Animal Models in Toxicology

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Second Edition

Animal Models in Toxicology

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Second Edition

Animal Models in Toxicology Edited by

Shayne C. Gad

Boca Raton London New York

CRC is an imprint of the Taylor & Francis Group, an informa business

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2007 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8247-5407-7 (Hardcover) International Standard Book Number-13: 978-0-8247-5407-5 (Hardcover) 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. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Animal models in toxicology / edited by Shayne C. Gad. -- 2nd ed. p. cm. Includes bibliographical references and index. ISBN 13: 978-0-8247-5407-5 (alk. paper) ISBN 10: 0-8247-5407-7 (alk. paper) 1. Toxicology-Animal models. 2. Toxicity testing. I. Gad Shayne C., 1948[DNLM: 1. Animals, Laboratory. 2. Toxicology-methods. 3. Models, Animal. 4. Toxicity Tests. QY 50 A59755 2006] RA1199.4.A54A52 2006 615.9--dc22

2005034787

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Preface Biomedical sciences’ use of animals as models to help understand and predict responses in humans, in toxicology and pharmacology in particular, remains both the major tool for biomedical advances and a source of significant controversy. On one hand, animal models have provided the essential components for research and serve as the source that has permitted the explosive growth of understanding in these fields, with a multitude of benefits to both humans and other animal species. At the same time, the benefits of such use, balanced against costs in terms of animal lives, potential suffering, and discomfort, have been subject to continuous criticism and questioning. The questioning has stimulated significant and continuous advances in the humane use of animals and understanding of the relevance of findings to what might happen in similarly exposed people. These advances are reflected in this new edition of Animal Models in Toxicology. Every section has been updated, and more guidance has been directed to specific uses of animals in toxicology units. Scientists have used animal models for so long that there is truth in the belief that many researchers employ animals primarily out of habit, with little or no thought as to what the best tools and the optimum ways of using them are. At the same time, although there are elements of poor practice that are real, by and large animals have worked exceptionally well as predictive models for humans—when properly used (Gad 1990; see also chapters 13 and 14 in this volume). Regulations governing the purchase, husbandry, and use of animals in research have continued to change over the course of the 21st century. Indeed, in some countries (and even cities in the United States), such use has been banned and some sources made unavailable. The real and most apparent problems underlying the failure of animal models arise primarily from selecting the wrong model, in not using an animal model correctly, or in extrapolating results to humans poorly. In addition, most graduate degree programs do not currently address these issues well (if at all) in their curricula. Indeed, broad training in animal model selection and use, and the techniques involved in such research, is currently available but not well utilized. This text originally was developed to address these needs. Indeed, it is essential to the performance of good science that the correct species be used as a model, and that data be analyzed appropriately. Chapter 1 presents a historical review of the use of animal models and an overview of broad considerations of metabolism and relevance to use in toxicology. The core of the book, however, is in chapters 2 through 10. Each of these chapters represents the joint efforts of experts in toxicology (addressing techniques for animal use and husbandry and peculiarities of the species as a toxicological model), toxicological pathology, and species-specific metabolism. For an investigator who is not well versed in the use of a particular species, each of these chapters provides an excellent introductory “course,” along with guidance to the literature for more detailed understanding. All the major species used (and strains or breeds within these species) are addressed in these core chapters. Chapter 11 presents the case for a range of species (fish, pigs, earthworms, etc.) that are not commonly used for safety assessment studies but that might provide useful alternative models for some specific endpoints. In chapter 12, Robert L. Hall presents and discusses the special considerations regarding the evaluation and interpretation of clinical pathology of the eight major model species. Chapter 13 addresses in detail the general case of how to select a model species and how to extrapolate the results to humans. Chapter 14 details the pitfalls in the process—the situations that cause either human or model to be significantly more sensitive than the other or totally irrelevant to each other in specific cases. Chapter 15 presents an overview of the regulations that govern how laboratory animals are obtained, maintained, and utilized. Such laws have become increasingly complex, and an understanding of what can (and cannot) be done is essential for the modern researcher.

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The Appendix provides a quick guide to the major commercial sources of laboratory animals, whether common (rats and mice) or harder to come by (Chinese hamsters and primates). Information on the selection and use of common anesthetics, drugs, and pharmacological agents for use in laboratory animals is available in Borchards’ Drug Dosage in Laboratory Animals: A Handbook (Telford Press). The aim of this volume is to provide a single source reference for the use of animal models in toxicology. Shayne C. Gad

REFERENCE Gad, S. C. (1990). Model selection in toxicology: Principles and practice. J. Am. Coll. Toxicol. 9, 291–302.

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The Editor Shayne C. Gad, Ph.D., DABT, has been the principal of Gad Consulting Services since 1994. He has more than 30 years of broad-based experience in toxicology, drug, and device development; document preparation; statistics; and risk assessment, having previously been director of toxicology and pharmacology for Synergen (Boulder, Colorado), director of medical affairs technical support system services for Becton Dickinson (Research Triangle Park, North Carolina), and senior director of product safety and pharmacokinetics for G.D. Searle (Skokie, Illinois). Dr. Gad is past president of the American College of Toxicology, a board-certified toxicologist (DABT), and fellow of the Academy of Toxicological Sciences. He is also a member of the Society of Toxicology, Teratology Society, Society of Toxicological Pathologies, Biometrics Society, and the American Statistical Association. Dr. Gad has previously published 29 books and more than 300 chapters, papers, and abstracts. He has contributed to and has personal experience with IND (he has successfully filed more than 75 of these), NDA, PLA, ANDA, 510(k), IDE, and PMS preparation, and has broad experience with the design, conduct, and analysis of preclinical and clinical safety and pharmacokinetic studies for drugs, devices, and combination products. He is also a retired Navy captain with extensive operational experience overseas.

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Contributors Gary B. Baskin Charles River Laboratories, Inc.

Karen M. MacKenzie Independent consultant

John C. Bernal Charles River Laboratories, Inc.

Daniel E. McLain Powderject

Byron G. Boysen Hazleton Wisconsin, Inc.

Glen K. Miller G. D. Searle and Company

Zuhal Dincer Scantox A/S

Joyce K. Nelson Charles River Laboratories, Inc.

Charles H. Frith Toxicology Pathology Associates

John C. Peckham Experimental Pathology Laboratories

Dawn G. Goodman Covance Inc.

Clare M. Salamon Takeda Global Research & Development Center

Gillian Haggerty Midwest Bio Research

Mette Tingleff Skaanild The Royal Veterinary and Agricultural University

Robert L. Hall Covance Laboratories

Ove Svendsen The Royal Veterinary and Agricultural University

Frederick G. Hess BASF Corporation

Mark D. Walker Charles River Laboratories, Inc.

Mark Johnson MPI

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Contents Chapter 1 Introduction ........................................................................................................................................1 Shayne C. Gad Chapter 2 The Mouse........................................................................................................................................19 Toxicology .................................................................................................................................24 Shayne C. Gad Pathology...................................................................................................................................72 Charles H. Frith, Dawn G. Goodman, and Byron G. Boysen Metabolism..............................................................................................................................122 Shayne C. Gad Chapter 3 The Rat...........................................................................................................................................147 Toxicology ...............................................................................................................................150 Mark D. Johnson Pathology.................................................................................................................................193 Shayne C. Gad Metabolism..............................................................................................................................217 Shayne C. Gad Chapter 4 The Hamster...................................................................................................................................277 Toxicology ...............................................................................................................................280 Shayne C. Gad Pathology.................................................................................................................................304 Frederick G. Hess Metabolism..............................................................................................................................312 Shayne C. Gad Chapter 5 The Guinea Pig ..............................................................................................................................333 Toxicology ...............................................................................................................................336 Shayne C. Gad Pathology.................................................................................................................................371 John C. Peckham Metabolism..............................................................................................................................400 Shayne C. Gad Chapter 6 The Rabbit......................................................................................................................................421 Toxicology ...............................................................................................................................424 Clare M. Salamon and Karen M. MacKenzie Pathology.................................................................................................................................449 John C. Peckham Metabolism..............................................................................................................................475 Shayne C. Gad

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Chapter 7 The Ferret.......................................................................................................................................493 Toxicology ...............................................................................................................................496 Daniel E. McLain Pathology.................................................................................................................................543 Sundeep Chandra Metabolism ..............................................................................................................................550 Shayne C. Gad Chapter 8 The Dog..........................................................................................................................................563 Toxicology ...............................................................................................................................566 Gillian C. Haggerty Pathology.................................................................................................................................588 John C. Peckham and Robert W. Thomassen Metabolism ..............................................................................................................................645 Shayne C. Gad Chapter 9 Primates..........................................................................................................................................663 Toxicology ...............................................................................................................................665 Mark D. Walker, Joyce K. Nelson, and John C. Bernal Pathology.................................................................................................................................706 Gary B. Baskin Metabolism ..............................................................................................................................716 Shayne C. Gad Chapter 10 The Minipig....................................................................................................................................731 Toxicology ...............................................................................................................................732 Shayne C. Gad Pathology.................................................................................................................................739 Zuhal Dincer and Ove Svendsen Metabolism..............................................................................................................................760 Mette Tingleff Skaanild Chapter 11 Alternative Species.........................................................................................................................773 Shayne C. Gad Chapter 12 Clinical Pathology of Laboratory Animals....................................................................................787 Robert L. Hall Chapter 13 Model Selection and Scaling .........................................................................................................831 Shayne C. Gad

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Chapter 14 Susceptibility Factors .....................................................................................................................863 Shayne C. Gad Chapter 15 Laws and Regulations Governing Animal Care and Use in Research .........................................901 Shayne C. Gad Appendix Commercial Sources of Laboratory Animals ................................................................................921 Shayne C. Gad Index...............................................................................................................................................927

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1 Introduction Shayne C. Gad Gad Consulting Services

CONTENTS Current Animal Studies......................................................................................................................2 Origins of Predictive Animal Testing ................................................................................................3 The “Lash Lure” Case..............................................................................................................3 The Elixir of Sulfanilamide Case ............................................................................................3 Thalidomide..............................................................................................................................3 Selecting an Animal Model ...............................................................................................................5 Husbandry and Care ..........................................................................................................................5 Caging.......................................................................................................................................5 Choosing Species and Strains............................................................................................................6 Dosing ................................................................................................................................................6 Animal Physiology.............................................................................................................................7 Background Incidence of Disease and Neoplasia ...................................................................7 Responses to Biologically Active Agents ................................................................................7 Absorption, Distribution, Metabolism, and Excretion of Chemicals......................................8 Absorption ....................................................................................................................8 Distribution .................................................................................................................10 Metabolism .................................................................................................................10 Xenobiotic Metabolism...............................................................................10 Enzymes Involved in Xenobiotic Metabolism ...........................................11 Excretion.....................................................................................................................14 Summary ..........................................................................................................................................15 References ........................................................................................................................................15

The use of animals in experimental medicine, pharmacology, pharmaceutical development, safety assessment, and toxicological evaluation has become a well-established and essential practice. Whether serving as a source of isolated organelles, cells or tissues, a disease model, or as a prediction for drug or other xenobiotic action or transformation in man, experiments in animals have provided the necessary building blocks that have permitted the explosive growth of medical and biological knowledge in the later half of the 20th century and into the 21st century (Meier and Stocker 1989; 1

2

ANIMAL MODELS IN TOXICOLOGY

Nevalainen et al. 1996). Animal experiments also have served rather successfully as identifiers of potential hazards to and toxicity in humans for synthetic chemicals with many intended uses. Animals have been used as models for centuries to predict what chemicals and environmental factors would do to humans. The earliest uses of experimental animals are lost in prehistory, and much of what is recorded in early history about toxicology testing indicates that humans were the models of choice. The earliest clear description of the use of animals in the scientific study of the effects of environmental agents appears to be by Priestley (1792) in his study of gases. The first systematic use of animals for the screening of a wide variety of agents was published by Orfila (1814), and was described by Dubois and Geiling (1959) in their historical review. This work consisted of dosing test animals with known quantities of agents (poisons or drugs), and included the careful recording of the resulting clinical signs and gross necropsy observations. The use of animals as predictors of potential ill effects has grown since that time.

CURRENT ANIMAL STUDIES The current regulatory required use of animal models in acute testing began by using them as a form of instrument to detect undesired contaminants. For example, miners used canaries to detect the presence of carbon monoxide, a case in which an animal model is more sensitive than humans (Burrell 1912). In 1907, the U.S. Food and Drug Administration (FDA) started to protect the public by the use of a voluntary testing program for new coal tar colors in foods. This was replaced by a mandatory program of testing in 1938, and such regulatory required animal testing programs have continued to expand until recently. The knowledge gained by experimentation on animals has undoubtedly increased both the length and quality of our lives, an observation that most reasonable people would find difficult to dispute, but it (as reviewed by Ewald and Gregg 1983) has also benefited animals. As is the case with many tools, animals have sometimes been used inappropriately. These unfortunate instances have helped fuel the actions of a vituperative animal “rights” movement. This movement has encouraged a measure of critical self-appraisal on the part of scientists concerning the issues of the care and usage of animals. The Society of Toxicology (SOT) and the American College of Toxicology (ACT) have both established Animals in Research Committees, and these have published guidelines for the use of animals in research and testing. In general, the purpose of these committees is to foster thinking on the four Rs of animal-based research: reduction, refinement, (research into) replacements, and responsible use. This new edition is, in part, a response to these continued concerns. The media frequently carry reports that state that most (if not at all) animal testing and research is not predictive of what will happen in people, and therefore such testing is unwarranted. Many animal rights groups also present this argument at every opportunity, and reinforce it with examples that entail seemingly great suffering in animals but add nothing to the health, safety, and welfare of society (e.g., Fano 1998). This is held to be especially the case for safety testing and research in toxicology. Animal rights activists try to “prove” this point by presenting examples of failure (e.g., thalidomide*). In light of the essential nature of animal research and testing in toxicology, this is equivalent to seeking to functionally disarm us as scientists. Our primary responsibility (the fourth R) is to provide the information to protect people and the environment, and without animal models we cannot discharge this responsibility. Confronted with this argument, all too many toxicologists cannot respond with examples to the contrary. Indeed, many might not even fully understand the argument at all. Very few are familiar enough with some of the history of toxicity testing to be able to counter with examples where it has not only accurately predicted a potential hazard to humans, but where research has directly benefited both people and animals. There are, however, many such examples. Demonstrating the * Where the lack of adequate testing (or interpretation of existing test results) prior to marketing is not pointed out.

INTRODUCTION

3

actual benefit of toxicology testing and research with examples that directly relate to the everyday lives of most people and not esoteric, basic research findings (which are the most exciting and interesting products to most scientists) is not an easy task. Examples that can be seen to affect neighbors, relatives, and selves on a daily basis would be the most effective. The problem is that toxicology is, in a sense, a negative science. The things we find and discover are usually adverse. If the applied end of our science works correctly, the results are things that do not happen, and therefore are not seen. If we correctly identify toxic agents (using animals and other predictive model systems) in advance of a product or agent being introduced into the marketplace or environment, generally it will not be introduced (or it will be removed) and society will not see death, rashes, renal and hepatic diseases, cancer, or birth defects, for example. As these things already occur at some level in the population, it would seem that seeing less of them would be hard to firmly tie to the results of toxicity testing that relies on animals. In addition, the fact that animals are predictive models for man is controversial.

ORIGINS OF PREDICTIVE ANIMAL TESTING The actual record of evidence for the predictive value of animal studies and how they have benefited man and domestic animals is reviewed in the following two sections. However, the negative image needs to be rebutted. First, it must be remembered that predictive animal testing in toxicology, as we now know it, arose largely out of three historical events. The “Lash Lure” Case Early in the 1930s, an untested eyelash dye containing p-phenylenediamine (Lash Lure) was brought onto the market in the United States. This product (as well as a number of similar products) rapidly demonstrated that it could sensitize the external ocular structures, leading to corneal ulceration with loss of vision and at least one fatality (McCally et al. 1933). The Elixir of Sulfanilamide Case In 1937, an elixir of sulfanilamide dissolved in ethylene glycol was introduced into the marketplace. One hundred and seven people died as a result of ethylene glycol toxicity. The public response to these two tragedies helped prompt Congress to pass the Federal Food, Drug, and Cosmetic Act of 1938 (Pendergrast 1984). It was this law that mandated the premarket testing of drugs for safety in experimental animals. The most compelling evidence that should be considered is “negative”: Since the imposition of animal testing as a result of these two cases, no similar occurrence has happened, even though society uses many more consumer products and pharmaceuticals today than during the 1930s. Thalidomide The use of thalidomide, a sedative-hypnotic agent, led to some 10,000 deformed children being born in Europe. This in turn led directly to the 1962 revision of the Food, Drug and Cosmetic Act, requiring more stringent testing. Current testing procedures (or even those at the time in the United States, where the drug was never approved for human use) would have identified the hazard and prevented this tragedy. In fact, it has not occurred in Europe or the United States except when the results of animal tests have been ignored. Table 1.1 presents an overview of cases in which animal data predicted adverse effects in humans, and table 1.2 provides some examples of known toxic reactions to substances in animals and humans.

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Table 1.1 Animal Models That Predicted Adverse Effects of Xenobiotics on Humans Agent

Effect

Animal Species

In Man

Phenacetin Thalidomide Accutane

Neurotoxicity, carcinogenicity Phocomelia Developmental toxicity of central nervous system (neural tube defects) Bone marrow depression Cleft palate Nephropathy Reversible immune response suppression (essential aid to organ transplantation) Hepatotoxicity Photosensitivity Anaphylactic shock/allergy Parkinsonism Hemorrhagic cystitis Encephalopathy Nephropathy Myelomonocytic leukemia Birth defects (?)/Litogen Behavioral disturbances and amnesia Phototoxicity Hemolytic anemia Development abnormalities Nephrotoxitcity, hepatotoxicity

Rat Rat Rat, rabbit, dog

Y N/Y Y

AZT Valproic acid Cyclosporine

Benoxaprofen (Oraflex) Zomepirac MPTP Cyclophosphamide Mercury* Diethylene glycol* Razoxin Benedictin Triazolam (Halcion) Quinalones Temafloxcine Diazepam (Valium) Fialuridine (FIAU)

Dog, rat, monkey Rat, mouse, rabbit Rat, dog Rat, monkey

Y Y

No Guinea pig No Monkey Rat, dog Rat, monkey Rat, dog Mouse No No Guinea pig, in No Rat

Y

vitro

Y Y Y Y Y Y Y Y ?/Y Y Y Y

* Not drugs.

Table 1.2 Selection of Toxic Reactions Occurring in Animals and Man Substance

Reaction

Substance

Reaction

Acetaminophen Acrylamide Aniline Asbestos Atropine Benzene Bleomycin

Hepatic necrosis Peripheral neuropathy Methemoglobinemia Mesothelioma Constipation Leukemia Pulmonary fibrosis

Isotretinoin, prenatal Kanamycin Methanol Methoxyflurane 8-Methoxypsoralen Methyl mercury Morphine

Carbon disulphide Carbon tetrachloride Cis-platinum Cobalt sulphate Cyclophophamide Cyclosporin A D & C Yellow Diethylene glycol Diethylaminoethoxyhexoestrol Doxorubicin Emetine Ethylene glycol Furosemide Gentamycin Hexacarbons Hexachlorophene

Nervous system toxicity Hepatic necrosis Nephropathy Cardiomyopathy Hemorrhagic cystitis Nephropathy Eczema Nephropathy Phospholipidosis of liver

MPTP Musk ambrette 2-Naphthylamine Neuroleptic drugs Nitrofurantoin Paraquat Phenformin Phenothiazine NP 207 Penicillamine

Multiple malformations Cochlear toxicity Blindness (monkey) Nephropathy (Fischer rat) Phototoxicity Encephalopathy Physical and psychological appearance Parkinsonism Photosensitivity Bladder cancer Galactorrhoea Testicular damage Lung damage and fibrosis Lactic acidosis Retinopathy (pigmented animals) Loss of taste

Cardiomyopathy ECG abnormalities Obstructive nephropathy Hypokalemia Nephropathy Peripheral neuropathy Spongiform encephalopathy Peripheral neuropathy Stenocardia Goiter

Pyridoxin Scopolamine Slow release potassium Thalidomide, prenatal Triothocresylphosphate Triparanol Vinyl/chloride

Sensory neuropathy Behavioral disturbances Intestinal ulceration Phocomelia (monkey, rabbit) Delayed neuropathy Cataract Angiosarcoma of the liver

Vitamin A Vitamin D

Osteopathy Nephrocalcinosis

Isoniazid Isoproterenol Isothiocyanates

INTRODUCTION

5

For example, birth defects have occurred with isotretinoin (Accutane) where developmental toxicity had been clearly established in animals and presented on labeling, but the drug has continued to be used by potentially pregnant women. Research into replacements such as cellular cultures, organs harvested from slaughterhouses, computer modeling, and physical and chemical systems has been extensive (Frazier 1990; Gad 2000). Although each of these have its own utility (Gad 1989, 2000), they will not replace animals for the foreseeable future. Some degree of animal use will continue. We hope that this book will assist the responsible investigator in designing and interpreting appropriate experiments (refinement) that will require fewer animals (reduction) in which the animals are appropriately husbanded and utilized (responsibility). SELECTING AN ANIMAL MODEL Choosing the appropriate animal model for a given problem is sometimes guesswork and too often a matter of convenience. One often uses a species with which one is most familiar, with little consideration as to whether the chosen species is actually the most appropriate for the problem at hand. For example, the rat is probably a poor model for studying the chronic toxicity of any new nonsteroidal anti-inflammatory drug (NSAID) because the acute gastrointestinal (GI) toxicity will probably mask any other toxic effects. The guinea pig is less sensitive to most NSAIDs than the rat, and would therefore be a more appropriate species for investigating the chronic (nongastrointestinal) toxicity of an NSAID. This practice of not rationally choosing an appropriate species for an experiment undoubtedly results in imprecise or questionable science. This alone should be considered a waste of animals and resources. It also results in additional, and sometimes duplicative, experiments. We hope that this book will contribute to the reduction and refinement of the use of animals by helping to alleviate this practice. The core chapters (2–11) include discussions of the strengths and weaknesses of each of the common laboratory species, and recommendations for potential appropriate uses. Chapter 12 directly addresses the issue of how to select the best practical model.

