6H5COCH2– C6H5COCH= (Cl4H9)– C6H5CH2CH2– C6H5O– C6H5– –C6H4– –OC6H4O– H2P– H2P(O)– O2P– (HO)2P(O)– 1,2-C6H4(CO–)2 2,4,6–(NO2)2C6H2– –OC(CH2)5CO– C5H10N– (C5H10N)– 3,4-(CH2O2)C6H3CH2– (CH3)3CCO– (CH3)2C=CHCH2– HC≡CCH2– CH2=CHCH2– (CH3)2C= CH3CH2CO– CH3CH2CH2O– CH3CH2CH2– (CH3)2CH– CH3CH2CH= C5H5N– (C5H4N)– (C3H4N)– 2–HOC6H4CO– HSe– HOCH2CH(NH2)CO– H3SiO– H3Si– H2Si= CH3CH=CHCH=CHCO–


TABLE 33.71 (Continued) Chemical Functional Groups Stearyl (from stearic acid) Styryl Suberyl (from suberic acid) Succinamyl Succinyl (from succinic acid) Sulfamino Sulfamyl Sulfanilyl Sulfeno Sulfhydryl (mercapto) Sulfinyl Sulfo Sulfonyl Terephthalyl Tetramethylene Thenyl Thienyl Thiobenzoyl Thiocarbamyl Thiocarbonyl Thiocarboxy Thiocyanato Thionyl (sulfinyl) Thiophenacyl Thiurain(aminothioxomethyl) Threonyl (from threonine) Toluidino Toluyl Tolyl (methylphenyl) α-tolyl Tolylene (methylphenylene) α-tolylene Tosyl [(4-methylphenyl) sulfonyl)] Triazano Trimethylene Triphenylmethyl (trityl) Tyrosyl (from tyrosine) Ureido Valeryl (from valeric acid) Valyl (from valine) Vinyl Vinylidene Xenyl (biphenylyl) Xylidino Xylyl (dimethylphenyl) Xylylene

CH3(CH2)16CO– C6H5CH=CH– –OC(CH2)6CO– H2NCOCH2CH2CO– –OCCH2CH2CO– HOSO2NH– H2NSO– 4-H2NC6H4SO2– HOS– HS– OS= HO3S– –SO2– 1,4–C6H4(CO–)2 –(CH2)4– (C4H3S)CH– (C4H3S)– C6H5CS– H2NCS– –CS– HOSC– NCS– –SO– C6H5CSCH2– H2NCS– CH3CH(OH)CH(NH2)CO– CH3C6H4NH– CH3C6H4CO– CH3C6H4– C6H5CH2– (CH3C6H3)= C6H5CH= 4–CH3C6H4SO2– H2NNHNH– –(CH2)3– (C6H5)3C– 4–HOC6H4CH2CH(NH2)CO– H2NCONH– C4H9CO (CH3)2CHCH(NH2)CO– CH2=CH– CH2=C= C6H5C6H4– (CH3)2C6H3NH– (CH3) 2C6H3– –CH2C6H4CH2–

From CRC Handbook of Chemistry and Physics, 73rd ed., Lide, D. R., Ed., CRC Press, Boca Raton, FL, 1992–1993. With permission.



Table 33.72 gives selected properties of 20-α-amino acids commonly found in proteins. The compounds are listed in alphabetical order by the three-letter symbols. Dissociation constants refer to aqueous solutions at 25°C. Mr = molecular weight; Tm = melting point; pKa = negative of the logarithm of the dissociation constant for the α-COOH group; pKb = negative of the logarithm of the dissociation constant for the α – NH3+ group; pKx = negative of the logarithm of the dissociation constant for any other group present in the molecule; pI = pH at the isoelectronic point; S = solubility in water at 25°C in units of grams per kilogram of water.

TABLE 33.72 Properties of Common Amino Acids Symbol Ala Arg Asn Asp Cys Glu Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Try Val

Name

Mol. form.