HUSBANDRY AND CARE The quality of an experiment often hinges on the details of animal husbandry and care. At one extreme, inappropriate handling could result in unhealthy animals and an experiment yielding variable and irreproducible results. All animals have optimal temperature, humidity, light cycle, light intensity, cage size and bedding, and dietary requirements. Rabbits, for example, have a different optimal temperature range than rats. Rats and ferrets have completely different dietary requirements. Albino rodents have very sensitive eyes, and lights of too high a candle power can cause incidental ocular damage, especially in those animals on the top row of a cage rack. Infrequent changing of indirect bedding materials can result in exposure of rodents to a high airborne concentration of ammonia, which can cause ocular damage. Recently, it has become clear that ad lib feeding of rodents in chronic or carcinogenicity studies both shortens their lives and alters the patterns of spontaneous tumors that occur. These are all examples of how inattention to the details of animal care can compromise an experiment, particularly a long-term one. A refined and responsibly designed experiment accommodates these details. It is hoped that this book will provide a convenient source of husbandry procedures for the more common animal species used in toxicological and pharmacological research. Caging Caging deserves special mention for two reasons. First, not all animals can be group housed. Hamsters, for example, are notoriously antisocial. Even breeding pairs cannot be left in the same

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small cage together for protracted periods. Guinea pigs, on the other hand, thrive when group housed. Obviously these factors need to be considered when designing an experiment. In modern toxicology practice, animals are seldom group housed during chronic studies to maintain identification, facilitate clinical observations, and ensure necropsy of moribund or dead animals (mice, in particular, are very cannibalistic). With short-term experiments, however, group housing of certain animals might not compromise a study and might decrease the amount of housing space needed. This book discusses appropriate housing, including instances when animals should or should not be group housed. Second, cage size is important because the animal rights movement has made it important. Although most investigators (and cage manufacturers) have long recognized that cages have optimal sizes, the 1989 proposed Animal Welfare Codes (which became law in 1991, and have been revised since) attempted to specify somewhat larger cages with several size cutoffs mediating cage changes. For example, there are three to four different cage specifications for guinea pigs depending on their age and/or weight. Many caging systems currently in use would no longer be permitted and their replacement would be very expensive. There is no scientific basis for believing that these changes will improve animal husbandry or quality of life. This is just an example of how the animal rights movement, and the resultant animal care laws, could affect the conduct of pharmacologists and toxicologists. This book contains in-depth discussion on current animal welfare laws (chapter 15). The investigator needs to be aware of not only the four Rs, but also the relevant laws and regulations governing animal experimentation.

CHOOSING SPECIES AND STRAINS Not only is it important to pick the correct species for an experiment, but sometimes the correct strain as well. For most of the species discussed in this book there are a handful of commonly used strains. In some cases, an inbred strain might provide qualitative and specific characteristics that make it a good disease model, such as the spontaneously hypertensive rat. There are other more quantitative strain-related differences such as size, color, temperament, and background disease. For example, the Fischer 344 rat is smaller than the Sprague-Dawley rat. The CD-1 mouse is shorter lived than the C57B6/F1 hybrid. These differences might make a particular strain more appropriate for one experiment than others. For example, the Fischer 344 rat has a high rate of spontaneous Leydig cell tumors as compared to the Sprague-Dawley rat, which would make the latter less appropriate for determining if a chemical is a testicular carcinogen. For these reasons, this book includes discussions of strainrelated differences. Rats and mice provide the greatest array of strains from which to choose, including outbred and some inbred. There are literally hundreds to choose from, but the majority are specializeduse animals, such as the athymic nude mouse. For the majority of generalized pharmacology and toxicology testing, a relatively small handful of rat and mouse strains are used and the emphasis in the relevant chapters is on those more commonly used strains. Many chapters include some mention of strain; however, the situation with dogs is somewhat different. All domestic dogs belong to the same family, which is subdivided by breed. Only the beagle breed is purposely raised for biomedical research; otherwise one uses mongrel or random-source dogs (obtained from pounds and used without regard to breed). Hence, the chapter on the dog focuses on the beagle. There might be supplier-related differences in beagles, but these have not been systemically studied.

DOSING To study the effects of a drug or other chemical in an animal, the two have to be brought together; that is, the animal has to be dosed. Dosing is the act of introducing a drug or chemical

INTRODUCTION

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into a living organism. It requires active interaction between man and animal. There are, however, passive dosing techniques that are also used frequently in which the chemical is placed in the animal’s air, water, or feed, and the animal doses itself by breathing, drinking, or eating. Hence, administering an antibiotic intravenously is active dosing; giving it in the feed is passive dosing. In the former case, dosimetry (i.e., calculating milligrams per kilograms of exposure) is generally intuitively simple (an exception being for the dermal route). In the latter case, other measurements must be taken (e.g., feed consumption) and a variety of formulas are used in dosimetry. The main routes used for active dosing are oral, intravenous, intraperitoneal, dermal, and subcutaneous. Other routes are sometimes used, and these are mentioned where appropriate (for a complete discussion of different routes, see Gad and Chengelis 1998, Chap. 10; Gad 2000, 2002). For oral dosing, for example, one might have a choice of using capsules or gavaging. However, capsules are rarely used with rats and gavage is seldom used with dogs. When necessary, a dog can be gavaged, but the technique is different from that used with rats. Intravenous dosing of ferrets is especially difficult, but can be done. This book presents the appropriate techniques, “tricks of the trade,” so that animals can be appropriately and humanely dosed. Second, some of the information (e.g., average feed consumption) and formulas needed to calculate or estimate dosimetry in passive dosing procedures are presented and discussed. With regard to dosing and dosimetry, it should be kept in mind that the terms dose and dosage are not synonymous. The dose is the total amount of test article given, such as 1,000 mg. The dosage is a rate term and is the dose divided by the weight of the test animal; for example, 1,000 mg/10 kg (for a dog) = 100 mg/kg. For some agents (particularly oncology drugs), this is presented in terms of quantity per meter squared (m2) of body surface area. In most instances, when one speaks of a dose–response curve, a dosage–response curve is being described.

ANIMAL PHYSIOLOGY All animal species and strains have their own distinctive physiology. As a result, values pertaining to blood pressure, breathing rates, ECGs, rectal temperatures, and normal clinical laboratory parameters often vary between species. Clearly, appropriate interpretation of an in vivo experiment requires a firm understanding of these baseline data. For example, there are well-established differences between species with regard to red blood cell size: What is normal for a dog would be high for a rat. The converse is true for breathing rates. This book provides a convenient source for these important background data. Background Incidence of Disease and Neoplasia All animals also have their own baseline, or natural incidence, or diseases that complicate the conduct and interpretation of chronic toxicity experiments. The background incidence of liver tumors in C57B6/F1 mice is quite high. It would, perhaps, be prudent to investigate a suspect hepatocarcinogen in a species with a lower spontaneous incidence than these. Ferrets in the United States are currently contaminated by the Aleutian mink virus, which could make this species inappropriate for chronic experiments. The background incidence of these diseases and pathological lesions are discussed to aid the investigator in choosing the most appropriate species for an experiment and in the interpretation of the results. Responses to Biologically Active Agents An animal’s responses to drugs or other biologically active agents might be just as important as the background incidence of disease, and species-related differences in sensitivity are important for two reasons. First, animals will often have to be anesthetized or receive other treatment such

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as antibiotics during an experiment. Appropriate dosages vary among species. Thus, this book presents the appropriate dosages of common anesthetics for the model species discussed here. Second, the other reason species-related differences are important is that in toxicity testing, these differences are the major hurdle in applying toxicity data to human hazard assessment. This is perhaps too broad a topic for a single book, but mention is made so that an investigator is aware of such differences. Cats, for example, are far more sensitive to digitoxin (LD50 ≅ 180 µg/kg po), than other species, such as the rat (LD50 ≅ 56 mg/kg po, as reported in National Institute of Occupational Safety and Health 1980). There can also be qualitative differences among species. Morphine, for example, is infamous for causing different clinical signs in different species: straub tail in mice, catatonia in rats, and extreme reactivity in cats. Some of the more frequent examples of these distinctions are mentioned in the core chapters. The salient message is that species often differ both quantitatively and qualitatively in their responses to drugs or chemicals. These differences must be investigated and considered in choosing a species for an experiment and in interpreting the results. Incidentally, cats (with the exception of veterinary products intended for use in cats) are seldom used in toxicity testing and are used in pharmacology mainly for acute, terminal neurophysiological experiments. For these reasons, an in-depth discussion of cats is not included in this book. Absorption, Distribution, Metabolism, and Excretion of Chemicals When studying the effects of drugs and other chemicals on intact animals, it is also vitally important to investigate the processes of absorption, distribution, metabolism, and excretion (ADME). These have been intensely studied widely, and space does not permit a review of this large body of work. Some basic degree of knowledge must be presumed. We have compiled a list of references to which the reader can refer if additional information is needed (table 1.3). For the remainder of this chapter, we touch on some basic principles that apply across all species. In each individual core chapter (2–10) of this book, some basic information on ADME is presented on a species-specific basis. The emphasis is on providing the information necessary to assist one in (a) the appropriate selection of an animal model, (b) the design of the experiment, (c) the interpretation of resultant data, and (d) the applicability of the results to humans. The principles that govern absorption and distribution apply fairly equally across all species (Pratt and Taylor 1990; Washington et al. 2001), and therefore are not discussed to any great extent on an animal-by-animal basis. It is most difficult to predict species differences in bioavailability (absorption across GI tract into the blood) or systemic bioavailability (bioavailability + first-pass metabolism) of a specific chemical. Species differences in gastric or intestinal pH, for example, may dictate species differences in GI permeability to specific chemicals, but will not account for differences in GI transit time or hepatic metabolism. Assumptions based solely on phylogenetic grounds can be quite misleading. We had recent experience with a drug found to be bioavailable in the rat and dog, but not at all absorbed in the monkey. In fact, the dog was the species most similar to the human. One needs to strive to ascertain test article bioavailability experimentally for any specific chemical, as general principles always come encumbered with exceptions. Absorption After dosing, a chemical must be absorbed to cause an effect. Absorption is the process of the chemical passing through a barrier to gain access to the general systemic circulation. The most common dosage routes are oral, inhalation, topical, intraperitoneal, intravenous (IV), subcutaneous (SC), and intramuscular (IM). Absorption is not generally a problem by the latter three routes as the test substance is introduced directly to the body. It is normally a foregone conclusion that drugs

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Table 1.3 Summary of General Reviews of Xenobiotic Metabolism Topic

Source

General reviews on process of drug metabolism and disposition

LaDu et al. (1972) Goldstein et al. (1974) Klaassen (1986) Sipes and Gandolfi (1986) deBethizy and Hayes (1989) Rozman (1988) Levy et al. (2000) Gonzales (1988) Black and Coon (1986) Kadlubar and Hammons (1987) Lewis (2001) Ziegler (1988) Tynes and Hodgson (1985) Seidegard and DePierre (1983) Oesch (1972) Jarina and Bend (1977) Pickett and Lu (1989) Boutin (1987) Mulder (1986) Siest et al. (1989) Singer (1985) Jacoby et al. (1984) Hirom et al. (1977) Lower and Bryan (1973) Leinweber (1987) Hawkins and Kalant (1972) Crabb et al. (1987) Klaassen and Watkins (1984) Levin (1978)

Cytochrome P-450

Flavin-dependent microsomal mixed function oxidase Epoxide hydrolase Glutathione S-transferase UDP-glucuronosyl transferase/glucronidation PAPS-Sulfotransferase/sulfate formation Amino acid conjugations Acetylations Esterases Alcohol metabolism Billiary excretion

so administered will reach the systemic circulation. Plasma concentrations will depend on rates of delivery (IV), or rates of diffusion (IM, SC). Although there are some technical concerns, the principles are either independent of species, or the species differences are obvious. For example, because of relative small muscle mass and rapid circulation time, drugs given intramuscularly will more rapidly equilibrate in rats than in monkeys. Via the intraperitoneal route, systemic availability will depend not only on the rates of diffusion, but also on the first-pass metabolism effect. There are no known species differences with regard to intraperitoneal absorption, but there are species differences with regard to rates of hepatic metabolism, which may dictate the degree of first-pass metabolism. Interestingly, first-pass effects are generally of greater concern in smaller species, such as rats and mice, where the intraperitoneal route is more commonly used. With regard to the oral, dermal, and inhalation routes, there are very real species differences. For example, thickness and length of the small intestine, size of cecum (if indeed there is one), and gut transit time all play a role in gastrointestinal absorption. Species differences in facilitated or active transport might also play a role in absorption. Whether an animal is an obligate nose breather or not, the structure of the nasal turbinates, respiration rate, and minute volume will all influence the size and number of particles reaching the alveoli by the inhalation route. The rat is a poor model for inhalation pharmacokinetic studies in extrapolating the results to humans for these reasons. There are well-described differences in skin structure that control dermal absorption and result in species differences. Such species differences vary with chemical class. This book might help one sort through this maze, but there are few scientifically sound generalizations. Our best recommendation is that investigators substantiate their assumptions on dermal

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or inhalation absorption before rendering any conclusion on studies conducted using these routes of administration. Distribution After gaining access to the systemic circulation, the toxin or drug is distributed among the organs. Distribution depends on these factors: • • • •

Blood flow to the organ Extent and avidity of binding to plasma proteins* The “natural” affinity a particular organ might have The degree and extent to which the chemical crosses barriers such as the blood–brain barrier or the placenta • The extent to which clearance (metabolism and/or excretion) competes with these processes

There are probably species differences with regard to all these processes, but not all have been vigorously explored. For example, there are few comparative studies on the blood–testes barrier, or comparisons on plasma protein binding of different chemicals in the monkey, so the database in this area is surprisingly small. A few transspecies comparisons of plasma protein binding have been done. As a broad generalization, binding is most extensive in humans and least extensive in mice. Such information is presented and discussed in the core chapters, but the reader should be aware of the gaps in the available knowledge. Metabolism In the area of ADME, the processes of metabolism or bioconversion are of greatest concern with regard to species-specific differences. Indeed, species differences in metabolism are a leading cause for species differences in toxicity. First, very few administered xenobiotics are excreted unchanged. Therefore, rates of metabolism often dictate the time length of a pharmacodynamic response. Second, metabolism of a xenobiotic might result in metabolites of similar potency or produce metabolites that are responsible for toxicity. For example, most genotoxic carcinogens require metabolic activation. Finally, because the metabolism of xenobiotics is an enzyme-based phenomenon, it shows a great deal of species differences. For example, Williams (1972) examined the metabolism of phenol, a relatively simple chemical, in 13 different species and found that no two species provided the same spectrum of metabolites. Species differences can be either quantitative (differing amounts of the same metabolites) or qualitative (different metabolites). Because of the importance of metabolism in toxicity testing, each individual animal chapter contains indepth discussion of xenobiotic metabolism. Xenobiotic Metabolism The area of species differences in xenobiotic metabolism is not new. Some of the efforts in this area are summarized in table 1.3. It is not the objective of this book to provide yet another interspecies comparison, but rather to present information on a species-specific basis. For example, what type of regimen is required to induce increases in microsomal multifunction oxidase (MMFO) activity in the dog? The metabolism of xenobiotics by mammals is a phenomenon that has been recognized since 1842 when Keller (Mandel 1972) identified that benzoic acid was excreted in the urine as the 1 cine conjugate (hi uric acid). As a modern science, drug (or xenobiotic) metabolism was formalized in the late 1940s when Williams (1947) published the first text on the * Interestingly, it has recently become clear that even proteins (such as some of the biotechnologically generated pharmaceutical agents) can be bound to plasma proteins.

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subject. Williams (see Williams 1972, 1974, for reviews) has been particularly instrumental in the area of species differences in metabolism. Early works in this area tended to concentrate on isolating and identifying various conjugations of simple chemicals given to intact animals. Mueller and Miller published on the importance of liver microsomes in xenobiotic metabolism in their studies on the oxidative metabolism of aminazodyes (Mannering 1972; Mueller and Miller 1949). The field has grown explosively since the mid-1950s, catalyzed by the studies of Brodie and colleagues in the United States (Brodie et al. 1955; Quinn et al. 1958) and Remmer in Germany (Mannering 1972; Remmer and Merker 1963). Their works confirmed the quantitative importance of the liver in xenobiotic metabolism, and that the major underlying enzymes were located in the microsomal fraction. It was during this period that the practice of naming an enzyme by its activity, such as aminopyrine demethylase or aniline hydroxylase, was adopted. It was only later that it was recognized that all these activities are catalyzed by the same enzymes (or family thereof); that is, the cytochrome P-450-dependent microsomal mixed function oxidase system (Gonzales 1988; Guengerich 1988). Cytochrome P-450 was discovered almost a decade after Mueller and Miller described microsomal metabolism requiring NADPH (Coon 1978; Klingenberg 1958; Mannering 1972). The importance of the identification and characterization of the cytochrome P450-dependent MMFO system to the fields of biochemistry, pharmacology, and toxicology cannot be understated. The reader is referred to any one of several reviews of the system (see table 1.1 and table 1.4). The process of xenobiotic metabolism has traditionally been divided into Phase I (oxidative) and Phase II steps. In general (as reviewed more extensively by deBethizy and Hayes [1989]), all mammalian processes are designed to convert lipophilic chemicals to more polar and more easily excreted metabolites. In reality, the process can be more complicated than two steps because the products of Phase I oxidation can be (a) further hydroxylated at different sites, (b) further oxidized at the same site (by a different enzyme such as alcohol dehydrogenase), or (c) conjugated with glutathione or glucuronic acid, sulfate, or one of several amino acids. This process is discussed in greater detail elsewhere (see table 1.1 and table 1.2). The result is that any one xenobiotic can have an astonishing spectrum of metabolites. For example, benzene is a relatively simple chemical, yet over 15 different metabolites have been described. Enzymes Involved in Xenobiotic Metabolism The main enzymes involved in xenobiotic metabolism are fairly uniform across species. In all mammalian species, the liver is quantitatively the most important site of xenobiotic metabolism, and the MMFO system is the most important enzyme. Although this system is ubiquitous, there are species differences in isozymic characteristics, substrate specificity, activity, and inducibility. More recently, Davin-dependent microsomal multifunction oxidase, which is distinct from the MMFO, has been identified (Ziegler 1988), and has been shown to play a role in the metabolism of many chemicals. There are also differences in Phase II enzyme activities and cosubstrate availability (Gregus et al. 1983). Seldom do two species dispose of the same chemical that way. Each species produces a spectrum of metabolite or chromatographic “fingerprints” that are often distinct. The characteristics of the MMFO for each of the most highly used species are discussed in this book in some detail. Other enzyme systems such as the flavin-dependent (noncytochrome P-450) monoxygenase can also be involved in xenobiotic oxidative metabolism, and are discussed where available information permits. The species characteristics of other important enzymes such as epoxide hydrolase and UDP-glucuronosyl transferase are also discussed. Some enzymes are ubiquitous, such as the alcohol dehydrogenase and carboxylesterase. All species metabolize primary alcohols to aldehyde, and subsequently to carboxylic acids. This is only discussed, therefore, when there is some species-specific characteristic. This is also true for esterases, as all species rapidly hydrolyze esters.

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Table 1.4 Compilation of Selected Papers That Compare Xenobiotic Metabolism in Different Species Species Compared Dog, guinea pig, rat, rabbit, monkey, human, mouse

Rat, mouse, guinea pig

Parameters Examined

Comments

References

Gastrointestinal differences that affect absorption; plasma and tissue binding; drug metabolism in liver and intestine GSH-T

Rhesus monkey best predictor for ADME in man Excellent bibliography

Rozman (1988)

Neal et al. (1987)

Rat, mouse, guinea pig

Induction of MMFO, GSH-T, EH

Rat, dog, monkey

Inducing effect of hexahydroindazole (P450, AP-demeth, ANOH)

Rat, mouse, guinea pig, dog, monkey Rats, rabbits, hamster, guinea pig, ferret Various: emphasis on dog, mouse, rat, rabbit, monkey

Conjugation reactions

With CDNB total activity mouse > guinea pig > rat. Parallel to AFT sensitivity. Quantitative differences in isoenzymes. In general, MMFO activity, rat had lowest, but BP activity most inducible. In some species, induction had no effect or decreased some activities. Hamster > rabbit = gp > mouse > rat. S-Sepharose elution patterns different. Dog > rabbit = mouse > rat with same order to LD50. Highest bioavailability in rats. More unchanged drug in dogs and monkeys. EH-M gp > rat > mouse, but gp is less inducible. For GSH-T: mouse > rat > gp and gp not induced. For MMFO: gp > mouse > rat. Increases in relative liver weights in all species. For P-450, monkey > rat > dog; for AP, monkey > rat = dog; for AN, rat = monkey > dog (gram basis, different if on protein). Best induction in dog. A review.

Metabolism of glyceryl trinitrite

Species differences in plasma halflife a function of body weight.

Ioannides et al. (1982)

Various aspects of metabolism covered: spectrum of metabolites, plasma half-lifes, developmental differences, inhibitors, inducers Changes in iron “spinstate” as determined by EPR induced by different binding spectra Different inducing agents

Excellent comprehensive review with emphasis on mixed function oxidase activity.

Kato (1979)

Proportion of high spin P-450 in vivo: rabbit > gp = hamster = mouse > rat

Kumaki et al. (1978)

Glucuronide formed only by liver, other organ involved in sulfation. Both produced in liver of rat, rabbit and gp, only sulfate formed by mouse. For all species, liver most active with lung and kidney 15%–40% of liver. No species superior in all activities, but hamster tended to have greatest activities.

Wong (1976)

Rat, hamster, mouse, guinea pig

Mouse, guinea pig, rabbit, hamster Rat, rabbit, dog, mouse Rat, dog, monkey

Rat, mouse, rabbit, hamster, guinea pig Rat, rabbit, guinea pig

Rat, mouse, rabbit, hamster, guinea pig

Induction of P-450 and MMFO activities (AP demethyl, BP-OH, ECdeethyl) by 2-AAF and 3-MC GSH-T (1-chloro-2,4dinetrobenzene) Chlorfenvinphos deethylation (in vitro) Metabolism and kinetics of Tolrestat

Protein, P-450 content, reductase concentrations, model substrates, GSH and UDPG transferase activities, lung, liver, kidney

Astrom et al. (1986)

Igarashi et al. (1986)

Hutson and Logan (1986) Cayen et al. (1985)

Thabrew and Emerole (1983)

Lan et al. (1983)

Caldwell (1982)

Litterst et al. (1975)

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Table 1.4 Compilation of Selected Papers That Compare Xenobiotic Metabolism in Different Species Species Compared

Parameters Examined

Comments

References

Rat, mouse, guinea pig, rabbit

Microsomal protein, BPOH, UDPG transferase, small intestine vs. liver

Haietanen and Vainio (1973)

Rat, mouse, guinea pig, hamster, rabbit, dog, pig, monkey

Metabolism of [3H]styrene oxide: EH and GSH-T in liver, lung, and kidney

Rat, mouse, guinea pig, rabbit, pig, monkey

P-450 b5, cytochrome c reduct, Km and Vmax of various substrates

Rat, mouse, guinea pig, hamster

Induction of EH UDPG transferase, and GSH-T by 2-AAF or 3-MC

Rat, mouse, guinea pig, hamster Rat, mouse, guinea pig, hamster

Effects of DDT on AHH activity and cytochrome P-450 Total GSH, γ-GTP, GSH synthetases, peroxidease, and reductase

Rat, mouse, rabbit, guinea pig

GSH-T activity with different substrates, different age animals, and with different inducing agents

Rat, mouse, hamster, guinea pig, monkey

Review on role of intestinal microflora in drug metabolism; excellent bibliography with references to primary articles and other reviews

No real differences in microsomal protein (30–35 mg/g). For BP, liver > gut for all species. For liver gp = mouse > rat > rabbit; for gut, gp > rabbit > rat = mouse; for UDPG liver, gp > rabbit = mouse > rat; for UDPGgut, rabbit > rat > mouse = gp. For EH, liver > kidney > lung. In general, mouse is lowest and primate is highest. For GSHT, liver > kidney > lung. GP is highest and primate is lowest. Includes lit comp. of EH activities. Not big differences in micro: P-450 ranges 0.38 (pig) to 0.75 nmol/mg (gp). b5 ranged 0.20 (mouse) to 0.49 nmol/mg (gp). Cytochrome c red ranged 115 (rabbit) to 136 (gp) nmol/min/mg Large species variation (3- to 12fold) in control activities. Except for EH in guinea pig, enzymes induced only in rat by 2-AAF. Except for GSH-T in hamster, enzymes induced only in rat by 3MC. Rats not representative of activities or inducibility of other species. Induction in hamsters, decreases in other species. Acute toxic effects depend on route and species. GSH lowest in guinea pig, highest in mouse. Synthesis lowest in hamster, highest in rat. γ-GT much higher in gp. GSH-T highest in gp, lowest in rat and not affected by fasting. GR lowest in rat = mouse, highest in hamster, fasting effects species dependent. Px highest in mouse = hamster, lowest in rat and mouse, dependent on substrate and fasting state Wide range of activities, depending on species and substrate. Agerelated changes evident in all species, peak (up to 120 days) varies with species. Activity inducible in all species, but extent depends on inducer and substrate. Rat, mouse most inducible. Gut flora (β-glucosidase and βglucuronidase; nitro, nitrite, and azo reductases) have large species difference, guinea pigs tend to have lowest amounts, mice the highest.

Pacific et al. (1981)

Amri et al. (1986)

Astrom et al. (1987)

Haietanen and Vainio (1976) Igarashi et al. (1983)

Gregus et al. (1985)

Rowland (1988)

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Table 1.4 Compilation of Selected Papers That Compare Xenobiotic Metabolism in Different Species Species Compared Rat, mouse, hamster, rabbit, guinea pig, dog, primate, others

Rat, rabbit, guinea pig, ferret

Rat, mouse, guinea pig, hamster, rabbits, dogs, primates

Rat, mouse, guinea pig, hamster, rabbits, dogs, primates Rat, mouse, rabbit, guinea pig, dog

Rat, mouse, guinea pig, rabbit, rat, dog, quail, trout Rat, mouse, guinea pig, rabbit, dog

Parameters Examined

Comments

References

Excellent review; qualitative differences (lack of specific enzymes) and quantitative differences; emphasis on in vivo data; species differences in the metabolism of [1-14cacetyl]phenacetin Case reviews on how species differences in drug metabolism lead to differences in toxicity

Examples: Only in the rat is aromatic hydroxylation the major route of amphetamine metabolism. Dogs and guinea pigs have a defect in N-acetylation. Guinea pigs do not make mercapturic acids.

Williams (1972, 1974)

Deacetylation highest in rat and ferret, aromatic hydroxylation high in ferret, low in others. Glucuronide formation dominant in rabbit, guinea pig, ferret; sulfation is dominant in rat. Example: Dog has increased risk of bladder cancer (in response to) aromatic amines because of ability to N-hydroxylate but limited capacity to acetylate.

Smith and Timbrell (1974)

Example: In general, protein binding is highest in primate and lowest in mouse.

Cayen (1987)

In general, most rapid half-life in mice, longest in dogs. Correlates with rates of microsomal demethylation.

Quinn et al. (1958)

A good basic comparison. Convenient source for species comparison.

Gregus et al. (1983)

Highest activities in hamster, lowest in dog.

Hadley and Dahl (1983)

Review chapter in monograph; species differences in biotransformation, plasma protein binding, biliary excretion, and pharmacokinetics

A citation classic; species and other factors, in the metabolism of four different chemicals explored Cytochrome P-450, MMFO, EH, UDPG transferase, PAPSsulfotransferase, Nacetyl transferase Drug metabolism by nasal tissue in vitro

Calabrese (1988)

Abbreviations: AP = demeti-aminopyrine demethylase; BP-OH = benzo(a)pyrene hydroxylase; EC = deethyl-7ethoxycoumain deethylase; 2-AAF = 2-acetylaminofluorene; 3-MC = 3-methylcholanthrene; EH = epoxide hydrolase; AN-OH = aniline hydroxylase; GSH = glutathione; UDPG = UDP-glucuronic acid; γ-GTP = γglutamyltranspeptidase; gp = guinea pig; GSH-T = glutathione s-transferase.

Excretion The processes of elimination is not dealt with in great detail in the core chapters. This is not to say, however, that excretion is not important. Like absorption, elimination can be both active and passive, and most xenobiotics are passively excreted. In most cases, conjugated metabolites are actively excreted. The process of xenobiotic metabolism can be viewed, to a certain extent, as packaging for the excretory process. Across species, the active excretion of a metabolite by the liver into the bile is probably (quantitatively) the most important active excretory process concerning xenobiotic disposition. Glucuronide conjugates (as reviewed by Levine 1978; Klaassen and Watkins 1984; Williams 1972) are actively excreted by the liver into the bile and ultimately into the feces. Amino acid conjugates, in contrast, tend to be excreted by the kidney into the urine. These are

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definite species-related differences in the molecular weight cut-off between 300 and 500 for the biliary transport of the metabolite that dictate whether a metabolite will end up in either the feces or the urine. Species differences do not follow any particular phylogenetic lines. For example, rats and dogs effectively excrete phenolphthalein glucuronide (50% in bile), whereas guinea pigs and monkeys do not (< 10%; Williams 1972). Although there are species differences in excretions, these tend to be overshadowed by the species differences in metabolism. That is, a particular species might not need an efficient biliary excretory process because with a particular chemical, it might produce glucuronides sparingly or not at all. This information might be of interest to the pharmacokinist in determining where to look for a metabolite. Generally, however, such information is of academic interest to the pharmacologist or toxicologist in interpreting an experiment because glucuronides are generally inactive end products, and it does not really matter whether they end up in the urine or feces. (As with any rule, there are exceptions: Mulder [1986] cites several examples of glucuronides being active metabolites; i.e. causing toxicity.) There are several instances, however, in which biliary excretion actually influences the toxicity of a chemical, such as with some of the cardiac glycosides or heavy metals where biliary excretion occurs without metabolism and biliary excretion is the “detoxification” mechanism (Klaassen and Watkins 1984). Cayen (1987) pointed out that species-related differences in indomethacin-induced intestinal damage directly correlates to the degree of exposure of the mucosa owing to biliary excretion and resultant enterohepatic circulation. Such instances are discussed on a species-specific basis where such data permit.

SUMMARY I have attempted to assemble a source of basic information on laboratory animals, and trust that this book will provide a convenient source of information for either the skilled or novice investigator to aid in the design and interpretation of in vivo pharmacological or toxicological studies.

REFERENCES Amri, H., Batt, A., and Siest, G. (1986). Comparison of cytochrome P-450 content and activities in liver microsomes of seven species including man. Xenobiotica 16, 351–358. Astrom, A., Maner, S., and DePierre, J. (1986). Induction of cytochrome P-450 and related drug-metabolizing activities in the livers of different rodent species by 2-acetylaminofluorene or by 3-methylcholanthrene. Biochem. Pharmacol. 35, 2703–2713. Astrom, A., Maner, S., and DePierre, J. (1987). Induction of liver microsomal epoxide hydrolase, UDPglucuronosyl transferase and cytosolic glutathione transferase in different rodent species by 2-acetylaminofluorene or 3-methylcholanthrene. Xenobiotica 17, 155–163. Black, S., and Coon, M. (1986). Comparative structure of P-450 cytochromes. In Cytochrome P450: Structure, mechanisms and biochemistry, ed. P. Oriz de Montellano, 161–216. New York: Plenum. Boutin, J. (1987). Indirect evidence of UDP-glucuronosyl-transferase heterogeneity: How can it help purification? Drug Metab. Revs. 18, 517–553. Boutin, J., Antoine, B., Batt, A., and Siest, G. (1984). Heterogeneity of hepatic microsomal UDP-glucuronyltransferase activities: Comparison between human and mammalian species activities. Chem.-Biol. Int. 52, 173–184. Brodie, B., Axelrod, J., Cooper, J., Gaudette, L., LaDu, B., Mitoma, C., and Udenfriend, S. (1955). Detoxification of drug and other foreign compounds by liver microsomes. Science 148, 1547–1554. Burrell, G. A. (1912). The use of mice and birds for detecting carbon monoxide after mine fire and explosions. Technical Paper 11, Department of Interior, Bureau of Mines, Washington, DC, 3–16. Calabrese, E. (1988). Comparative biology of test species. Environ. Health Perspec. 77, 55–62. Caldwell, J. (1981). The current status of attempts to predict species differences in drug metabolism. Drug Metab. Revs. 12, 221–237.

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Caldwell, J. (1982). Conjugation reactions in foreign-compound metabolism: Definition, consequences, and species variations. Drug Metab. Revs. 13, 745–777. Cayen, M. (1987). Retrospective evaluation of appropriate animal models based on metabolism studies in man. In Human risk assessment—The role of animal selection and extrapolation, eds. M. Roloff, A. Wilson, W. Ribelin, W. Ridley, and F. Ruecker, 99–112. New York: Taylor & Francis. Cayen, M., Hicks, D., Ferdinandi, E., Kraml, M., Greselin, E., and Dvomik, D. (1985). Metabolic disposition and pharmacokinetics of aldose reductase inhibitor tolrestat in rats, dogs and monkeys. Drug Metab. Dispos. 13, 412–419. Coon, M. (1978). Oxygen activation in the metabolism of lipids, drugs and carcinogens. Nutr. Rev. 36, 319–328. Coon, M., and Vaz, A. (1987). Mechanism of microsomal electron transfereactions. Chem. Scripta 27A, 17–19. Crabb, D., Borson, W., and Li, T. K. (1987). Ethanol metabolism. Pharmacol. Ther. 34, 59–73. deBethizy, J., and Hayes, J. (1989). Metabolism: A determinant of toxicity. In Principles and methods of toxicology, ed. A. Hayes, 29–66. New York: Raven. Dubois, K. P., and Geiling, E. M. K. (1959). Textbook of toxicology, 11–12. New York: Oxford University Press.Ewald, B., and Gregg, D. (1983). Animal research for animals. Annl. NY Acad. Sci. 406, 48–58. Fano, A. (1998). Lethal laws. New York: Zed Books. Frazier, J. M. (1990). In vitro toxicology. New York: Marcel Dekker. Gad, S. C. (1989). A tier testing strategy incorporating in vitro testing methods for pharmaceutical safety assessment, humane innovations and alternatives in animal experimentation 3, 75–79. Gad, S. (2000). In vitro toxicology (2nd ed.). Philadelphia: Taylor & Francis. Gad, S. (2002). Drug safety evaluation. New York: Wiley. Gad, S., and Chengelis, C. (1998). Acute toxicity testing; Perspectives and horizons (2nd ed.). San Diego, CA: Academic Press. Goldstein, G.W., et al. (1974). Pathogenesis of lean encephalopathy: Uptake of lead and reaction of brain capillaries. Arch. Neuro. 31, 382–389. Gonzales, F. (1988). The molecular biology of cytochrome P-450s. Pharmacol. Rev. 40, 243–288. Gregus, Z., Varga, F., and Schmelas, A. (1985). Age-development and inducibility of hepatic glutathione Stransfersase activities in mice, rats, rabbits and guinea-pigs. Comp. Biochem. Physiol. 80C, 85–90. Gregus, Z., Watkins, J., Thompson, T., Harvey, M., Rozman, K., and Klaassen, C. (1983). Hepatic phase I and phase II biotransformations in quail and trout: Comparison to other species commonly used in toxicity testing. Toxicol. Appl. Pharmacol. 67, 430–441. Guengerich, F. (1988). Minireview; cytochromes P-450. Comp. Biochem. Physiol. 89C, 1–4. Hadley, W., and Dahl, A. (1983). Cytochrome P-450 dependent monooxygenase activity in nasal membranes. Drug Metab. Dispos. 11, 275–276. Haietanen, E., and Vainio, H. (1973). Interspecies variations in small intestinal and hepatic drug hydroxylation and glucuronidation. Acta Pharmacol. Toxicol. 33, 57–64. Haietanen, E., and Vainio, H. (1976). Effect of administration route on DDT acute toxicity and on drug biotransformation in various rodents. Arch. Environ. Contam. Toxicol. 4, 201–216. Hawkins, R., and Kalant, H. (1972). The metabolism of ethanol and its metabolic effects. Pharmacol. Rev. 24, 67–157. Hirom, P., Idle, J., and Millburn, P. (1977). Comparative aspects of the biosynthesis and excretion of xenobiotic conjugates by non-primate mammals. In Drug metabolism: From microbes to man, eds. D. Park and R. Smith, 299–329. Philadelphia: Taylor & Francis. Hutson, D., and Logan, C. (1986). Detoxification of the organophosphorus insecticide chlorfenvinphos by rat, rabbit and human liver enzymes. Xenobiotica 16, 87–93. Igarashi, T., Satoh, T., Ueno, K., and Kitagawa, H. (1983). Species difference in glutathione level and glutathione related enzyme activities in rats, mice, guinea pigs and hamsters. J. Pharm. Dyn. 6, 941–949. Igarashi, T., Tomihari, N., and Ohmori, S. (1986). Comparison of glutathione S-transferase in mouse, guinea pig, rabbit, and hamster liver cytosol to those in rat liver. Biochem. Int. 13, 641–618. Ioannides, C., Parke, D., and Taylor, I. (1982). Elimination of glyceryl trinitrate: Effects of sex age, species and route of administration, Br. J. Pharmacol. 77, 83–88. Jacoby, W., Duffel, M., Lyon, E., and Ramasway, S. (1984). Sulfotransferases active with xenobiotics—Comments on mechanism. In Progress in drug metabolism, Vol. 8, eds. J. Bridges and L. Chasseaud, 11–33. London: Taylor & Francis.

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Jarina, D., and Bend, J. (1977). Glutathione S-transferases. In Biological reactive intermediates: Formation, toxicity and inactivation, eds. D. Jallow, J. Kocsis, R. Snyder, and H. Vanio, 207–236. New York: Plenum. Kadlubar, F., and Hammons, G. (1987). The role of cytochrome P-450 in the metabolism of chemical carcinogens. In Mammalian cytochrome P450, Vol. 1, ed. F. Guengerich, 1–54. Boca Raton, FL: CRC Press. Kato, R. (1979). Characteristics and differences in the hepatic mixed function oxidases of different species Pharmacol. Ther. 6, 41–98. Klaassen, C. (1986). Distribution, excretion and absorption of toxicants. In Casarett and Doull’s toxicology: The basic sciences of poisons, eds. C. Klaassen, M. Amdur, and J. Doull, 33–63. New York: Macmillan. Klaassen, C., and Watkins, J. (1984). Mechanisms of bile formation, hepatic uptake, and biliary excretion. Pharmacol. Rev. 36, 1–67. Klingenberg, M. (1958). Pigments of rat liver microsomes. Arch. Biochem. Biophys. 75, 376–386. Klinger, W. (1982). Biotransformation of drugs and other xenobiotics during postnatal development. Pharmacol. Ther. 16, 377–429. Kumaki, K., Sato, M., Kon, H., and Nebert, D. (1978). Correlation of type I, type II, and reverse type I, difference spectra with absolute changes in spin state of hepatic microsomal cytochrome P-450 iron from five mammalian species J. Biol. Chem. 253, 1048–1058. LaDu, B., Mandel, H., and Way, E. (eds.). (1972). Fundamentals of drug metabolism and drug disposition. Baltimore: Williams & Wilkins. Lan, S., Weinstein, G., Keim, G., and Migdalof, B. H. (1983). Induction of hepatic drug-metabolizing enzymes in rats, dogs, and monkeys after repeated administration of an anti-inflammatory hexahydroindazole. Xenobiotica 13, 329–335. Leinweber, F. (1987). Possible physiological roles of carboxylic ester hydrolases. Drug Metab. Revs. 18, 379–440. Levine, W. (1978). Biliary excretion of drugs and other xenobiotics. Ann. Rev. Pharmacol. Toxicol. 18, 81–96. Levy, R. H., Thummel, K. E., Tragen, W. F., Hanslen, P. D., and Eichelbaum, M. (2000). Metabolic drug interactions. Philadelphia: Lippincott, Williams & Wilkins. Lewis, D. F. V. (2001). Guide to cytochrome P450. Philadelphia: Taylor & Francis. Lower, G., and Bryan, G. (1973). Enzymatic N-acetylation of carcinogenic aromatic amines by liver cytosol of species displaying different oran susceptibilities. Biochem. Pharmacol. 22, 1581–1588. Litterst, C., Mimnaugh, E., Reagan, R., and Gram, T. (1975). Comparison of in vitro drug metabolism by lung, liver, and kidney of several common laboratory species. Drug Metab. Dispos. 3, 259–265. Mandel, H. (1972). Pathways of drug biotransformation: Biochemical conjugation. In Fundamentals of drug metabolism and drug disposition, eds. B. LaDu, H. Mandel, and E. Way, 149–186. Baltimore: Williams & Wilkins. Mannering, G. (1972). Microsomal enzyme systems which catalyze drugs. In Fundamentals of drug metabolism and drug disposition, eds. B. LaDu, H. Mandel, and E. Way, 206–252. Baltimore: Williams & Wilkins. McCally, A. W., Farmer, A. G., and Loomis, E. C. (1933). Corneal ulceration following use of lash lure. J.A.M.A. 101(20), 1561. Meier, J., and Stocker, K. (1989). Review article: On the significance of animal experiments in toxicology. Toxicon 27, 91–104. Miura, T., Shimada, H., Ohi, H., Komori, M., Kodama, T., and Kamataki, T. (1989). Interspecies homology of cytochrome P-450: Inhibition by anti-P-450-male antibodies of testosterone hydroxylases in liver microsomes from various animal species including man. Jpn. J. Pharmacol. 49, 365–374. Mueller, G., and Miller, J. (1949). The reductive cleavage of 4-dimethylaminoazobenzene by rat liver: The intracellular distribution of the enzyme system and its requirement for triphosphoropyridine nucleotide. J. Biol. Chem. 180, 1125–1136. Mulder, G. (1986). Sex differences in drug conjugation and their consequences for drug toxicity, sulfation, glucuronidation, and glutathione conjugation. Chem.-Biol. Int. 57, 1–15. Mulder, G., Meerman, J., and van den Goorbergh, J. (1986). Bioactivation of xenobiotic by conjugation. In Advances in xenobiotic conjugation chemistry, 282–300. Washington, DC: American Chemical Society Symposium Series.

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National Institute of Occupational Safety and Health. (1980). 1979 Registry of toxic effects of chemical substances, Publication number 80-111. Washington, DC: National Institute of Occupational Safety and Health. Neal, G., Nielsch, U., Judah, D., and Hulbert, P. (1987). Conjugation of model substrates or microsomallyactivated aflatoxin bl with reduced glutathione, catalyzed by cytosolic glutathione-S-transferase in livers of rats, mice and guinea pigs. Biochem. Pharmacol. 36, 4269–4276. Nevalainen, T., Han, J., and Sarvikarju, M. (1996). Frontiers in laboratory animal science. Scandinavian J. Lab. Animal Sci. 23, Supp. 1. Oesch, F. (1972). Mammalian epoxide hydrases: Inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and olefinic compounds. Xenobiotica 3, 305–340. Orfila, M. J. B. (1814). Traite de toxicologie. Paris, J. de Chimie Medicale. Pacifici, G., Boobis, A., Brodie, M., McManus, M., and Davies, D. (1981). Tissue and species differences in enzymes of epoxide metabolism. Xenobiotica 11, 73–79. Pendergrast, W. (1984). Biological drug regulation in the seventy-fifth anniversary commemorative volume of food and drug law, 293–305. Washington, DC: The Food and Drug Law Institute. Pickett, C., and Lu, A. (1989). Glutathione S-transferases: Gene structure, regulation, and biological function. Ann. Rev. Biochem. 58, 743–764. Pratt, W. B., and Taylor, P. (1990). Principles of drug action: The basis of pharmacology (3rd ed.). New York: Churchill Livingston. Priestley, J. (1792). On different kinds of air, philosophical transactions. Quinn, G., Axelrod, J., and Brodie, B. (1958). Species, strain and sex differences in metabolism of hexobarbitoane, amidopyrine, antipyrine, and aniline. Biochem. Pharmacol. 1, 152–159. Remmer, H., and Merker, H. (1963). Drug-induced changes in liver endoplasmic reticulum: Association with drug-metabolizing enzymes. Science 142, 1567–1568. Rowland, I. (1988). Role of the gut flora in toxicity and cancer. New York: Academic Press. Rowland, I., Mallett, A., and Bearne, C. (1986). Enzyme activities of the hindgut of microflora of laboratory animals and man. Xenobiotica 16, 519–523. Rozman, K. (1988). Disposition of xenobiotics: Species differences. Toxicol. Pathol. 16, 123–129. Seidegard, J., and DePierre, J. (1983). Microsomal epoxide hydrolase: Properties, regulation, and function. Biochim. Biophys. Acta 695, 251–270. Siest, G., Magdalou, J., and Burchell, B. (eds.). (1989). Cellular and molecular aspects of glucuronidation, Colloque Inserm, Vol. 173. London: John Libbey Eurotext. Singer, S. (1985). Preparation and characterization of the different kinds of sulfotransferases. In Biochemical pharmacology and toxicology, Vol. I, eds. D. Zakim and D. Vessay, 95–159. New York: Wiley Interscience. Sipes, I., and Gandolfi, A. (1986). Biotransformation of toxicants. In Casarett and Doull’s toxicology: The basic sciences of poisons, eds. C. Klaassen, M. Amdur, and J. Doull, 64–98. New York: Macmillan. Smith, R., and Timbrell, J. (1974). Factors affecting the metabolism of phenacetin: I. Influence of dose, chronic dosage, route of administration and species on the metabolism of [1-14C-acetyl]phenacetin. Xenobiotica. 8, 489–501. Thabrew, M., and Emerole, G. (1983). Variation in induction of drug metabolizing enzymes by trans-stilbene oxide in rodent species. Biochem. Biophys. Acta. 756, 242–246. Tynes, R., and Hodgson, E. (1985). Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: Characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat. Arch. Biochem. Biophys. 240, 77–93. Washington, N., Washington, C., and Wilson, C. (2001). Physiological pharmaceutics (2nd ed.). Philadelphia: Taylor & Francis. Williams, R. T. (1947). Detoxification mechanisms: The metabolism of drugs and allied organic compounds. New York: Wiley. Williams, R. T. (1972). Species variation in drug biotransformations. In Fundamentals of drug metabolism and drug disposition, eds. B. LaDu, H. Mandel, and E. Way, 187–205. Baltimore: Williams & Wilkins. Williams, R. T. (1974). Inter-species variations in the metabolism of xenobiotics. Biochem. Soc. Transat. 2, 359–377. Wong, K. (1976). Species differences in the conjugation of 4-hydroxy-3-methoxypheylethanol. Biochem. J. 158, 33–37. Ziegler, D. M. (1988). Flavin-containing monooxygenase: Catalytic mechanism and specificities. Drug Metab. Rev. 19, 1–32.