Mr

tm/°C

pKa

pKb

Alanine Arginine Asparagine Aspartic acid Cysteine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine

C3H7NO2 C6H14N4O2 C4H8N2O3 C4H7NO4 C3H7NO2S C5H9NO4 C5H10N2O3 C2H5NO2 C6H9N3O2 C6H13NO2 C6H13NO2 C6H14N2O2 C5H11NO2S C9H11NO2 C5H9NO2 C3H7NO3 C4H9NO3 C11H12N2O2 C9H11NO3 C5H11NO2

89.09 174.20 132.12 133.10 121.16 147.13 146.15 75.07 155.16 131.17 131.17 146.19 149.21 165.19 115.13 105.09 119.12 204.23 181.19 117.15

297 244 235 270 240 160 185 290 287 284 293 224 281 283 221 228 256 289 343 315

2.33 2.03 2.16 1.95 1.91 2.16 2.18 2.34 1.70 2.26 2.32 2.15 2.16 2.18 1.95 2.13 2.20 2.38 2.24 2.27

9.71 9.00 8.73 9.66 10.28 9.58 9.00 9.58 9.09 9.60 9.58 9.16 9.08 9.09 10.47 9.05 8.96 9.34 9.04 9.52

pKx 12.10 3.71 8.14 4.15

6.04

10.67

10.10

pI

S/g kg–1

6.00 10.76 5.41 2.77 5.07 3.22 5.65 5.97 7.59 6.02 5.98 9.74 5.74 5.48 6.30 5.68 5.60 5.89 5.66 5.96

165.0 182.6 25.1 4.95 v.s. 8.61 42 250.9 43.5 34.2 22.0 5.8 56 27.9 1623 421.7 98.1 13.2 0.46 88.5

Mr — Molecular weight Tm — Melting point pKa, pKb, pKc, pKd — Negative of the logarithm of the acid dissociation constants for the COOH and NH2 groups (and, in some cases, other groups) in the molecule (at 25°C) pI — pH at the isoelectric point S — Solubility in water at 25°C in units of grams of compound per kilogram of water; when quantitative data are not available, the notations sl.s. (for slightly soluble and v.s. (for very soluble) are used.



D. MATERIAL SAFETY DATA SHEETS TABLE 33.73 Information Disclosed on a Material Safety Data Sheet (MSDS) SECTION 1.

SECTION 2.

SECTION 3.

SECTION 4.

SECTION 5.

SECTION 6. SECTION 7.

SECTION 8.

SECTION 9.

Chemical Product and Company Identification Product Name Generic Names/Synonyms Product Use Manufacturer’s Name and Address Name and Phone Number of the Person/Group Who Prepared the MSDS Date MSDS was Prepared Emergency Phone Number Composition/Information on Ingredients Ingredient Name(s) CAS Number(s) Percent by Weight Hazards Identification Potential Human Health Hazards: To Skin (irritancy, sensitization) To Eyes (irritancy) via Inhalation (acute effects) via Ingestion (acute effects) Delayed Effects (chronic effects) Carcinogenicity, reproductive and developmental effects, mutagenicity, other First Aid Measures Specific First Aid Measures for Various Routes of Exposure Notes to Physician Including Antidotes and Medical Conditions Affected by the Product Fire Fighting Measures Flammable Properties (flashpoint, autoignition temperature, etc.) Extinguishing Media Hazardous Combustion Products Explosion Hazards Firefighting Precautions and Instructions Accidental Release Measures Procedures to be Followed in Case of Spill or Other Release Handling and Storage Normal Handling Procedures Storage Recommendations Exposure Controls/Personal Protection Engineering Controls Personal Protective Equipment Exposure Guidelines (TLV, PEL, other) Physical and Chemical Properties Appearance Boiling Point Physical State Melting Point Odor Vapor Pressure Specific Gravity Vapor Density Solubility Evaporation Rate pH % Volatiles



TABLE 33.73 (Continued) Information Disclosed on a Material Safety Data Sheet (MSDS) SECTION 10.

SECTION 11.

SECTION 12.

SECTION 13. SECTION 14.

SECTION 15.

SECTION 16.

Stability and Reactivity Stability Conditions Incompatibilities Hazardous Decomposition Products Hazardous Polymerization Toxicity Information Acute Effects (LD50, LC50) Subchronic and Chronic Effects Irritancy Sensitization Neurotoxicity Reprotoxicity Developmental Toxicity Mutagenicity Ecological Information Aquatic Toxicity Terrestrial Toxicity Bioaccumulation Potential Biodegradability Microbial Toxicity Disposal Information Shipping Information D.O.T. Hazard Class D.O.T. I.D. Number Regulatory Information TSCA Inventory Status Other Federal, State, Local Foreign Regulatory Information Information not covered in the other 15 sections

REFERENCES Fasman, G. D., Ed., Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Boca Raton, FL, 1989. Hinz, H. J., Ed., Thermodynamic Datafor Biochemistry and Biotechnology, Springer-Verlag, Heidelberg, 1986. Smith, E. L. et al., Principles of Biochemistry, 7th ed., McGraw Hill, New York, 1983.


Handbook of toxicology

Handbook of Toxicology, Second Edition

Poisoning and Toxicology Handbook

Preclinical Development Handbook: Toxicology