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CHAPTER

2

The Mouse Toxicology:

Shayne C. Gad Gad Consulting Services

Pathology:

Charles H. Frith Toxicology Pathology Associates

Dawn G. Goodman PATHCO, Inc.

Byron G. Boysen Hazleton Wisconsin, Inc.

Metabolism: Shayne C. Gad Gad Consulting Services

CONTENTS Toxicology........................................................................................................................................24 History ....................................................................................................................................24 Choice of the Mouse in Toxicological Research...................................................................25 Normal Physiological Values .....................................................................................25 Species Differences ....................................................................................................27 Strain Differences.......................................................................................................27 Husbandry...............................................................................................................................28 Facilities......................................................................................................................28 Temperature and Relative Humidity...........................................................30 Lighting .......................................................................................................30 Ventilation ...................................................................................................31 Noise............................................................................................................32 Construction Parameters .............................................................................32 Caging.........................................................................................................................33 Cage Type....................................................................................................34 Cage Size ....................................................................................................35 Population....................................................................................................35 Bedding .......................................................................................................36 Animal Identification..................................................................................................36 Cage Cards ..................................................................................................37 19


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Ear Tags.......................................................................................................37 Ear Notching or Punching ..........................................................................37 Tattoos .........................................................................................................38 Color Coding of Skin or Hair.....................................................................38 Toe Clipping................................................................................................39 Implantable Electronic Microchips.............................................................39 Food ............................................................................................................................39 Nutritional Requirements............................................................................39 Selection ......................................................................................................40 Provision of Feed and Feeders ...................................................................43 Analysis.......................................................................................................45 Water...........................................................................................................................45 Water Bottles with Sipper Tubes ................................................................46 Automatic Watering Systems......................................................................46 Water Quality ..............................................................................................46 Prevention of Infectious Diseases ..............................................................................47 Study Design ..........................................................................................................................48 Acute Toxicity Studies ...............................................................................................48 Short-Term Toxicity Studies ......................................................................................50 Chronic Toxicity Studies (26 Weeks to 2 Years).......................................................51 Carcinogenicity Studies (18–24 Months) ..................................................................51 Teratology Studies ......................................................................................................53 Segment II Teratology Studies....................................................................54 Genetic Toxicity Studies ............................................................................................54 Mouse Micronucleus Assay ........................................................................54 Heritable Translocation Assay ....................................................................55 Microbial Host-Mediated Assay .................................................................56 Special Studies ...........................................................................................................57 Mouse Ear Swelling Test ............................................................................57 Dermal Carcinogenicity (Skin Painting) Study..........................................57 Dosing Techniques .................................................................................................................58 Oral Administration....................................................................................................59 Gavage.........................................................................................................59 Dietary Admixture ......................................................................................60 Drinking Water............................................................................................61 Intravenous Injection ..................................................................................................62 Description of Technique............................................................................63 Intraperitoneal Injection .............................................................................................64 Description of Technique............................................................................65 Intramuscular Injection...............................................................................................65 Description of Technique............................................................................66 Subcutaneous Injection...............................................................................................66 Description of Technique............................................................................66 Intradermal Injection ..................................................................................................67 Description of Technique............................................................................67 Topical Administration ...............................................................................................67 Description of Technique............................................................................68 Inhalation ....................................................................................................................68 Chamber (Whole Body)..............................................................................69 Head/Nose Exposure (Head Only/Nose Only)...........................................69


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Data Collection Techniques ...................................................................................................69 Clinical Observations and Physical Examinations ....................................................70 Clinical Laboratory Evaluations.................................................................................71 Postmortem Procedures..............................................................................................71 Summary.................................................................................................................................72 Pathology..........................................................................................................................................72 Cardiovascular System ...........................................................................................................74 Vessels ........................................................................................................................74 Nonneoplastic Lesions ................................................................................74 Neoplastic Lesions ......................................................................................78 Heart ...........................................................................................................................78 Nonneoplastic..............................................................................................78 Proliferative Lesions ...................................................................................79 Digestive System ....................................................................................................................79 Salivary Glands...........................................................................................................79 General ........................................................................................................79 Sexual Dimorphism.....................................................................................79 Nonneoplastic Lesions ................................................................................80 Neoplastic Lesions ......................................................................................80 Pancreas ......................................................................................................................81 Nonneoplastic Lesions ................................................................................81 Neoplastic Lesions ......................................................................................81 Esophagus...................................................................................................................81 Nonneoplastic..............................................................................................81 Neoplastic Lesions ......................................................................................81 Stomach ......................................................................................................................81 Normal Anatomy.........................................................................................81 Nonneoplastic Lesions ................................................................................82 Neoplastic Lesions ......................................................................................82 Intestine ......................................................................................................................82 Noneoplastic Lesions ..................................................................................82 Neoplastic Lesions ......................................................................................83 Endocrine System...................................................................................................................83 Adrenal Gland ............................................................................................................83 Nonneoplastic Lesions ................................................................................83 Neoplastic Lesions ......................................................................................85 Pituitary Gland ...........................................................................................................86 Nonneoplastic Lesions ................................................................................86 Neoplastic Lesions ......................................................................................86 Thyroid Gland ............................................................................................................86 Nonneoplastic Lesions ................................................................................86 Neoplastic Lesions ......................................................................................87 Parathyroid..................................................................................................................87 Pancreatic Islets ..........................................................................................................87 Nonneoplastic Lesions ................................................................................87 Neoplastic Lesions ......................................................................................88 Female Genital System ..........................................................................................................88 Ovary ..........................................................................................................................88 Nonneoplastic Lesions ................................................................................88 Hyperplastic Lesions...................................................................................89 Neoplastic Lesions ......................................................................................89


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Uterus, Uterine Cervix ...............................................................................................90 Nonneoplastic Lesions ................................................................................90 Neoplastic Lesions ......................................................................................90 Vagina .........................................................................................................................91 Induced Neoplasms .....................................................................................91 Clitoral Gland .............................................................................................................91 Nonneoplastic Lesions ................................................................................91 Neoplastic Lesions ......................................................................................92 Mammary Gland.........................................................................................................92 Nonneoplastic Lesions ................................................................................92 Neoplastic Lesions ......................................................................................93 Induction of Mammary Tumors in Mice....................................................93 Hematopoietic System............................................................................................................93 Nonneoplastic Lesions ...............................................................................................93 Thymus........................................................................................................93 Spleen ..........................................................................................................94 Lymph Nodes ..............................................................................................95 Neoplastic Lesions .....................................................................................................96 Malignant Lymphoma .................................................................................96 Follicular Center Cell Lymphoma ..............................................................96 Immunoblastic Lymphoma .........................................................................97 Plasma Cell Lymphomas ............................................................................97 Lymphoblastic Lymphoma..........................................................................97 Small Lymphocyte Lymphomas .................................................................97 Other Neoplasms.........................................................................................97 Male Genital System ..............................................................................................................98 Testes ..........................................................................................................................98 Nonneoplastic Lesions ................................................................................98 Neoplastic Lesions ......................................................................................99 Accessory Sex Glands................................................................................................99 Nonneoplastic Lesions ................................................................................99 Neoplastic Lesions ......................................................................................99 Integument ............................................................................................................................100 Nonneoplastic Lesions .............................................................................................100 Alopecia ....................................................................................................100 Amyloidosis ..............................................................................................100 Atrophy......................................................................................................100 Ulcers ........................................................................................................100 Dermatitis ..................................................................................................100 Neoplastic Lesions ...................................................................................................100 General ......................................................................................................100 Fibroma .....................................................................................................101 Fibrosarcoma.............................................................................................101 Fibrous Histiocytoma................................................................................101 Benign Fibrous Histiocytoma ...................................................................101 Malignant Fibrous Histiocytoma ..............................................................101 Neurofibroma and Neurofibrosarcoma .....................................................101 Benign Schwannoma.................................................................................101 Malignant Schwannoma............................................................................102 Sarcomas NOS ..........................................................................................102 Mast Cell Tumor .......................................................................................102 Malignant Melanoma ................................................................................102


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Epithelial Neoplasms................................................................................................102 Basal Cell Adenoma .................................................................................102 Basal Cell Carcinoma ...............................................................................102 Sebaceous Cell Adenomas and Carcinoma ..............................................102 Trichoepithelioma .....................................................................................103 Basosquamous Tumor ...............................................................................103 Keratoacanthoma.......................................................................................103 Squamous Cell Papilloma.........................................................................103 Squamous Cell Carcinomas ......................................................................103 Liver and Biliary System .....................................................................................................103 Liver..........................................................................................................................103 Nonneoplastic Lesions ..............................................................................103 Neoplastic Lesions ....................................................................................106 Gallbladder ...............................................................................................................108 Nonneoplastic Lesions ..............................................................................108 Neoplastic Lesions ....................................................................................109 Nervous System....................................................................................................................109 Nonneoplastic Lesions .............................................................................................109 Cerebral Mineralization ............................................................................109 Hydrocephalus...........................................................................................109 Vacuolization of the White Matter, Central Nervous System..................109 Infarct ........................................................................................................109 Developmental Abnormalities...................................................................110 Neoplastic Lesions ...................................................................................................110 Oligodendroglioma....................................................................................110 Astrocytoma ..............................................................................................110 Meningioma ..............................................................................................110 Metastatic Tumors.....................................................................................111 Respiratory System...............................................................................................................111 Upper Respiratory Tract (Nasal Cavity, Larynx, Trachea)......................................111 Lung..........................................................................................................................111 Nonneoplastic Lesions ..............................................................................111 Neoplastic Lesions ....................................................................................112 Alveolar/Bronchiolar Carcinoma ..............................................................113 Special Senses ......................................................................................................................113 Eye ............................................................................................................................113 Nonneoplastic Lesions ..............................................................................113 Neoplastic Lesions ....................................................................................113 Ear.............................................................................................................................114 Vestibular Syndrome .................................................................................114 Harderian Gland .......................................................................................................114 Nonneoplastic Lesions ..............................................................................114 Neoplastic Lesions ....................................................................................114 Urinary System.....................................................................................................................114 Kidney.......................................................................................................................114 General ......................................................................................................114 Degenerative Lesions ................................................................................115 Neoplastic Lesions ....................................................................................116 Ureter ........................................................................................................................117 Nonneoplastic Lesions ..............................................................................117


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Urinary Bladder........................................................................................................117 Nonneoplastic Lesions ..............................................................................117 Neoplastic Lesions ....................................................................................118 Musculoskeletal System .......................................................................................................118 Bone..........................................................................................................................118 Nonneoplastic Lesions ..............................................................................118 Neoplastic Lesions ....................................................................................118 Skeletal Muscle ........................................................................................................119 Multiple Systems..................................................................................................................119 Amyloidosis..............................................................................................................119 Morphology...............................................................................................120 Genetically Engineered Mice in Toxicology .......................................................................120 p53+/- Knockout Mouse ..........................................................................................120 Tg.AC Mouse ...........................................................................................................121 rasH2 Transgenic Mouse..........................................................................................122 Metabolism.....................................................................................................................................122 References ......................................................................................................................................130

TOXICOLOGY History The domesticated mouse of North America and Europe (Mus musculus) is the most widely used animal in medical research. The mouse is a member of the order Rodentia, family Muridae, and subfamily Murinae. The use of the mouse in biomedical research has been chronicled for several hundred years. William Harvey (1578–1657) published the results of his work on animal reproduction and blood circulation based in part on his work with mice (Harvey 1616, cited in Morse 1981). Joseph Priestly (1775) used mice in exploring the phlogiston theory, and Antoine Lavoisier (1777) used mice in his studies of the physiology of respiration (both cited in Morse 1981). Mice were selectively bred for coat color for many centuries, but in the early 1900s efforts turned to breeding strains of mice that might mimic human disease states. Subsequently, inbred strains were derived that were particularly susceptible or resistant to various types of cancers and viruses. A strain is considered to be inbred when it has been derived by brother × sister matings for 20 or more consecutive generations (F20), and can be traced to a single ancestral breeding pair in the 20th or subsequent generations. Certain other breeding systems (e.g., parent × offspring) can be substituted as long as the inbreeding coefficient achieved is at least equal to that at F20 (Lyon 1981). The genetic groundwork was laid for most of the strains of inbred mice currently in use by researchers such as William E. Castle, Clarence C. Little, and Leonell C. Strong during the period of about 1900 to 1930 (Morse 1981). Although highly inbred strains have proven invaluable in fields such as genetics and histocompatibility research, a school of thought developed that random bred or specifically outbred strains might more closely represent man in many areas of medical research. A random breeding program attempts to achieve a level of genetic variability similar to the initial (noninbred) population, and in so doing, preserve the “hybrid vigor” associated with heterozygosity. Many of the inbred and outbred strains of mice currently in use are referred to as Swiss strains. All of these Swiss strains are traceable to a group of two male and seven female albino mice obtained from the noninbred stock of Dr. A. de Coulon of Lausanne, Switzerland, and imported


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into the United States by Dr. Clara Lynch of the Rockefeller Institute in 1926 for use in cancer research (Lynch 1969, cited in Hill 1983). Choice of the Mouse in Toxicological Research As discussed in chapter 1, the choice of a species for toxicity testing is based on consideration of a range of variables. Ideally, if toxicity testing is intended to provide information on the safety of a test article in or by humans, the species chosen for testing should be most similar to the human in the way it handles the test article pharmacodynamically. Substantial differences in absorption, distribution, metabolism, or elimination (ADME) between test species and the target species (e.g., the human) will reduce the predictive value of the test results. From a practical standpoint, often the pharmacokinetics are unknown in humans or the variety of available test species at the time of species selection. For this reason, testing is usually conducted in at least two species. Generally, one of those species is usually a rodent and one a nonrodent. The two most commonly used rodent species are mice and rats, and often toxicity testing is conducted in both of those species. Mice have many advantages as test animals for toxicity testing. They are small; relatively economical to obtain, house, and care for; and generally easy to handle. Mice are generally more economical than rats in these respects. Although mice might attempt to escape or bite handlers, with regular, gentle handling they are easily managed. Other advantages of the species include a short gestation period and a short natural life span. These characteristics allow studies that include evaluation of reproductive performance or exposure to a test article for periods approaching the expected life span (e.g., evaluation of carcinogenic potential) to be conducted in a practical time frame. Highquality, healthy mice are available from reliable commercial suppliers. Many genetically well-defined, highly inbred, specifically or randomly outbred strains are available. Mice have been used in biomedical research for hundreds of years, and because of this, many technical procedures have been developed for use with the species, and a vast body of historical data is available for most strains. This historical database includes information on optimal nutritional and housing requirements in addition to data such as the expected background incidence of various diseases and types of tumors in untreated animals, and it is continuously being added to (Blackwell et al. 1995). There are some disadvantages to using mice, and most are related to the small size of the animal. The smaller size and higher metabolic rate compared to the rat renders the species a bit less hearty than rats. Deviations in environmental conditions such as an air conditioning failure or failure in an automatic watering system typically have more severe effects on smaller species such as mice than they do on rats. Owing to their high level of natural activity, most strains of mice will not become as docile or easy to handle as rats that have received equivalent handling. Small size often precludes or renders more difficult a number of procedures that are commonly conducted in toxicity testing, such as the collection of large samples or repeated samples of blood and urine, electrocardiographic evaluation, and some necropsy evaluations. This section provides brief summaries of some of the normal physiological values and salient features of the species and some of the specific strains that might be useful in selecting an appropriate species and strain for toxicity testing. Normal Physiological Values Selected normal physiological values for mice are shown in table 2.1 and table 2.2. Median survival of a number of groups of Charles River CD-1 outbred mice is shown in table 2.3. These normal values will vary depending on the strain of mouse, supplier, condition at arrival, type of feed, environmental and housing conditions, and in some cases, time of year. These data should be considered as a reference, but will not necessarily represent experience in any particular laboratory.


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Table 2.1 Normal Physiological Values: General and Reproductive General Life span Average Maximum reported Adult weight Male Female Surface area Chromosome number (diploid) Food consumption Water consumption Body temperature Oxygen consumption Reproductive Age, sexual maturity Male Female Breeding season Estrus cycle Gestation period Average Range Litter size Average Range Birth weight Age begin dry food Age at weaning

1–3 years 4 years 20–40 g 18–40 g 0.03–0.06 cm2 40 4–5 g/day 5–8 ml/day ad libitum 36.5°C 1.69 ml/g/hr

50 days (20–35 g) 50–60 days, (20–30 g) Continuous, cyclic 4–5 days 19 days 17–21 days 12 1–23 1.5 g 10 days 16–21 days (10–12 g)

Source: Data derived from Jacoby and Fox (1984), and from the Animal Diet Reference Guide, Purina Mills, Inc. (1987) Table 2.2 Normal Physiological Values: Cardiovascular and Respiratory Cardiovascular Heart rate Average Range Blood pressure Systolic Diastolic Blood volume Plasma Whole Hematocrit RBC life span RBC diameter Plasma pH Respiratory Rate Average Range Tidal volume Average Range Minute volume Average Range

600/min 320–800/min 133–160 mm Hg 102–110 mm Hg 45 ml/kg 78 ml/kg 41.5% 20–30 days 6.6 microns 7.2–7.4

163/min 84–230/min 0.18 ml 0.09–0.38 ml 24 ml/min 11–36 ml/min

Source: Data derived from Jacoby and Fox (1984), and from the Animal Diet Reference Guide, Purina Mills, Inc. (1987)


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Table 2.3 Median Survival of 16 Groups of Control Mice Sex Male Female

Period of Time on Study (Months) 6 12 18 21 98% 98%

91% 95%

63% 74%

46% 68%

Data represent median survival of Charles River CD-1 outbred albino mice enrolled in 24-month chronic toxicity studies at pharmaceutical or contract toxicology laboratories. Source: Adapted from Lang (1989b).

Species Differences Mice are similar to other common laboratory animal species and to humans in many ways, yet the differences should not be underestimated. Mice have a high metabolic rate compared to other species. This fact alone could result in increased or decreased toxicity of a test article, depending on the specific mechanism of intoxication. In many cases, high metabolic rate may be associated with rapid ADME of a test article. Mice are obligate nose breathers, and have more convoluted nasal passages than humans. This might result in an excess of respirable test article deposited in the nasal passages, causing either increased or decreased relative toxicity, depending on the most critical site of absorption. The small size of the mouse compared to other common laboratory species offers a significant advantage if the test article is expensive or in short supply. As an approximation, a mouse weighs about 10% as much as a rat, about 5% as much as a guinea pig, about 1% as much as a rabbit, and less than 1% as much as a dog or primate. Material requirements to administer equivalent dose levels are usually proportional to body weight, so the test article savings associated with the mouse are evident. The small size of a mouse results in high surface area to body mass ratio, which in turn causes the mouse to be relatively intolerant of thermal and water balance stresses. The kidneys of a mouse have about twice the glomerular filtering surface per gram of body weight as a rat, and owing to the specific architecture of the murine kidney, they are capable of producing urine that is about four times as concentrated as the highest attainable human concentrations (Jacoby and Fox 1984). These characteristics of renal architecture and function might be important to the toxicity of some test articles. Mice differ from most species by the formation of a persistent vaginal plug after mating. The presence of a vaginal plug is easily detected, is considered evidence of mating, and is a useful characteristic during the conduct of reproductive studies. It is also frequently the case in pharmaceutical research and development that the nonclinical efficacy model for a new drug is in the mouse, making it the natural choice for rodent evaluation of the drug. Strain Differences In addition to differences between mice and other species, there are important differences among different strains of mice. The appropriate choice of a strain of mice for a particular toxicity study should consider the specific objectives of the study and the specific characteristics of candidate strains that might assist or hinder in achieving those study objectives. One difference among strains is in the normal body weights of various strains at different ages. These differences are summarized for selected strains available from the Charles River Breeding Laboratories in table 2.4. Outbred strains tend to be larger at maturity than inbred strains, with the CD-1 strain reaching the highest mean weights at 56 days of age of those strains in table 2.4. The CF-I strain has been reported to be highly resistant to mouse typhoid and to be relatively resistant to salmonellosis (Hill 1981). Nude or athymic strains of mice are more sensitive to tumor development than heterozygous strains. These sensitive strains develop the same types of tumors as those


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Table 2.4 Normal Body Weights in Grams of Selected Strains of Mice Age (days) 21 28 35 42 49 56

Outbred Strains CD-1 CF-I CFW M F M F M F

C3H M F

12 20 27 30 33 35

— 17 18 20 24 27

11 18 22 24 26 27

12 18 24 27 28 30

11 17 21 22 24 26

9 16 19 24 27 28

9 13 17 20 22 23

— 16 17 18 23 26

Inbred Strains C57BU6 BALB/c M F M F — 14 17 19 21 22

— 13 14 16 17 18

— 16 17 18 20 21

— 14 16 17 18 19

Hybrid B6C3FI M F — 16 20 22 24 26

— 14 17 18 19 21

Source: Data derived from Charles River Growth Charts, Charles River Laboratories, ca. 1975.

seen in more conventional strains, but the incidences are higher and the latency periods shorter. There is a wide spectrum of susceptibility to spontaneous lung tumors in various strains of mice, and evidence suggests that there is a high correlation between spontaneous incidence and chemical inducibility in those various strains (Shimkin and Stoner 1975). The inbred strain A mouse appears to be the most susceptible to lung tumors, and forms the basis of a lung tumor bioassay, with tumors inducible within 8 weeks or less of treatment. Susceptibility of various strains to the initiation or promotion of skin tumors has also been shown to differ greatly (Chouroulinkov et al. 1988; Steinel and Baker 1988). The incidences of selected spontaneously occurring neoplastic lesions in CD-1 (outbred) and B6C3F1 (hybrid) strains are compared in table 2.5. The number of strain-related differences in susceptibility to various test articles and environmental conditions exceeds the scope of this chapter, but additional information is available (Nebert 1981). Husbandry Facilities Facilities used to conduct toxicology studies in mice must have separate and adequate areas for the required laboratory procedures and for the housing and treatment of study mice. This discussion is limited to facilities for the housing and treatment of mice. Several characteristics for an adequate facility for the conduct of toxicity studies intended to support regulatory approval of new drugs are listed in the Good Laboratory Practice regulations (CFR 1988, para. 58.43). Such a facility should include enough animal rooms or areas to properly separate different species and projects. In addition, there should be facilities for the quarantine of incoming or sick animals, and for the isolation of any studies that involve use of hazardous materials. In practice, animal rooms used for toxicity studies in mice should not contain any other species of animals. Isolation of individual projects is generally interpreted to mean that an animal room should be dedicated to a single toxicity study. One exception to the requirement to conduct only one study per animal room is the case of acute or very short-term toxicity studies, each of which is limited to a small number of animals. Another exception is dermal carcinogenesis, or “skin painting” studies, which generally involve a relatively small number of animals treated for 30 to 40 weeks. A number of acute or dermal carcinogenesis studies can be run concurrently in a single room. For practical reasons, adequate isolation for acute or dermal carcinogenesis studies is typically interpreted to mean separate cage racks for each study or isolation of multiple studies within the room by geographical location within the room. The intent of the requirements to isolate species and, in most cases, studies is to reduce the probability of cross-contamination between studies with the various test chemicals or disease entities, and to minimize the opportunity for accidental administration of the incorrect test substance to a group of animals. The concept of quarantining incoming animals can be met by having new animals delivered into a sanitized room that contains no other animals, then allowing the new mice an


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Table 2.5 Incidence of Spontaneously Occurring Neoplastic Lesions Lesions Occurring at Spontaneous Incidence of ~1% in Either Sex of Charles River CD-1 or B6C3F1 Mice Strain CD-1 Location and lesion Lymphoreticular tumors Lymphosarcoma Lymphocytic leukemia Lymphoma Histiocytic lymphoma Histiocytic sarcoma Lymphoblastic lymphoma Lymphocytic lymphoma Reticulurn cell sarcoma Skin/subcutis Fibrosarcoma Mammary gland Adenocarcinoma Lung Bronchiolar/alveolar adenoma Bronchiolar/alveolar carcinoma Alveolar type 11 carcinoma Alveolar type 11 adenoma Adenoma Liver Nodular hepatocellular prolif. Hepatocellular adenoma Hepatocellular carcinoma Hemangioma Hemangiosarcoma Reproductive system Ovary Cystadenoma Uterus Endometrial stromal polyp Endometrial sarcoma Leiomyorna Leiomyosarcoma Hemangioma Pituitary Adenoma Thyroid gland Follicular cell adenoma Adrenal Cortical adenoma Harderian gland Cystadenoma Adenoma

M

F

B6C3F1 M F 6.0 1.4

12.0

1.1 2.6 2.6

1.3 9.9 0.5 0.1 1.7 1.7 5.1

1.4 1.4 0.2 0.6 0.4

0.1 1.7 0.2

0.2

0.5

1.0

0.5

3.7

1.7 4.0 3.5 11.7

2.9 3.1 13.9

1.2

0.7

5.4 5.6 7.3 1.0 1.0

1.7 0.8 1.0 1.2 0.2

8.3 1.9 0.2 2.5

3.3 0.6 0.1 1.2

0.5 17.2 13.2 0.7 0.5

0.1 7.1 2.4 0.3 0.1

1.1

0.3

3.3 1.9 1.0 1.0 1.0

2.9 0.6

3.4 0.2 8.6

0.9

1.0

0.3 0.9 0.3

7.9

0.8

2.4

0.4

0.3

1.9 1.5

1.6 0.6

Source: Data from Charles River Breeding Laboratories, compiled from control animals on 24-month studies completed between 1978 and 1986.

adequate acclimatization period prior to initiation of the toxicity study. This procedure minimizes the risk of exposure of either new or existing animals to incoming or endemic diseases. The physical conditions in an animal room are referred to as the macroenvironment. These conditions include such things as the temperature, relative humidity, lighting, ventilation, and concentrations of various gases (e.g., CO2, ammonia). Many of the macroenvironmental parameters


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are routinely monitored in facilities that conduct toxicology studies. Abnormal fluctuations in some of these parameters can have a deleterious effect on the validity of toxicity data generated. Temperature and Relative Humidity Mice are quite sensitive to variations in temperature, and respond to those variations with important physiological changes. This sensitivity is caused by the large surface area to body weight ratio in mice, which causes them to radiate heat quickly in a cold environment. Mice respond to low temperature by nonshivering thermogenesis, and resting mice can generate heat at a rate about triple their basal metabolic rate. Group-housed mice can compensate for low temperatures by huddling in a group, a practice that is more effective in a solid-bottom cage containing bedding. Mice have a limited capacity to compensate for excessive heat, and do so primarily by vasodilation of the ears to increase heat loss, and by increasing body temperature by several degrees. In the wild, mice adapt to excessive temperatures by moving to cool burrows. Mortality is often observed if the ambient temperature reaches 37°C or higher. The range of environmental temperatures where an animal’s oxygen consumption is minimal and virtually independent of changes in ambient temperature is called the thermoneutral zone. The thermoneutral zone for mice is one of the narrowest of species studied, and is about 30.0°C ± 0.5°C. Mice seem to be generally healthier at a temperature of about 21°C to 25°C than they are within the thermoneutral zone (Jacoby and Fox 1984). The recommended dry-bulb temperature for a mouse housing room is 18°C to 26°C (ILAR 1985). Variations in environmental temperature can affect the results of toxicity studies in unpredictable ways. Muller and Vernikos-Danellis (1970) found that the acute toxicity of dextroamphetamine was reduced by tenfold when the temperature was reduced to 15°C from a normal of 22°C, but was increased by two to threefold when the temperature was increased to 30°C. Conversely, the acute toxicity of caffeine was increased when the temperature was altered either up or down from 22°C. Food consumption is inversely related to ambient temperature, and the secondary effects of changes in food consumption can be complex. The relative humidity in a mouse room is a significant factor in thermoregulation for the mice housed. At constant temperature, mice are more active at lower relative humidity than at higher humidity. This difference is believed to be a function of the mouse’s ability to better dissipate heat under conditions of lower relative humidity (Stille et al. 1968). Low ambient relative humidity has been associated with increased transmission of a disease called ringtail in mice. High relative humidity leads to increased production of ammonia in the urine and feces. Increased ammonia concentrations have been associated with the development of respiratory diseases in rodents (Broderson et al. 1976). The recommended relative humidity for a room housing mice is 40% to 70% (ILAR 1985). Lighting Any description of lighting in an animal facility must include a description of the intensity, wavelength or spectrum, and photoperiod of the light. Light intensity is expressed in footcandles (ftc) (lumen/ft2) or lux (lumen/m2). Historically, lighting intensity was selected for convenience of the researchers, based on the assumption that what was good for people was good for mice. In fact, the Guide for the Care and Use of Laboratory Animals as recently as 1978 recommended light intensity, of 100 to 125 ftc. Mice are nocturnal, however, so it is not surprising that their eyes are better adapted to lower light levels. Continuous exposure to light at 100 to 125 ftc for 6 days has been found to cause loss of 90% of the photoreceptors in albino-beige mice (Robison and Kuwabara 1978). Light levels of 25 ftc or lower have been recommended for mice (Robison et al. 1982). In addition, the specific design and location


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of individual cages within an animal room can have a substantial effect on the light level at the animal’s level. Owing to the nocturnal nature of mice and the attenuation of light by the structure and location of individual cages, there is probably little or no actual injury to an animal housed in a room lighted to 100 to 150 ftc on a 12-hr light, 12-hr dark cycle. The wavelength or spectrum of light found in a mouse room is generally a function of the type of fluorescent lighting in common use at the animal facility. Although the lighting spectrum might not be of the highest concern in designing toxicity studies, Spalding et al. (1969) showed that mice exhibited the highest level of voluntary wheel running activity under red light or in darkness; an intermediate level of activity under yellow; and the lowest under green, blue, or daylight. Recalling the nocturnal characteristics of mice, the proximity of red light to dusk or darkness and of blue light to ultraviolet and daylight on the visible spectrum is probably not a coincidence. The photoperiod for mouse rooms used for toxicity studies is typically diurnal, with a cycle of about 12 hr light to 12 hr darkness. The photoperiod influences circadian rhythms, and is probably most often thought of in the context of influence on the estrus cycle. Whereas the estrus cycle of the rat is synchronized by the photoperiod, the estrus cycle of the mouse is less easily influenced by photoperiod (Campbell et al. 1976). Ventilation Ventilation in an animal housing facility should be designed to provide sufficient fresh air to remove the thermal load generated by the animals, lights, and equipment; maintain acceptable levels of dust and odor; provide adequate oxygen; and contain any biohazards or intercurrent disease in the animal colony. A common practice for animal facilities has been to design the heating, ventilation, and air conditioning system to provide 10 to 15 air changes per hour. In reality, this approach can be quite misleading because it depends on the total volume of the animal room rather than the biological and thermal load. An animal room with 12-ft ceilings would require 50% more cubic feet of fresh air per minute to achieve 15 air changes per hour than would a room with 8-ft ceilings. Clearly the more important factor in this example is the number of cubic feet of fresh air per mouse (or kilogram of animals) housed rather than the number of air changes per hour. As a guide, an adequate ventilation rate for a mouse room is about 0.147 ft3/min of fresh air per mouse, with a heat removal capacity of about 0.6 BTU per hour per mouse (Runkle 1964). If at all possible, supplied air should be 100% fresh outside air, introduced into the animal room at ceiling level, with exhaust air picked up at or near the floor to eliminate a maximum amount of heavier-than-air ammonia vapors. Supply air should minimally be drawn through a particle filter, and many facilities now include high-efficiency particle (HEPA) filters on supply air. Addition of HEPA filtration to an existing system usually requires a substantial increase in air conditioning blower power or capacity to overcome the added resistance of the filters. Supply of 100% fresh air at the ventilation rates discussed entails significant energy costs for heating and cooling. These costs can be reduced by the installation of a heat (cool) recovery system between the exhaust air and the incoming fresh air. The efficiencies of such a system are accrued during both the heating and the cooling seasons. If, for some reason, 100% fresh air cannot be supplied, any recycled air must be passed through a complex filtration system to prevent reintroduction of (and cross-contamination by) biological or chemical contaminants and odors. Relative air pressure between animal rooms and corridors should be considered as a prime mechanism for control of cross-contamination and communication of disease between animal rooms. In theory, if all animal rooms are either at positive pressure relative to the corridor, or if all are negative relative to the corridor, there should be no communication of airborne contamination between rooms. If any hazardous substances or human pathogens will be used within the animal rooms, those rooms should be maintained negative to the corridor to prevent contamination of people.


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Noise There are no specific regulations or guidelines governing noise to which mice can or should be exposed. There are, however, numerous reports in the literature of adverse or abnormal effects of “stressful” noise on mice, including reduction in body weight (Fink and Iturrian 1970); changes in immune response and tumor resistance (Jensen and Rasmussen 1970); audiogenic seizures (Mokler 1973), which are seen in genetically susceptible mice and can also be induced in normal mice; unexpected responses to certain drugs (Iturrian and Johnson 1975); actual hearing impairment; and others. In view of the variety of effects associated with noise, control of environmental noise in a facility used to conduct toxicity studies in mice should be addressed. Noise should be described in terms of two dimensions: intensity and frequency. Sound intensity (sound pressure level or loudness) is measured in decibels (dB) and frequency (pitch) is measured in hertz (Hz). Hertz is the standard unit for cycles per second. Although a “safe” intensity of sound has not been described, intensities of 90 to 100 dB have produced adverse effects, including inner ear damage, and it has been recommended that noise levels be maintained below 85 dB (Anthony 1962). It is especially important to consider the frequency of environmental noise because the frequency spectrum for hearing in mice differs substantially from that in humans. High-frequency noise that is inaudible to humans can be disruptive or injurious to mice. The average human ear can hear sounds in the frequency range of about 20 Hz to 20 kHz, with maximum sensitivity at about 2 kHz. In contrast, mice cannot hear sounds with frequencies as low as 1 kHz, clearly hear sounds at 50 kHz, and probably have an upper limit in the range of 60 to 70 kHz (Clough 1982). Mice emit sound in these upper frequencies, apparently as a means of communication between mothers and young, and associated with mating and aggression. Environmental noise near these frequencies might disrupt normal behavior. Devices that emit sound in this spectrum include ultrasonic motion detectors (used for door openers and intrusion alarms), ultrasonic cleaning and mixing equipment, high-speed homogenizers, dropped or banged metallic devices (e.g., cages, pans, covers), and many others. Dogs and nonhuman primates create noise of an intensity and frequency that can be disruptive to mice. Environmental noise should be controlled at two levels: the design and selection of facilities and equipment and establishment of procedures for animal husbandry and the conduct of laboratory activities that might produce noise at a disruptive intensity or frequency. As the noise of dogs and nonhuman primates is disruptive to rodents (ILAR 1985), mice should not be housed in close proximity to these species. Loud noises (e.g., barking of dogs) can be transmitted from room to room, sometimes significant distances, through the ventilation system ductwork. This transmission can be reduced by installing labyrinthine configurations and commercially available acoustic attenuators in the ductwork that services noisy rooms. Mice should not be housed in close proximity to essentially noisy operations such as cage washing. Intense, high-frequency noise can be generated by the movement of cage racks, equipment carts, and so on, in hallways adjacent to animal rooms. A variety of design considerations can improve this situation. A cage rack moving on rubber or synthetic cushioned wheels with well-lubricated bearings over a monolithic flooring will be much quieter than a similar rack on steel wheels with squeaky bearings moving over quarry tile or even concrete flooring. Procedures should be devised and personnel should be trained with an appreciation for the deleterious effects excessive noise could have on the well-being of mice, and consequently on the results of toxicity studies conducted with those animals. Construction Parameters Clearly a detailed discussion of the architectural and engineering aspects of a facility intended to house laboratory mice is beyond the scope of this text. Our experience, however, has suggested two areas that should receive top priority when new construction, renovation, or even routine maintenance is required.


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Cleanability. Animal rooms are exposed to a wide variety of soiling agents on a continuing basis. Sources include such things as feed dust, spilled water, animal waste, bedding, parasites (e.g., mouse pinworms), and various bacterial and viral strains that might infect the species housed. Consequently, animal rooms must be swept and mopped frequently, typically using a chemical detergent and disinfectant, and should be sanitized at least before each new study goes into a room, and on a regular basis if a long-term study is in progress. One of the most effective processes for sanitization uses a pressure sprayer to clean ceilings, walls, and floors with an effective disinfectant. This process requires that all surfaces (ceilings, walls, and floors) be coated with a durable, waterproof, chemical-resistant finish. That finish should withstand impacts, such as cage racks colliding with walls, and various objects being dropped on floors without damage to the flooring. In addition, all lighting fixtures, electrical outlets, switches, computer connections, thermostats, and so on, should be either of waterproof construction, or should be equipped with waterproof covers that can be closed during sanitization. Vermin Resistance. Animal rooms are notoriously attractive to insects such as cockroaches, flies, and other pests. The ready access to feed and water that is essential for the mice is an ideal environment for insect infestation as well. The use of toxicants to control pests in rooms housing animals for toxicity studies is discouraged for several reasons. Many common pesticides are known to induce the synthesis of hepatic microsomal enzymes, which in turn could alter the apparent toxicity of the substance being tested. As most toxicity studies are conducted on test substances with unknown or incompletely understood pharmacology, the possible interactions with a particular pesticide are unpredictable. Pests might consume a toxicant, then enter animal cages prior to dying, and be ingested by the study mice. This could lead to indirect intoxication of the study animals. One environmental requirement for a successful insect population explosion is the availability of a concealed harborage for breeding. We have had remarkable success in controlling insect infestation in our facility by the simple process of sealing, caulking, closing, or eliminating every crack, crevice, hole, wall penetration, electrical outlet, and floor drain in our animal rooms. For this practice to be successful, contractors and maintenance people who work in these rooms must understand the purpose and importance of their task. Cracks in a wall behind a sink are just as useful as harborages as cracks in the middle of the wall, so each contractor and maintenance person must watch for and eliminate these problems when they are found. If elimination of harborage has not been implemented or is incomplete, it might be necessary to employ a toxicant on a carefully controlled basis. The study toxicologist should participate in the selection of a suitable toxicant, factoring in all that is known about the pharmacology of the substance being tested in the toxicity study and providing a best estimate of possible interactions with the toxicant. Accurate records should be kept of what toxicant was used, where and how much was applied, and how often it was applied. These factors should be reviewed and considered when the toxicity study is completed and the results are being interpreted. Caging The physical conditions in an animal cage (primary enclosure) are referred to as the microenvironment. These conditions (e.g., temperature, relative humidity, lighting, ventilation, concentrations of various gases [e.g., CO2, ammonia]) might differ substantially from the conditions in the macroenvironment, depending on the specific design and placement of the cage within the animal room. Microenvironmental parameters should be evaluated for various cage designs and locations within animal rooms, but are not routinely monitored in facilities that conduct toxicology studies. Any caging used for mice in toxicology studies should be designed to provide adequate space for freedom of movement and a comfortable environment in terms of temperature, humidity, and ventilation that will minimize stress on the animals. The caging should be cleaned regularly to allow the mice to remain clean and dry. Caging should be as resistant to escape as possible to


34


preserve the integrity of the study as well as the health and safety of the animals. Even if an escaped animal can be recovered and returned to its cage, the health of the animal and resultant impact on the integrity of the toxicity data generated remains in question, as the animal might have contacted toxic or interfering substances during its travels. Cage Type The two types of mouse caging most commonly used for toxicology studies are wire-bottom cages and solid-bottom cages. Wire-Bottom Cages. Wire-bottom cages are typically suspended in rows on a movable rack with between 10 and 70 cages arranged on a side. Racks can be single sided or double sided, depending on the configuration of the animal room and the number of animals that need to be housed. Most contemporary wire-bottom cages are fabricated of stainless sheet steel backs and sides, with stainless steel wire mesh fronts and floors. Sides and backs might have holes or slots stamped into them at manufacture to allow improved ventilation and access to an automatic watering system if one is available. Mesh fronts allow observation of the animals. Wire-bottom cages are also fabricated of polycarbonate or other rigid plastic products. Transparent plastic cages provide easier observation of animals, but typically offer reduced ventilation through the cage itself. Mesh floors allow urine, feces, and spilled food to drop through, typically to a waste pan or absorbent paper, which can be cleaned or replaced easily and regularly. Waste pans should be cleaned or papers replaced at least three times a week. A wire-bottom design has the advantage of keeping animals relatively free of contamination from urine and feces, and also providing ease of cleaning. Wire-bottom cages should be rotated out of use and washed at least once every 2 weeks. This washing procedure should be monitored regularly, and should be adequate to produce negative results on microbiological swab testing. A procedure that achieves good microbiological test results for cages with average levels of soil first requires removal (or emptying) of feeders, waste pans, and water bottles (if present). Then the racks with the suspended wire cages still in place are passed through a commercial rack washer that operates much like an automatic dishwasher. All cycle times are variable, but an effective combination for average soil is a wash cycle of about 10 min, followed by initial and final rinse cycles of about 3 min each. The wash cycle includes an effective disinfectant detergent (e.g., PRL-18, manufactured by Pharmacal Research Laboratories, Inc., Naugatuck, Connecticut). All water temperatures (wash, initial, and final rinse) are maintained at or above 82°C (180°F), but this temperature is most important for the final rinse. Solid-Bottom Cages. Solid-bottom cages, often referred to as shoebox cages, can be suspended in a rack, like wire-bottom cages, or can be supplied with tops, and arranged on shelves, which are typically movable as a rack. Shoebox cages are typically constructed of polycarbonate, which allows convenient observation of the animals; polypropylene, which is a translucent plastic; or sheet metal, such as stainless steel. Shoebox cages provide a secure base for animals, and are essential if animals are to be allowed to deliver and suckle live litters. Solid-bottom cages can be provided with filter tops, which can significantly reduce airborne contamination of the environment within the cage. Filter tops do have a negative impact on ventilation within the cage, and levels of CO2 and ammonia have been found to be substantially higher in cages with filter tops than in those with stainless steel rod tops (Serrano 1971). There are a number of disadvantages associated with solid-bottom cages. These cages must be provided with some form of low-dust or dust-free absorptive bedding. This bedding must be changed regularly, which is labor intensive. If matings are conducted, vaginal plugs are often difficult or impossible to find in the bedding. As mice engage in coprophagy, a toxic substance or metabolite that is eliminated in the feces may be “recycled,” thereby leading to an overestimate of the true toxicity of the substance. In addition, mice commonly ingest various types of bedding, such as wood chips, which renders impossible any serious estimate


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of food consumption. If an automatic watering system is in use, solid-bottom cages have the potential to fill with water if there is a malfunction, drowning the inhabitant(s). In routine toxicology studies, solid-bottom cages, covers, feeders, and so on, should be rotated out of use and washed once or twice a week, and their supporting racks should washed at least once per month. This cage-cleaning schedule should not be followed if reproductive procedures (mating, delivery, and suckling of young) are being conducted, as the constancy of home-cage odor is critical to reproductive efficiency and reducing the likelihood of cannibalization of young. Cage washing might need to be suspended completely during pregnancy and lactation. (See “Bedding” section for special practices used with reproduction procedures.) The cage and accessory washing procedure should be monitored regularly, and should be adequate to produce negative results on microbiological swab testing. A procedure that achieves good micrcobiological test results in our facility for cages with average levels of soil employs passage through a tunnel washer on a steel mesh belt. The total transit or cycle time is typically about 3 min, with about 15 sec for prewash, and just under 1 min each for main wash, rinse, and drying cycles. The wash cycle includes an effective disinfectant detergent (e.g., Clout, manufactured by Pharmacal Research Laboratories, Inc., Naugatuck, Connecticut). The rinse water is heated to at least 820°C (1,800°F), and the drying cycle consists of exposure to high-temperature forced air. The belt speed in a machine such as this can be reduced (total transit time lengthened) for heavily soiled cages or increased (transit time shortened) for lightly soiled cages, but the single criteria that governs the minimum length of the transit time is the maintenance of negative results on the microbiologic monitoring of the clean cages. Cage Size Mouse caging must be of adequate size to allow free movement and to avoid overcrowding. Minimum space requirements for mice are provided in the Guide for the Care and Use of Laboratory Animals (ILAR 1985) on the basis of body weight. Those requirements are listed in table 2.6. As a practical matter, to avoid the need for multiple-sized caging and frequent (e.g., perhaps weekly) changes in the size of caging occupied, the minimun specified for adults can be used for all ages. Population Mice can be housed singly (one to a cage) or in groups (several animals of the same treatment group caged together) for toxicity testing. It has long been recognized, however, that group housing can substantially alter the toxicity of some substances. Chance (1946) demonstrated that the acute toxicity of a group of sympathomimetic amines was increased by two- to tenfold in mice that were group housed compared to mice housed singly. In addition, increased population in solid-bottom cages has been shown to lead to substantially higher levels of CO2 and ammonia within the cage, even when open stainless steel rod tops were used, but especially if filter tops were in place (Serrano 1971). Increased levels of ammonia have been associated with hepatic microsomal enzyme induction, which can alter expected metabolism in a toxicity study. Table 2.6 Minimum Cage Space Requirements for Mice Body Weight (g) < 10 10–15 15–25 > 25

Floor Area per Mouse in2 cm2 6.0 8.0 12.0 15.0

38.7 51.6 77.4 96.8

Cage Height in cm 5 5 5 5

12.7 12.7 12.7 12.7


36


Single Occupancy. Single housing of mice used in toxicity studies offers many advantages. Of paramount importance is the reduced likelihood of mistaking the identity of individuals when conducting various study procedures (weighing, dosing, collecting various observational or other data). Singly housed mice are more quickly and easily identified and captured throughout the study. In addition, the risk of injury or cannibalism is eliminated in the event that one member of a group becomes debilitated. The biggest disadvantage to single housing is cost. Purchase price for individual caging for large numbers of mice is much higher than the cost for group housing. Individual caging requires much more floor space in the animal rooms than group caging for an equivalent number of animals, and the cost to provide animal care (food, water, cage cleaning, and sanitization) is much higher for single caging. Group Housing. The biggest advantage to group housing is cost savings. Disadvantages include increased probability of mistaken identity, increased stress on animals as a result of establishment and testing of dominance hierarchy, and difficulty with individual animal identification systems. Group-housed mice tend to tear out each other’s ear tags, and tattooed markings might be obliterated as a result of repeated fighting for dominance. Measurement of food or water consumption is generally not useful for group-housed mice, as the distribution of food and water among animals of different hierarchical positions tends to be quite uneven. Group housing in solid-bottom cages effects microenvironmental parameters within the cage such as temperature, humidity, various gas concentrations (CO2, ammonia), and others. Bedding Some form of bedding material is required in solid-bottom cages to allow mice to remain clean, dry, and free of urine and feces. The Good Laboratory Practice (GLP) regulations (CFR 1988, para. 58.90) states that bedding should “not interfere with the purpose or conduct of the study” and should “be changed as often as necessary to keep the animals dry and clean.” Appropriate bedding should absorb urine effectively, be as free of dust as possible to minimize pulmonary complications, be free of contaminating chemicals, and have no unacceptable effect on the normal physiology or metabolism of the mice. Inhalation of aromatic hydrocarbons from cedar and pine bedding has been shown to cause induction of hepatic microsomal enzymes, which could seriously compromise the results of a toxicity study (Vesell 1967; Wade et al. 1968). Opinions differ on whether the use of cedar wood shavings as bedding material contributes to the increased incidence of mammary gland tumors and hepatomas (Heston 1975; Sabine et al. 1973). One of the most commonly used bedding materials for toxicity studies is hardwood chips derived from woods such as maple, birch, and beech (e.g., Absorb-Dri hardwood chips, Maywood, New Jersey). This selection is probably based on the properties of high absorption and low dust, coupled with an absence of proof of harmful interactions. Bedding material should be changed at least once or twice weekly when cages are washed. More frequent bedding changes might be required if mice are group housed. An exception to this practice for bedding change occurs when reproductive procedures (mating, delivery, and suckling of young) are involved in the study. A continuity of home-cage odor reduces maternal stress, is critical to reproductive efficiency, and reduces the likelihood of cannibalization of young. Therefore, during reproductive procedures, a portion (but not all) of the soiled bedding should be removed at regular intervals and replaced with clean bedding. Animal Identification Reliable identification of each animal used in a toxicity study is essential to the integrity of the study data and to the accurate interpretation of study results. The GLP regulations (CFR 1988, para. 58.90) state that


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animals, excluding suckling rodents, used in laboratory procedures that require manipulations and observations over an extended period of time or in studies that require the animals to be removed from and returned to their home cages for any reason (e.g., cage cleaning, treatment, etc.), shall receive appropriate identification (e.g., tattoo, toe clip, color code, ear tag, ear punch, etc.).

And further, that “All information needed to specifically identify each animal within an animal-housing unit shall appear on the outside of that unit.” The GLP regulation was amended (Federal Register 1989) to eliminate toe clipping from the methods listed (previously), and to discourage the use of toe clipping in nonclinical laboratory studies, as other, more humane methods are available. One new method not listed in the GLP regulation, but which appears to offer some advantages, is the implantable electronic microchip. Cage Cards In practice, the comprehensive information already specified for the outside of an animal housing unit typically appears on a cage card. This information is relatively straightforward as long as animals are housed singly. The cage card should include complete identification of the study (study number is adequate if a unique coding system is used)and identification of or references to the species, strain, sex, source, age, treatment group (e.g., substance tested and dose group), and individual animal number of the mouse (or mice) housed. The identification that appears on the mouse itself is limited to a few code letters and or numbers that can be cross-referenced to the cage card and raw data record for the study to obtain all of the information available about that individual mouse. Ear Tags Ear tags are durable, typically made of a noncorroding metal such as Monel, with letters or numbers stamped into the surface. The tags are quick and easy to apply, using a special pliers-like applicator, and are easily read over a long period of time. One disadvantage to ear tags is that they can be torn out of the ear if they get caught on something in the cage, or especially if mice are group housed. Group-housed mice seem to find each other’s ear tags especially vulnerable to pulling and hierarchical conflict, and the incidence of tag loss in group-housed mice has proven unacceptably high in our facility. Conversely, the incidence of tag loss among singly-housed mice (the norm for toxicity testing at our facility) is remarkably low, especially after the first few days of adaptation. When tags are lost from singly-housed mice, they are typically found in or under the cage, so the tag number can be confirmed and a replacement tag installed. One technique to increase tag retention is to avoid the edge of the ear, installing the piercing portion of the tag as close to the center of the ear as possible. A useful tag for mice is a small (5/16 in. long) self-piercing Monel tag (V shaped prior to application, crimped to a flattened O by installation) such as Style 4-1005, Size I from National Band and Tag (Newport, Kentucky). These tags can be ordered custom stamped with at least three numbers or letters on one surface, and four on the other surface (i.e., up to 10 million different numbers, and more combinations if letters are used as well). Ear Notching or Punching Ear notching or punching is a system of holes punched or notches cut into the edges of the ears based on a predetermined numeric code. Figure 2.1 is an example of an ear-marking code (from Leard 1984). Notching has the advantage of not offering a hard object that can be caught or pulled from the ear, such as an ear tag. Disadvantages include the requirement for substantially more time and care in initial identification of the animals. Clearly it takes longer and inflicts more pain on the mouse to punch and clip a precise pattern of from one to five holes and notches into the two


38


Left

Right

Left

Right

10

1

40

4

20

2

50

5

30

3

60

6

70

7

200

100

80

8

90

9

Figure 2.1 Example of an ear marking code which can be used for animal identification. (From Leard 1984, used with permission.)

ears of a mouse than to crimp on a single ear tag. Imprecisely positioned punches or notches may be difficult to read for the duration of the study. Group-housed mice might chew at each other’s ears, especially when the notches are fresh, rendering the code more difficult to read. Finally, most codes allow at most a few hundred nonrepeating numbers, meaning that patterns will have to be repeated within most facilities in a short time. Tattoos Mice can be tattooed on the tail or feet as a means of identification. Tattoos are permanent unless they are obliterated by chewing or injury. The process of applying alphanumeric tattoos to the tails of mice is more time-consuming than ear tagging or notching, but the tails are more accessible than the feet. Another practical consideration is the size of a mouse’s tail. Very small letters are both more difficult and time-consuming to apply to a mouse tail, and more difficult and time-consuming to read at each subsequent identity check throughout the study. The size of the mouse tail and practical considerations of legibility limit the tattoo to four or five alphanumeric characters on the dorsal (easily readable) surface of the tail. It is possible to tattoo an additional four to five alphanumeric characters on the ventral surface of the tail if necessary, but this surface is less easily read. Although tattooing is a viable method of identification for longer toxicity studies because of its permanence, the amount of time required to both apply and read the tattoos makes this method impractical for shorter studies. Color Coding of Skin or Hair The skin or fur of a mouse can be quickly color coded using a variety of indelible felt-tip markers. A variety of combinations of colored markings on the tail or on the fur of the back are easily read without disturbing the animal. This procedure is particularly useful for short toxicity studies. Owing to the fastidious grooming habits of mice, there is a tendency for colored marks to be groomed off quite quickly. This problem is exacerbated if the mice are group housed. A number of different brands and types of markers should be tested, as there is wide variability in their durability or resistance to grooming. This problem can be reduced somewhat by locating the color code in a place that is more difficult to groom, such as the top or back of the head. One substance that confers a durable stain on


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the fur is Bouin’s fixative, but the color is limited to yellow, so the identity code must be based on the location of the stain rather than the color. The number of possible easily distinguished colors and combinations of colors available for a color code and the maximum number of different sites that can be easily coded on a mouse limit the number of unique codes to a few hundred. In general, color coding is a useful procedure for identifying mice in short studies. The toxicologist should evaluate each mouse and be prepared to “touch up” the color code on a daily basis. Toe Clipping Toe clipping for purposes of identifying mice involves amputation of various combinations of toes using a surgical scissors, nail clipper, or other suitable device to establish an identity code. Toe clipping has the advantage of permanence, but an individual mouse might become “unreadable” through accident or injury that compromises unclipped toes. Toe clipping has many disadvantages, and no clear advantages over other methods such as ear tagging or notching. Most important, toe clipping is more traumatic than other methods, and has an increased likelihood of resulting in infection because it involves the feet. The method is time-consuming to perform and the codes are difficult to read through the duration of the study because the mouse must be picked up and often the toes of each foot must be spread to facilitate accurate reading. As stipulated at the beginning of this section on animal identification, the Food and Drug Administration (FDA) has amended the GLP regulations to eliminate toe clipping from the list of procedures recommended for identifying animals. Toe clipping might have some utility in neonatal (or very young) mice, in which the nervous system is not as well developed, and more conventional methods are impractical. Implantable Electronic Microchips The current preferred approach to animal identification is the implantable electronic microchip. The microchip is a transponder that has been sealed into a glass capsule about 1.0 cm in length and about 1.5 mm in diameter. This device is suitable for subcutaneous implantation in mice. The device is “energized” by one of a variety of portable or stationary readers that emits a low-power radiofrequency signal. The transponder is stimulated to transmit its unique identification number back to the reader, where it can be both displayed and linked directly to a computer system. The microchips are easy to install, resistant to loss, and should perform well over the course of a longer term study. The device has a capacity for up to 34 billion different numbers. Preliminary data from several laboratories suggest that the devices are well tolerated, and do not produce problematic histological changes at the implantation site. One disadvantage is that the electronic reading equipment must read the devices. The implication is that in the event of equipment failure, there is no means for manual decoding or reading of the chips. Possession of multiple readers should provide reasonable redundancy for most facilities. Food Nutritional Requirements The explicit nutrient requirements for mice have not been extensively studied nor defined. What is known of these requirements has been estimated on the basis of a number of studies that have had other objectives. Some studies have focused on the effects of specific dietary deficiencies, and others have looked at acceptable performance in growth and reproductive parameters as evidence of nutrient adequacy. Mice have different nutrient requirements for growth, reproduction, and maintenance, and the many diverse genetic strains differ in their minimal requirements as well. Estimated minimum nutritional requirements for proper growth and reproduction of conventional mice have been compiled in table 2.7 from Nutrient Requirements of Laboratory Animals (National Research Council [NRC] 1978).


40


Table 2.7 Estimated Nutrient Requirements of Mice: National Research Council (1978) Nutrient Linoleic acid Protein (growth) Protein (reproductive) L-Amino acids Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Minerals Calcium Chloride Magnesium Phosphorus Potassium Sodium Chromium Copper Fluoride Iodine Iron Manganese Selenium Vanadium Zinc Vitamins A D E K1 equivalent Biotin Choline Folacin Inositol(myo-) Niacin Pantothenate (Ca) Riboflavin Thiamine Vitamin B6 Vitamin B12

Unit

Requirement

% % %

0.3 12.5 18.0

% % % % % % % % % %

0.3 0.2 0.4 0.7 0.4 0.5 0.4 0.4 0.1 0.5

% 0.4 (required, but not quantified) % 0.05 % 0.4 % 0.2 (required, but not quantified) mg/kg 2.0 mg/kg 4.5 (status uncertain for mice) mg/kg 0.25 mg/kg 25.0 mg/kg 45.0 (required, but not quantified) (status uncertain for mice) mg/kg 30.0 IU/kg 500.0 IU/kg 150.0 IU/kg 20.0 mg/kg 3.0 mg/kg 0.2 mg/kg 600.0 mg/kg 0.5 (bacterial synth. usually adequate) mg/kg 10.0 mg/kg 10.0 mg/kg 7.0 mg/kg 5.0 mg/kg 1.0 mg/kg 0.01

Selection The choice of a specific diet to be used in a mouse toxicity study should take into account all of the objectives and requirements of the study as well as the convenience, efficiency, cost, and availability of a particular diet. In general, diets obtained from well-known commercial suppliers with established procedures for quality control and documentation will be worth any added cost. One of the basic choices is whether to use pelleted diet or meal. Pelleted diet is generally easier and neater to handle, both for the people and the mice involved. The meal form of a diet is usually easier to use if the study design requires mixing of a test chemical with the diet or if food consumption will be measured.


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Mice can obtain adequate nutrition from a variety of types of diets. Diets are classified on the basis of the amount of refinement of the ingredients (NRC 1978). Three types of diets are described here. The types are natural ingredient diets, purified diets, and chemically defined diets. Natural Ingredient Diets. These types of diets contain grains such as corn, wheat, oats, beet pulp, and other ingredients that have been subjected to minimal processing such as fish meal, soybean meal, wheat bran, and a variety of vitamin and mineral supplements. They also have been referred to as a cereal-based, unrefined, nonpurified, or stock diets. Natural ingredient diets are the most widely used and are relatively economical. The principal objection to such diets is that they have had a tendency to vary widely in terms of nutrient and contaminant content (Newberne 1975; Rao and Knapka 1987; Wise 1982; Wise and Gilburt 1980). Data were compiled on key nutrient and contaminant concentrations for all of the lots of a commercially available natural ingredient rodent diet (Purina Certified Rodent Chow 5002, Purina. Mills, Inc., St. Louis, Missouri) received at our facility over a period of 1 year. The variability of these concentrations from lot to lot is relatively low, as seen in table 2.8 and table 2.9. Table 2.8 Content of Several Key Nutrients in Different Lots of a Closed Formula, Natural Ingredient Rodent Diet (Purina Certified Rodent Chow 5002)

Pellets

Means Meal

Means Combined means Range

Protein (%)

Fat (%)

Fiber (%)

Calcium (%)

Phosphorus

20.80 20.30 20.30 21.60 20.30 21.90 20.80 20.20 20.10 21.00 20.20 20.80 20.60 20.50 20.30 20.70 20.30 20.80 20.80 20.65

5.75 4.64 5.08 5.45 5.40 4.93 5.00 5.73 5.82 6.07 5.94 5.89 5.80 5.60 5.74 5.68 5.90 5.50 6.17 5.58

4.46 4.20 4.14 4.76 4.28 4.11 4.60 4.09 3.77 3.97 4.36 4.39 3.95 4.08 3.75 3.86 3.87 3.74 3.81 4.12

0.781 0.708 0.684 0.834 0.893 0.758 0.774 0.710 0.760 0.786 0.768 0.831 0.841 0.682 0.763 0.853 0.726 0.754 0.860 0.777

0.680 0.650 0.633 0.666 0.661 0.695 0.588 0.629 0.607 0.683 0.599 0.745 0.592 0.592 0.606 0.622 0.536 0.588 0.600 0.630

21.30 21.90 21.50 21.70 21.40 20.30 20.30 20.70 21.10 21.10 21.20 21.20 21.10 21.50 20.50 20.40 21.08

5.76 5.45 5.40 4.82 4.92 5.74 5.91 6.43 5.83 6.02 5.76 6.15 5.75 6.33 6.16 5.81 5.77

4.47 4.45 4.26 3.84 4.07 4.00 4.31 3.68 4.13 4.03 4.45 3.99 3.67 3.93 4.20 3.97 4.09

0.850 0.924 0.715 0.877 0.718 0.632 0.903 0.741 0.809 0.681 0.817 0.880 0.849 0.832 0.732 0.790 0.797

0.589 0.716 0.542 0.696 0.647 0.561 0.566 0.676 0.701 0.587 0.732 0.576 0.581 0.605 0.561 0.602 0.621

20.84 20.1–21.9

5.67 4.64–6.43

4.10 3.67–4.76

0.786 0.632–0.924

0.626 0.536–0.745


42


Table 2.9 Content of Several Key Contaminents in Different Lots of a Closed Formula, Natural Ingredient, Rodent Diet (Purina Certified Rodent Chow 5002) (in ppm) Arsenic Pellets

Cadmium

Lead

Mercury

Selenium

Lead

0.334 30.0 4.8 0.0 2.4

35.7 26.3 17.1

52.4 63.2 56.1

7.1 10.5 24.4

The cut surface of a normal testis bulges. The color of the testis in young beagles is light pink to tan (depth of color increases with age due to increased pigmentation of the interstitial cells). The testis is subdivided into numerous lobules by delicate connective tissue septa that extend from a core of connective tissue (mediastinum testis) to the capsule of the testis (tunica albuginea). Ducts, blood vessels, lymphatics, and nerves enter and leave the testis through the mediastinum. Each of the lobules contains one or more convoluted sperm-producing tubules (seminiferous tubules) that empty at both ends into straight tubules (tubuli recti) that connect with a series of epithelial-lined channels (rete testis) in the mediastinum. Spermatozoa are swept out of the seminiferous tubules, through the rete testis, and into the epididymis in a fluid secreted by the sustentacular cells (Sertoli cells). The interstitial cells (Leydig cells) are the endocrine (testosterone-producing) cells of the testis. Epididymis. The epididymis consists of the ductuli efferentes and the much longer and highly tortuous ductus epididymidis. The ductuli efferentes emerge from the mediastinum to connect the rete testis with the ductus epididymidis. The epididymis lies along the dorsolateral surface of the testis. For descriptive purposes the epididymis is divided into three portions. The initial portion located near the cranial extremity of the testis and into which the ductuli efferentes empty is called the head of the epididymis (caput epididymis). The portion of the head with the ductuli efferentes is called the initial segment. The main length of the epididymis is the body (corpus epididymis), and the segment attached to the caudal extremity of the testis is the tail (cauda epididymis). The epididymis is the maturation and storage site for the spermatozoa. The ductus epididymidis is subdivided further, with each subdivision apparently fulfilling a specific function in the maturation process of the sperm. Ductus Deferens. The ductus deferens (deferent duct) is a thick-walled tube that is continuous with the tail of the epididymis and extends to the prostatic urethra. Its initial portion is located within the spermatic cord and surrounded by the veins of the pampiniform plexus, arteries, lymph vessels, nerves, and smooth muscle. Prostate Gland. The prostate gland (the only accessory male sex gland in the dog) is an ovoid musculoglandular organ that completely surrounds the proximal portion of the ureter. The prostate is composed of two portions: external and internal. The external portion forms most of the dog’s prostate and consists of two large bilateral lobes. The internal portion consists of a few small glands scattered along the urethra. Trabeculae divide the two external lobes into lobules in which the glandular elements are most prevalent near the periphery. Secretion from prostate glands enters the urethra by way of numerous excretory ducts. The paired ductus deferens enter the dorsal surface of the prostate and run caudoventrally on either side of the median plane to open into the dorsal surface of the prostatic urethra. James and Heywood (1979) found incomplete prostate development in beagles 9 to 10 months of age and demonstrated a wide range of prostate weights in control beagles on short-term toxicity studies (table 8.14). Penis. The male copulatory organ (penis) is composed of three principal parts: root, body, and distal free part (glans penis). The glans penis is mostly enveloped in stratified squamous epithelium and, when not erect, is entirely withdrawn into a tubular sheath of integument (prepuce). The

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Table 8.14 Prostate Weight in Beagles Under 2 Years of Age Number of Dogs

Mean Age (Weeks)

Mean Body Weight (kg)

Mean Prostate Weight (g)

< 5.0

42 38 41

37 ± 4 46 ± 2 73 ± 4

11.9 ± 1.8 15.6 ± 2.0 12.4 ± 2.0

3.3 ± 1.6 6.5 ± 2.3 8.1 ± 2.9

84.4 23.4 9.8

% of Dogs with Prostate Weight in Range (g) 5.1–10.0 10.1–20.0 15.6 66.1 63.4

0.0 10.5 26.8

> 20.0 0.0 0.0 0.0

mucosa of the penile urethra is lined by transitional epithelium except near the external urethral opening, where it changes to stratified squamous epithelium similar to that covering the penis. Female Genital Organs The female genital organs consist of the ovaries, oviducts, uterus, vagina, and vulva. The ovaries, oviducts, and uterus are attached to the walls of the abdominal and pelvic cavities by folds of peritoneum called broad ligaments. Each broad ligament contains an ovary, oviduct, and uterine horn (plus vessels, nerves, and fat). Ovary. The ovary is an ovoid gland situated within a fossa of the peritoneum (ovarian bursa) and supported by the mesovarium (cranial portion of the broad ligament) and the suspensory ligament of the ovary. The ovary is completely enclosed by the ovarian bursa except for a narrow slit, 2 mm to 15 mm long, located on the medial side. The structure of the ovaries varies with age and the phase of the sexual cycle. The ovary is composed of a cortex and medulla that contains a prominent rete ovarii. The cortex consists of a connective tissue stroma that contains blood vessels, lymphatics, follicles, and corpora lutea. The surface of the cortex is smooth in immature ovaries. Oviducts. The oviducts (uterine tubes) are tortuous structures that extend from the region of the ovary to the uterine horns. In the bitch, the oviduct almost completely encircles the ovary. The large funnel-shaped ovarian end of the duct is called the infundibulum and is located near the slit in the ovarian bursa. Uterus. The uterus is a Y-shaped tubular organ that communicates with the oviducts cranially and the vagina caudally. The uterus consists of a neck (cervix), body (corpus), and two horns (cornua). In the bitch, the uterine body is short and the horns relatively long and straight. The right horn is usually longer than the left. The size and shape of the uterus varies according to age and the stage of the sexual cycle. The uterine horns unite at the body. The body extends from the point of convergence of the uterine horns to the cervix. The wall of the uterus consists of three layers: mucosa (endometrium), muscularis (myometrium), and serosa (perimetrium). The endometrium is the thickest of the three layers and consists of three zones: crypt, intermediate, and basal (McEntee 1990). The crypt zone has numerous short, epithelial-lined recesses. The intermediate zone contains uterine glands, but it is predominantly connective tissue. The uterine glands branch, coil, and terminate in the basal zone. Vagina. The vagina is the highly dilatable musculocutaneous canal extending from the uterus to the vulva. Cranially, the vagina is limited by the cervix, which can protrude up to 1 cm into the vagina. Caudally, the vagina ends just cranial to the urethral opening. Flat longitudinal folds extend throughout the length of the vagina. The tunica mucosa is nonglandular stratified squamous epithelium. Vulva. The vulva is the external genitalia of the bitch and consists of the vestibule, clitoris, and labia. The vestibule is the space connecting the vagina with the external genital opening and is the

630


largest part of the vulva. The vestibule is the common opening for the genital and urinary tracts. The clitoris (homologue of the male penis) is located in the extreme caudal region of the vestibule near the ventral commissure of the vulva. The labia (lips) form the external boundary of the vulva. The vestibule is lined by stratified squamous epithelium and contains small, mucus-producing vestibular glands. Numerous lymph follicles are present in the vestibular mucosa and might be large enough to be seen grossly. The labia are covered with stratified squamous epithelium and are rich in sebaceous and tubular sweat glands. The Estrous Cycle. Puberty is the age at which first estrus occurs in the bitch and varies among breeds and within breeds. The range is usually given as 6 to 12 months. The average age for first estrus in the laboratory beagle appears to be about 12 months, with a range of 10 to 14 months (Andersen 1970; Sekhri and Faulkin 1970; Sokolowski 1973). Most female beagles will be sexually immature when started on toxicology studies. At termination of short-term studies, they might still be sexually immature or be in any stage of their first estrous cycle. The sexual stage of the bitch at termination of a study impacts the gross and microscopic appearance of the sex organs (and mammary gland) and the weights of the ovaries, uterus, and conceivably the hypophysis and adrenal glands. Traditionally, the cyclical changes occurring in the reproductive system of the bitch have been divided into four phases: proestrus, estrus, metestrus, and anestrus. This classification has deemphasized the important extended luteal phase of the cycle and obscured several events that occur during the estrus phase of the cycle. Recently, several workers (Cupps et al. 1969; Holst and Phemister 1974; McDonald 1969) have reexamined the traditional classification and suggested modification. Phemister (1974) summarizes the revised view as follows. Proestrus remains the phase of rapid follicular growth and rising estrogen levels when there is swelling of the vulva, generalized congestion of the genital tract, and a sanguineous vaginal discharge. As a rule, proestrus lasts about 9 days. Estrus is the period of sexual receptivity, usually lasting 7 to 10 days. In most bitches the initial day of estrus coincides approximately with a surge of luteinizing hormone (LH) from the pituitary gland. The LH surge is followed by ovulation 2 days later, usually on about the 3rd day of estrus. Metestrus, as the term is used for other species, defines the brief period when the corpus luteum is being formed and becoming functional. In the bitch this phase lasts for about 4 days and occurs entirely within the period of acceptance (estrus). By 4 days after ovulation, the genital system is dominated by luteal progesterone. By definition this phase of progesterone dominance is diestrus. Its onset is signaled by an abrupt change in vaginal cytologic characteristics: from predominantly large comified, superficial squamous cells to noncomified, small intermediate and parabasal cells. On an average, diestrus begins 2 to 3 days before the end of estrus, and based on hormonal data, lasts for 2 to 3 months, or even longer if morphologic data are used. Following diestrus, the bitch enters a period of reproductive quiescence, anestrus, which lasts for 3 months or more.

Metestrus in the revised classification describes the period more in accordance with its use in other species, and emphasizes the fact that corpora lutea form and function for comparable periods (2–3 months) in a bitch whether she is pregnant or not pregnant. In other words, every nonpregnant bitch experiences a period of pseudopregnancy (pseudocyesis) to some degree. When the bitch’s appearance and behavior closely mimic pregnancy, the pseudopregnancy becomes a clinical problem. Necropsy and Laboratory Techniques Because of the large functional reserve of the kidney, renal diseases might or might not be associated with renal dysfunction. Two-thirds to three-fourths of the renal parenchyma must be functionally impaired before clinical signs develop in chronic renal failure (Osborne et al. 1983). Urinary enzymes have been useful in dogs for detecting acute renal damage, but less helpful for chronic damage. Increases in brush border enzymes, including gamma-glutamyl transferase and

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alkaline phosphatase, have been associated with proximal tubular damage, whereas increases in N-acetyl-beta-D-glucosaminidase have been observed in the early stages of renal papillary necrosis (Clemo 1998). Functional abnormalities of the kidney can be clinically manifested in a number of ways, including polyuria (formation and elimination of large quantities of urine), oliguria (decrease in the rate of formation or elimination of urine), anuria (lack of urine formation or elimination), polydipsia (excessive thirst), weight loss, anorexia, vomiting, diarrhea, dehydration, edema, stomatitis, weakness, and depression. Cloudy urine or hematuria might indicate disease of the urinary bladder. It is unlikely that signs of a reproductive system disorder would be detected in short-term toxicology studies. At postmortem, the initial examination of the urogenital system should be done with all organs of the system in place. Kidney The kidneys should be approximately equal in size. The normal cortex is brownish red and the medulla white to pink. The kidney should be cut into multiple transverse sections and the surfaces of each examined. One or two sections (4–5) should be taken from the midpyramidal region and placed in fixative. Proximal tubular epithelium autolyzes rapidly and kidneys should be placed into fixative as early as possible. Both the immature and mature kidney of the beagle have been studied in detail and much is known about normal morphometry of the organ (Eisenbrandt and Phemister 1977, 1978 1979, 1980; Jaenke et al. 1980; Stuart et al. 1975). Ureters and Urinary Bladder Usually, the ureters need only to be examined externally in situ. The urinary bladder should be removed, incised longitudinally, everted, and the mucosal surface examined. The entire bladder should be fixed. Usually, the bladder contains some urine at necropsy. Uncontaminated urine specimens can be obtained by puncturing the undisturbed bladder with a hypodermic needle (avoiding blood vessels) and withdrawing urine into a syringe. Prostate, Scrotum, and Epididymis The prostate should be removed and its internal surface examined by one or more transverse cuts (some prostates are so small that bisection is adequate). At least two sections should be fixed. The scrotum should be incised, the scrotal ligaments severed, the testes removed, and the vaginal tunics surrounding the spermatic cords incised to the level of the abdominal wall. After examining the structures of the spermatic cords and the surfaces of the testis and epididymis, the testis and epididymis are freed. The epididymis should be dissected from the testis before the testis is weighed. The epididymides can be fixed intact. The testis should be sliced before fixation to verify that the cut surface bulges. (Failure to bulge indicates degeneration of seminiferous tubules.) The incisions should be made with a very sharp knife. McEnree (1990) also recommends samples be taken from the head extremity, middle part, and caudal extremity. Bouin’s is the recommended fixative for the testis. Ovaries and Uterus The ovaries and uterus are usually weighed in toxicology studies. As with the testis and prostate, the weight of the female organs varies according to sexual maturity, and, additionally, on the phase of the estrous cycle. Reproductive organs should be examined in detail in studies of compounds with known or potential effects on the reproductive system. Less attention might be paid to the reproductive tract in other studies; nevertheless, it seems appropriate that some observations be

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made to establish the probable sexual state of a bitch at necropsy and to have these observations to correlate with microscopic findings and organ weight data. Gross and Microscopic Appearance of Female Genital Organs at Necropsy The gross and microscopic appearance of the female genital organs of the beagle during the various phases of the estrous cycle have been described (Andersen 1970; Sokolowski 1977; Sokolowski et al. 1973). Vulva. The external genitalia swell and discharge a sanguineous fluid at the onset of proestrus. (The sanguineous discharge of proestrus and estrus is not due to mucosal hemorrhage but to extravasation of erythrocytes by diapedesis through the endometrium and into the lumen of the uterus and vagina.) The thickening of the labia and wall of the vestibule is due to congestion and edema that account for the warm feeling of the external genitalia from which the expression “in heat” is derived. The vestibular mucosa remains unwrinkled during proestrus and estrus, but lymph follicles might become prominent. Vagina. The vaginal mucosa shows extensive wrinkling beginning at proestrus and continuing into estrus. (The normal longitudinal folds are exaggerated and in turn irregularly subdivided by numerous transverse wrinkles.) The wrinkling decreases later in estrus and by early metestrus (diestrus) the folds have lost their wrinkled appearance. The cervix protrudes prominently into the vagina during estrus, and its surface is marked by numerous folds. The cervical canal is patent throughout estrus and early diestrus. By the end of diestrus and throughout anestrus, the cervical canal is almost completely sealed. Uterus. Enlarged blood vessels appear in the broad ligaments and in the perimetrium with proestrus and are present throughout estrus. The uterine wall thickens owing to congestion, edema, and proliferation of uterine glands. The uterus attains maximum nonpregnant size 20 to 30 days postovulation and exhibits a characteristic corkscrew appearance. At 60 days postovulation, the uterus is about the same size as in proestrus. The uterus never returns to the size seen in immature bitches. Oviduct. During estrus, a small mass of red tissue protrudes from the slitlike opening of the bursal sac. The tissue is hyperemic and edematous fimbriae that almost completely close the slit. Ovary. The ovaries of immature bitches are small, smooth, and have no follicles larger than 1 mm in diameter. A definite cortex and medulla are present at 6 months. On the first day of proestrus, follicles can be up to 4 mm in diameter and up to 14 mm in diameter just prior to ovulation. Ovarian weight is greatest at approximately the time of ovulation (presumably due to the size of follicles and the luteinizing that is occurring). Luteinizing begins immediately after follicles rupture, and it is not unusual to find follicles of varying size and corpora lutea in the same ovary at estrus. Corpora lutea are most numerous and fully developed about 10 days after ovulation. Corpora lutea begin to degenerate at about 20 days postovulation and by 60 days postovulation have undergone fatty degeneration and appear nonfunctional. Corpora lutea are bright salmon pink from the time of ovulation until 10 days after ovulation. They then gradually yellow and are light tan about 60 days after ovulation. No remnants of corporea lutea (corpora albicantia) are seen in ovaries during the first estrous cycle. Mammary Gland. Gross changes are imperceptible in the mammary gland until shortly after ovulation, when a bluish plaque can be observed at the base of each teat. Thereafter the mammary glands enlarge and regress during the metestrus (diestrus) stage of the estrous cycle.

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Pathology Many changes have been reported for the kidney and male genital organs. In contrast, few observations have been made for the female genitalia. Nonneoplastic Findings: Spontaneous Kidney. Inflammation of the kidney is a frequent finding in dogs. The inflammation can be either primary or secondary and affects various tissue components resulting in many disease entities being reported. Inflammation of the kidney without identifying specific affected components can also be termed nephritis. Minor focal nonspecific inflammation occurred in 76 (12%) of 647 dogs (Hottendorf and Hirth 1974). Leukocytic foci were observed in 14% of young beagles (Glaister 1986). Focal embolic nephritis was reported in 1 of 647 dogs (Hottendorf and Hirth 1974). Inflammation of the glomerulus can also be termed glomerulitis, glomerulonephritis, and glomerulosclerosis. Glomerulitis and glomerulonephritis were reported in 2 (0.3%) of 647 dogs (Hottendorf and Hirth 1974). Local or diffuse mesangial proliferation and thickened and wrinkled glomerular basement membranes are not an unusual finding in clinically healthy, nonproteinuric laboratory beagles (Stuart et al. 1975). Periglomerular sclerosis might also be present. With time, the intercapillary sclerosis generally increases in severity and could be associated with intermittent or persistent proteinuria. A progressive alteration of the renal glomerulus called progressive intercapillary glomerulosclerosis (ISG) was described by Guttman (1970). This change is seen as early as 6 months of age and consists of thickening of basement membranes and increases in the mesangial matrix. Inflammation of the interstitium, usually termed interstitial nephritis or chronic interstitial nephritis, is common. Interstitial nephritis was reported by Oghiso et al. (1982). Focal interstitial nephritis was recorded in 6% of young beagles (Glaister 1986). Chronic interstitial nephritis occurred in 6 (0.9%) of 647 dogs (Hottendorf and Hirth 1974). Inflammation of the pelvis, also termed pyelitis and pyelonephritis, is another common finding. Pyelitis, usually with minor degrees of mononuclear infiltration of the lamina propria and epithelium of the renal pelvis, was observed in 4% to 7% of young beagles (Glaister 1986). Pyelonephritis is a nephritis that arises in the pelvis and spreads upward to adjacent areas of the kidney. Pyelonephritis was reported by Oghiso et al. (1982). Pyelitis or pyelonephritis occurred in 12 (2%) of 647 dogs (Hottendorf and Hirth 1974). Pyelitis and pyelonephritis were also reported by Pick and Eubanks (1965). Granulomatous inflammation or granulomas are frequent findings. Barron and Saunders (1966) reported the kidney as a common site for Toxocara granuloma. Cortical Toxocara granulomas were reported in 4% of young beagles (Glaister 1986). Cortical granulomas were observed in 2 (5%) of 37 dogs (Pick and Eubanks 1965). Granulomas occurred in 11 (2%) of 647 dogs (Hottendorf and Hirth 1974). Toxocara granuloma were also recorded in the kidney by Maita et al. (1977). Mineralization of the kidney is very common in dogs and is usually seen in the form of clumps of basophilic granules adjacent to and in the lumen and lining of collecting tubules. The incidence is probably higher than the reported 50%. Collecting tubule calcification was reported by Pick and Eubanks (1965) and calcification by Oghiso et al. (1982). Microcalculi of the renal medulla were found in almost 50% of the 647 males and females (Hottendorf and Hirth 1974). Mineralization of renal papilla was observed in both males and females (Glaister 1986). Degenerative changes of the glomeruli and tubules were mentioned but these changes were not described by Oghiso et al. (1982), who also reported albuminous urinary casts. Occasionally, one or more lobules of the glomerular tuft will be filled with large foam cells, which, with appropriate stains, are shown to contain fat. The condition is known as glomerular lipidosis and has no known functional significance.

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Hottendorf and Hirth (1974) reported the occurrence of hydronephrosis in their 647 dogs. Acidophilic rectangular or cubic crystalline intranuclear inclusions (identical in appearance to those seen in hepatocytes) are also commonly found in nuclei of cells lining proximal and distal tubules (Oghiso et al. 1982). Unilateral renal agenesis is occasionally found in laboratory beagles (Hottendorf and Hirth 1974; Robbins 1965; Vymetal 1965) and was observed in adult breeding colony beagles (Fritz et al. 1966, 1967). Four (0.4%) of 1,000 dogs were affected (Hottendorf and Hirth 1974). This condition is not detected during routine physical examinations. The ureter might or might not be missing. The developed kidney is usually about twice normal size (weight) and histologically of normal appearance, suggesting that the increased size is due to an absolute increase in the number of nephrons. Postnatal nephrogenesis is a characteristic of the canine kidney. Eisenbrandt and Phemister (1979) have shown that nephrogenesis continues through the first 8 to 10 days of life in the beagle, a fact that probably accounts for the normal histological appearance of the single enlarged kidney seen in unilateral renal agenesis. Urinary Bladder. Calculi and cystitis were seen infrequently in the urinary bladder, buut more often in males (Hottendorf and Hirth 1974). Cystitis occurred in 27 (23 males and 4 females) of 647 dogs (Hottendorf and Hirth 1974). Catheterization might be the cause of some instances of prostatitis and cystitis. Detrusor myopathy is a condition characterized by degenerative lesions in the urinary bladder muscule tunics that occurred in 59 of 449 (13%) young beagles. Both sexes were affected equally. Arteritis and periarteritis were reported in 10 of the 59 dogs with detrusor myopathy (Cain et al. 2000). Calcification of the round ligament of the bladder (remnant of the umbilical artery of the fetus) was seen, but not listed as a significant finding (Hottendorf and Hirth 1974). Calculi were reported as an uncommon finding in the urinary bladder. Five animals were grossly affected in 1,000 dogs (Hottendorf and Hirth 1974). Urethra. Calculi are an unusual finding in the urethra, with 1 animal grossly affected in 1,000 dogs (Hottendorf and Hirth 1974). Testis. Focal atrophy of seminiferous tubules was reported in 11 (3%) of 326 dogs by Hottendorf and Hirth, (1974) and in 5% of young beagles by Glaister (1986). Testicular degeneration occurs naturally in the dog and can be focal or diffuse, unilateral or bilateral. Early degenerative lesions consist of loss of primordial germ cells that might appear within the lumen of the seminiferous tubule as individual cells or as multinucleated giant cells. As the degeneration advances, more germinal cells are lost and tubules might be lined only by sustentacular cells (Sertoli cells). Intratubular giant cells were observed in the seminiferous tubules of 9 (3%) of 326 untreated young beagles (Hottendorf and Hirth 1974). Lymphocytic orchitis has been observed in beagle colonies and occurs in laboratory beagles, usually associated with lymphocytic thyroiditis. The lymphocytic infiltration can be diffuse, aggregated, or nodular (with germinal centers) and is commonly associated with focal or diffuse degeneration and atrophy of seminiferous tubules. The epididymis might be involved (Fritz et al. 1976). Orchitis can be caused by Brucell canis infections (Jubb et al. 1992). Interstitial cell hyperplasia is rarely seen in untreated young beagles. It was noted in 1 (0.3%) of 326 dogs (Hottendorf and Hirth 1974). Epididymis. Inflammation of the epididymis was reported in 1 of 326 male dogs by Hottendorf and Hirth (1974). Epididymitis can be caused by Brucella canis infections (Jubb et al. 1992). Lymphocytic epididymitis often associated with lymphocytic orchitis and thyroiditis is characterized by lymphocytic infiltrations (Fritz et al. 1976). Spermatic granulomas are inflammatory lesions that occur in the efferent ductules and epididymis. Intratubular granulomas were observed in the caput epididymis of three clinically normal dogs that were associated blind-ending efferent ducts. Although spermatic granulomas can be induced by trauma, infection, or toxins, spontaneous

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granulomas due to blind-ending ductules should be considered in the differential (Foley et al. 1995). James and Heywood (1979) report spermatocoele granuloma (spermatic granuloma) and inflammation for the epididymis. It is normal to find intranuclear, eosinophilic, periodic acid-Schiff positive inclusions in the epididymal epithelium of the dog (McEntee 1990). The significance of the inclusions is unknown. Normal epididymal cells also contain granular, yellow to yellow-brown pigment. Prostate. Subclinical prostatitis is common in the dog. The inflammation is usually minimal in young adult beagles. Prostatitis was reported in 124 (38%) of 326 males from 39 studies (Hottendorf and Hirth 1974). Chronic inflammation of the prostate was also reported by Maita et al. (1977) and James and Heywood (1979). Collection of urine by catheterization could be the cause of some prostatitis. Prostatitis can be caused by Brucella canis infections (Jubb et al. 1992). Minor accumulations of lymphocytes (leukocytic foci) to large lymphoid aggregates with germinal center formation accompanied by interstitial fibrosis and epithelial atrophy (prostatitis) were observed in 12% of young beagles (Glaister 1986). Atrophy was reported in the absence of inflammation in 7 (2%) of 326 males by Hottendorf and Hirth (1974). Focal cystic hyperplasia was reported in 15 (5%) of 326 males by Hottendorf and Hirth (1974). Prepuce. Mild balanoposthitis (inflammation of the glans penis and prepuce) is common and can result in slight mucopurulent preputial discharge. Ovary. Occasionally, clear cysts can be found in the vicinity of the ovaries of young adult beagles. The cysts have one of several origins (McEnree 1990). The finding of a parovarian cyst is not a specific diagnosis. Parovarian cysts were observed grossly in the ovaries of 2 (0.4%) of 499 dogs (Hottendorf and Hirth 1974). Uterus. Distention of the uterus was observed in 11% of young beagles (Glaister 1986). Myometrial cysts have been reported in the uterus with 1 affected animal in 499 females (Hottendorf and Hirth 1974). Inflammation of the uterus or endometritis caused by Brucella canis infections can result in abortion during the third trimester of pregnancy (Jubb et al. 1992). Neoplastic Findings: Spontaneous Kidney. Spontaneous tumors are rare in the kidney of young dogs. A renal carcinoma was reported in 1 of 1,000 laboratory dogs (Hottendorf and Hirth 1974). A benign mixed mesenchymal tumor was detected in the kidney of an 11-month-old beagle (Robison et al. 1997). Nervous System Anatomy and Histology The nervous system is the chief coordinating system of the body. The body’s other major coordinating system, the endocrine system, is controlled by the nervous system and a major part of it (the hypothalamus) is also a major part of the nervous system. The overall function of the nervous system is to produce the proper reaction of the organism to changes in both the external and internal environment (Jenkins 1978). This function depends directly on the neuron. Neurons, along with supportive cells (neuroglia), make up the nervous system. The nervous system is divided into the central nervous system (CNS) and the peripheral nervous system (PNS), more for discussion purposes than on the basis of structure or function. The CNS consists of the brain and spinal cord (neuraxis). The PNS consists of cranial and spinal nerves, sensory and motor ganglia, sensory and motor nerve endings, and the autonomic nervous system.

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The autonomic nervous system is that part of the nervous system concerned with motor innervation of smooth muscle, cardiac muscle, and glands. On anatomical, pharmacological, and functional bases, the autonomic nervous system is divided into a sympathetic portion and a parasympathetic portion. The general function of the sympathetic portion is to prepare the body for a state of emergency. The general function of the parasympathetic portion is to restore the body to a normal state of quiescence. The hypothalamus is the key to autonomic regulation. The rostral hypothalamic area controls the parasympathetic function and the caudal hypothalamic area controls the sympathetic function. Divisions of the Nervous System: Structure and Function All divisions of the nervous system are structurally and functionally connected. The PNS transmits impulses toward the CNS by way of sensory (afferent) neurons and away from the CNS by way of motor (efferent) neurons. The typical spinal nerve and the majority of cranial nerves have both types of functional neurons and are referred to as mixed nerves. A typical peripheral nerve consists of an outer connective tissue sheath (epineurium) enclosing bundles (fasciculi) of nerve fibers. Each fasciculus is surrounded by its own connective tissue sheath (perineurium) and is made up of individual nerve fibers (microscopic in size) surrounded by their connective tissue sheaths (endoneurium). Individual nerve fibers consist of an axon and an enclosing sheath formed by lemmocytes (Schwann cells). The axon is the elongated cytoplasmic extension (process) of the nerve cell. The surface membrane of the axon is the axolemma. Most peripheral axons are surrounded by an insulating sheath of lipoprotein (myelin). Myelin is the nonnucleated plasma membrane of the Schwann cell. The nucleated portion of the Schwann cell is the neurilemma (regeneration of axons is possible in Wallerian degeneration owing to survival of the nucleated neurilemma). The terms axolemma and neurilemma are not synonymous, and neither neurilemma nor myelin is synonymous with the total Schwann cell. The terms axon and axis cylinder are synonymous. Unfortunately, the total nerve fiber is also referred to as the axon. Nerves of the CNS The nerves attached to the brain are referred to as cranial nerves. There are 12 pairs, and except for the first pair (olfactory), all are attached to the brain stem. Nerves attached to the spinal cord are referred to as spinal nerves. There are usually 36 pairs of spinal nerves in the dog, derived from 36 spinal cord segments: 8 cervical, 13 thoracic, 7 lumbar, 3 sacral, and 5 coccygeal. With the exception of the first cervical nerve, all spinal nerves pass through intervertebral foramina. Because the spinal cord is shorter than the spinal column (in the embryo, the vertebral column grows more rapidly than the spinal cord, resulting in a relative caudal migration of vertebrae) there is not a one-to-one relationship between the spinal cord segments and the vertebrae. As a result, spinal nerves generally exit caudal to their segment of origin and vertebrae of one region might contain spinal cord segments of another region. The actual spinal cord segment and vertebral body correlations must be kept in mind when spinal cord segments are chosen for histological examination. The nerve distribution to the fore- and hindlimbs allows the spinal cord to be divided into functional units. The location of the units within vertebrae is shown in table 8.15 (Bailey and Morgan 1983). The CNS is protected, supported, and nourished by three sheetlike connective tissue coverings called the meninges (singular: meninx). The outermost (and toughest) meninx is the dura mater, The dura mater of the cranial cavity serves a dual function as meningeal covering for the brain and endosteal (or periosteal) lining for the bones of the calvarium. The spinal dura mater is separated from the periosteum of the vertebrae by the epidural cavity. The second meninx (arachnoid) is much thinner and more delicate than the dura mater. The arachnoid is separated from the dura mater by an almost nonexistent subdural cavity. The arachnoid connects with the pia mater (the third meninx) by delicate trabeculae, but it is separated from the

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Table 8.15 Vertebrae and Spinal Cord Segments Cord Segment

Nerve/Tract Distribution

Cervical, C1–C5

Nerve fiber tracts ascending from forelimbs and hindlimbs and descending from brain Origin of nerves to forelimbs Nerve fiber tracts ascending from hindlimbs and descending from brain Origin of nerves to hindlimbs except S, contribution to the sciatic nerve Origin of nerves to the anus, urinary bladder, and perineum. S, contribution to sciatic nerve Origin of nerves to tail caudally

Cervicothoracic, C6–TI/T2 Thoracolumbar, T2–L3 Caudal lumbar, L4–L7 Sacral, S1–S3 Coccygeal, C1–C5

Vertebrae CI–C4 C5–C7 T2–L3 L3–L4 L5 L5

pia mater by a large underlying space (subarachnoid space) filled with cerebrospinal fluid. The cerebrospinal fluid (CSF) pushes the arachnoid peripherally to contact the dura mater, forming a fluid envelope to cushion and protect the brain and spinal cord. The pia mater is closely adherent to the brain and extends deeply into the sulci of the cerebral hemispheres and between the folia of the cerebellum. The pia mater is the vascular meninx and mainly concerned with the nutrition of the CNS. The pia mater and arteries combine to form the tela choroidea of the choroid plexuses, which produce the major portion of the CSF. The cerebrospinal fluid is a clear, colorless fluid that fills the ventricular system of the brain, the subarachnoid space of both the brain and spinal cord, and the central canal of the spinal cord. The ventricular system of the brain and the central canal of the spinal cord are lined by a layer of closely packed cuboidal or columnar epithelial cells known collectively as the ependyma. In the ventricles of the brain, the ependyma is modified to form the special secretory epithelium of the choroid plexcus. There are four ventricles in the brain: two lateral and single midline third and fourth ventricles. The lateral ventricles communicate with the third, the third with the fourth, and the fourth with a dilatation of the subarachnoid space (cisterna magna) between the cerebellum and medulla oblongata. Necropsy and Laboratory Techniques As noted in preceding sections, disease in another organ system can directly or indirectly involve the nervous system. Some of the more prominent signs of neurological disease include depression (dull, lethargic, inattentive), disorientation (loss of proper bearings, mental confusion), stupor (partial unconsciousness, can arouse with stimulation), coma (unconsciousness from which animal cannot be aroused with powerful stimuli), hyperexcitable (excessive response to normal stimuli), tilting of head, twisting of bod, tremors, paresis (incomplete loss of voluntary motor function) or paralysis (complete loss of voluntary motor function) of one or more limbs, ataxia (incoordination of gait), abnormal reflexes, loss of control or urination and defecation and hyperesthesia, among others (Greene and Oliver, 1983). If in-life observations indicate that a neurological problem exists, thorough physical and neurological examinations should be done to fully characterize the clinical signs, to localize the disease process (brain, spinal cord, peripheral nervous system), and, if possible, to define more precisely where in the brain, spinal cord, or PNS the problem might be. Based on these (and clinical laboratory) findings, the pathologist can modify the postmortem procedures as needed to assure that all appropriate areas are examined and all appropriate tissues are taken. To minimize the handling of the brain, the head should be removed from the body. First, reflect the skin from the head and neck, sever neck muscles at their attachment to the posterior aspect of the skull, and cut completely through the spinal cord at the atlanto-occipital articulation. The cut is made by passing a thin, narrow blade through the dorsal atlanto-occipital membrane into the cisterna magna and then through the spinal cord. As the cisterna magna is entered, a quick appraisal

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of the cerebrospinal fluid (CSF) should be made. Expose the calvarium by removing the temporalis muscles. Remove the head by completing the disarticulation of the atlanto-occipital joint. (The disarticulation can be done quickly if the prosector is methodical in cutting the joint capsule and the various ligaments.) Three cuts are made through the calvarium. One cut is made transversely through the frontal bone at the anterior limit of the cranial cavity slightly rostral to the zygomatic processes. The incision includes the frontal sinuses. Two identical cuts are made on each side of the calvarium, just dorsal to the widest part of the brain case. The lateral cuts connect the transverse cut with the foramen magnum. The foramen magnum should be entered at its widest point slightly dorsal to the occipital condyles. The cuts through the calvarium require practice and a sense as to when the saw is about to leave bone and enter the brain. Pull the calvarium upward and backward. Generally, the dura mater will strip from the calvarium and remain with the brain. After examining the dura mater, cut it (and the arachnoid) parallel to the bone incisions. Two large folds of the dura mater will also need to be freed from their insertions between parts of the brain. The falx cerebri is the midsagittal fold between the cerebral hemispheres. The tentorium cerebelli is the transverse fold between the occipital poles of the cerebral hemispheres and the cerebellum. Failure to completely remove these folds will interfere with removal of the brain. The brain is extremely fragile and should be removed from the skull mostly by gravitational force and a little gentle traction. This is accomplished by holding the head upside down in one hand and freeing the brain by severing the cranial nerves, posteriorly rostrally, as they come to view. In the process, the brain gradually falls into the palm of the supporting hand. The hypophyseal stalk (infundibuluni) is severed as soon as it is seen so that the hypophysis remains intact in the sella turcia. The optic nerves are cut and as much of the olfactory lobes are removed as possible. Examine the ventral surface of the brain as it lies in the palm of the hand. Place the brain in a weighing boat and examine the dorsal surface. The brain need not be grasped, but can be slid from weighing boat to weighing boat or weighing boat to fixative. The removal of the hypophysis has been described in the endocrine system. The inner surface of the calvarium and ventral surface of the cranial cavity should be examined. The entire spinal cord, or segments of it, can be removed by either a ventral or dorsal approach. Before attempting to remove a segment, always completely transect the cord cranially and caudally to prevent stretching and twisting of the spinal cord as the spinal column is manipulated. The spinal cord should be removed by grasping the dura mater. If the entire spinal cord is to be collected, it should be fixed in a fully extended position in an adequately long container. Cervical and thoracolumbar segments of the spinal cord are easily removed from vertebrae C1–C4 and T2–L4, respectively. These segments sample ascending and descending tracts of all the limbs and the nerves of origination to the hindlimbs. Traditionally, a portion of a sciatic nerve is collected as the representative sampling of the PNS. The sciatic nerve can be removed with minimal trauma and is large enough to provide adequate samples (5 cm or longer). The sciatic nerve contains both afferent and efferent nerve fibers (mixed nerve). If a sensory nerve (afferent fibers) is required, a branch of the sciatic nerve, the caudal (lateral) cutaneous sural nerve, can be taken. The caudal cutaneous sural nerve has been studied in healthy adult beagles and morphometric and electrophysiological data exist for it (and the ulnar and saphenous nerves; Illanes et al. 1988). Artifacts in nervous tissue can be as troublesome as artifacts in muscle tissue. The makeup of nervous tissue makes it unusually sensitive to autolysis, rough handling, and the chemical effects of fixative. Primary fixation by whole body perfusion with buffered glutaraldehyde addresses most of the problems and can be performed as successfully in the dog as in smaller laboratory animals. However, whole body perfusion has practical application only in selected studies, leaving the problem of tissue artifacts in the brain, spinal cord, and peripheral nerves to be dealt with on a daily basis in routine studies. With the brain, the choice is between the artifacts of handling and distortion from slicing a brain in an unfixed state and the artifacts of autolysis in brains fixed by immersion. The choice is usually to fix the brain in toto and examine for gross internal lesions when the brain is sectioned for processing. Full transverse (coronal) sections are preferred for microscopic examination. Cross-sections and longitudinal

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sections of the spinal cord should be prepared. Specimens of peripheral nerves should be at least 5 cm in length and removed with great care. Peripheral nerves should be fixed in a gently stretched state on cardboard or corkboard. Ten percent neutral buffered formalin is the traditional fixative for nervous tissue. Brains should be fixed in large volumes of fixative that should be changed frequently. The investigator might wish to treat peripheral nerve specimens as surgical specimens and follow a nerve biopsy protocol (Asbury and Johnson 1978), which provides for plastic or paraffin embedding; routine, thick, or thin sections; and special stains and nerve fiber microdissection (nerve teasing, teased specimen). Routine sections of peripheral nerves should always include transverse and longitudinal sections. H&E is the most universally used stain; however, it does not stain specific nerve components and special stains are required for myelin, Nissl substance, neurofibrils, neuroglia, and so on. The writers fully agree with Jenkins (1978): “Neurostaining is a special technique which ideally should be reserved only for the experienced histotechnician who has the time and interest to devote to the subject.” Microscopists who are unfamiliar with the artifacts commonly encountered in peripheral nerves are referred to Asbury and Johnson (1978). Readers interested in contemporary neuropathological methods in toxicology are referred to Spencer and Bischoff (1982). Pathology Hydrocephalus is a common nervous system finding in laboratory dogs. It was observed grossly in 14 (1%) of 1,000 dogs on 39 studies (Hottendorf and Hirth 1974). Hydrocephalus (ventricular dilatation) associated with a sponge-like alteration in the surrounding brain tissue was observed by Oghiso et al. (1982). Several beagles with hydrocephalus were reported by Fritz et al. (1967). Hydrocephalus is characterized by abnormal accumulations of CSF. It is usually manifested as dilatation of one or more ventricles of the brain (internal hydrocephalus), but ventricles might be unaffected and it is the subarachnoid space that is dilated with the excess CSF (external hydrocephalus). If excess CSF is present in both locations, the condition is referred to as communicating hydrocephalus (Sullivan 1985). Although hydrocephalus appears to be fairly common in laboratory beagles, it is likely that minor degrees of ventricular dilatation are unrecognized and the real incidence of the condition is greater than reported. Lateral ventricles of surprising size (some with thinning or rupture of the septum pellucidum) are found incidentally while trimming brains of dogs that showed no neurological signs. At necropsy, the ventral surface of the brain should be closely examined, as the piriform area will dimple under slight pressure even in mild hydrocephalus. Hydrocephalus in laboratory beagles is probably a congenital condition. Inflammation of the nervous system was also frequently reported. Chronic focal meningitis was observed in brains from 44 (7%) of 630 dogs in 33 studies and focal encephalitis in 5 (0.8%) of these 630 dogs (Hottendorf and Hirth 1974). Focal myelitis was observed in spinal cords from 2 (0.3%) of 647 dogs (Hottendorf and Hirth 1974). Toxocara granulomas in the brain and spinal cord (cauda equina) were reported by Barron and Saunders (1966). Hemorrhage involving the brain was reported by Pick and Eubanks (1965). They did not elaborate on the location or extent of the hemorrhage. Hottendorf and Hirth (1974) observed, but did not report as lesions, small subependymal collections of glial cells usually seen around the anterior parts of the lateral ventricles. Spherical, eosinophilic granular structures have been observed in the medulla oblongata, pons, or anterior cervical cord. They seem to be most common in the gracilis tract and nucleus. Newberne et al. (1960) and Innes and Saunders (1962) believe the structures are degenerating axis cylinders. They apparently have no neurological or pathological significance. Spontaneous degenerative lesions of the peripheral nerves occur in the beagle as in other laboratory animals. Although commonly considered an aging change, occasionally digestion chambers and myelin bubbles are seen in the sciatic nerves of young beagles.

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During examination of peripheral nerves, cylindrical, loosely textured, whorled, cell-sparse structures are occasionally observed within the nerves. These are Renaut bodies and were well known to histologists of the late 19th century, but they have gradually been forgotten (Asbury 1973; Asbury and Johnson 1978). Renaut bodies are particularly well developed in the horse and donkey, but are less conspicuous in the dog and humans. Their purpose is unknown. It is important that they be recognized as normal structures and not misdiagnosed as nerve infarction or necrotizing angiopathic neuropathy, as has been done in the past (Asbury 1973). Eye and Ear Anatomy and Histology Eye The eye is the organ of vision. It is composed of the eyeball (globe), the optic nerve, and accessory structures including the eyelids, conjunctiva, lacrimal apparatus, and ocular muscles. The canine eye is relatively large for the size of the animal. The human eye is approximately 2.5 cm in diameter (Kuwabara and Cogan 1977); the eye of an adult beagle is approximately 2.2 cm in diameter (Andersen 1970). The canine eye is also placed well forward in the head, giving the dog a large field of binocular vision. The eyeball is roughly spherical in shape, with the rostral curvature of the cornea making the anterioposterior diameter the greatest diameter of the eye. To facilitate description and orientation of the eye, special designations as to side, direction, and position are used. Oculus dexter (OD) designates the right eye; oculus sinister (OS) designates the left eye; and oculus unitus (OU) designates both eyes. The side of the globe nearest the nose is the nasal, or medial, side; the opposite side is the temporal, or lateral, side. The dorsal side is superior; the ventral side is inferior. The corneal pole is distal, or anterior; the cerebral pole is proximal, or posterior. The line connecting the two poles is the anatomical or optic axis. The equator is the greatest expansion of the eyeball perpendicular to the anatomical axis. A horizontal plane through the poles divides the eyeball into an upper half and a lower half. A vertical plane through the poles divides the eyeball into a nasal half and a temporal half. Eyeball. The eyeball consists of an inner coat, or tunic, of neural light-sensitive tissue (retina) held in shape by surrounding coats that protect it (corneoscleral coat, outer fibrous coat) and nourish it (uvea, middle vascular coat). The outer fibrous coat is subdivided into a larger, tough, white posterior portion (sclera) that covers about 75% of the globe and a smaller, transparent anterior portion (cornea). The transition from the opaque sclera to the transparent cornea is comparatively abrupt and occurs at the corneoscleral (sclerocorneal) junction, or limbus. The limbal area of the sclera is pigmented laterally and medially but not dorsally or ventrally. Sclera. The thickness of the sclera varies. At the equator, it is so thin as to be semitransparent and the dark color of the uvea shows through. It is thick at the ciliary region where extraocular muscles insert and around the optic nerve. Cornea. The cornea is transparent, colorless, and nonvascular; however, it possesses dense nerve fiber plexuses and is highly sensitive. Opposite to what exists in humans, the cornea of the dog is thicker in the center than at the periphery (Getty 1967). The transparency of the cornea is due to a nonkeratinized and nonpigmented surface epithelium, lack of blood vessels and lymphatics, cell-poor stroma composed of thin collagen fibrils arranged in orderly lamellae, and a sodiumpotassium pump in the cell membrane of the corneal endothelium that maintains a high degree of stromal dehydration (Wilcock 1985). The composition of the cornea is notably uniform and consists of six or seven layers (six in the dog). The outermost and often overlooked layer is the tear film

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(not seen in histological sections). The outer stratified squamous epithelium is a continuum of the conjunctival epithelium. The basement membrane of the epithelium is the third layer. According to Dellmann (1976b), the dog does not have an anterior limiting membrane (Bowman’s membrane). However, Prince et al. (1960) indicate that an anterior limiting membrane is present, but it is extremely thin (1.5 mcm vs. 30 mcm in humans). Shively and Epling (1970), in their study of the fine structures of the beagle eye, found no consistent layer of randomly oriented collagen fibers that would constitute an anterior limiting membrane. The stroma constitutes the bulk of the cornea. Descemet’s membrane is present (a required constituent, as it is the basement membrane of the innermost layer, the endothelium). The endothelial layer should probably be known as mesothelium based on its structural characteristics and its probable origin (Shively and Epling 1970). Uvea. The uvea is the highly pigmented and vascular coat of the eye. It consists of the iris, ciliary body, and choroid. Iris. This the most anterior portion of the uveal tract and is the diaphragm of the eye. Ciliary Body. This is a ring of tissue that extends from the base of the iris to the neuroserisory retina posteriorly. The main components of the ciliary body are the ciliary processes and the ciliary muscle. The ciliary processes are actually linear folds that appear from the rear as multiple radiating ridges. The ciliary processes are highly vascular and are thought to be the main sites for formation of the aqueous humor (an arrangement comparable to the formation of CSF by the choroid plexuses of the brain). In accommodating for near vision, the ciliary muscle contracts, pulling the ciliary body forward, which allows the supporting fibers (zonule fibers) of the lens to relax, leading to relaxation and an anterior-posterior thickening of the lens. Choroid. The bulk of the uvea is formed by the choroid, which consists mainly of blood vessels and melanocytes. Externally, it blends with the sclera; internally, it is bounded by a basal lamina called Bruch’s membrane; and dorsally (above the optic disc) it displays a peculiar structure known as the tapetum lucidum. Tapetum. Many animals have one of two types of tapeta: tapetum lucidum fibrosum or tapetum lucidum cellulosum. The dog has a tapetum lucidum cellulosum. The tapetum lucidum is a lightreflecting layer (responsible for the luster of eyes) that is situated in the dorsal half of the choroid. The tapetum is triangular to semicircular in shape, extending about halfway to the periphery of the choroids, and has a horizontal base that just contacts the top of the optic disc. Although the tapetum appears to be immediately adjacent to the pigment epithelium cells of the retina, there is a layer of capillaries (choriocapillaris) between the tapetum and Bruch’s membrane. The tapetum is made up of flat cells varying in number from 9 to 10 layers centrally to 1 or 2 layers peripherally (Prince et al. 1960). Retina. The innermost tunic of the eye is the retina. It is divided into a sensory portion (pars optica) that rests on the choroid and a nonsensory portion that rests on the ciliary body (pars ciliaris) and the posterior surface of the iris (pars iridica). The sensory and nonsensory portions join posterior to the ciliary body at a scalloped border known as the ora serrata. The sensory retina is firmly attached at the ora serrata and optic disc, but it is loosely attached over the choroid. Under normal circumstances, the pressure of the vitreous is sufficient to hold the retina in contact with the choroid. The nonsensory retina consists of a simple layer of pigmented cuboidal epithelial cells that continues at the ora serrata to form the outermost of the 10 layers of the sensory retina. Not all of the pigment epithelium is pigmented; the cells overlying the tapetum lucidum are not. According to Kuwabara and Cogan (1977), two pigments can be found in the pigment epithelium: melanin and lipofuscin. The

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pigment epithelium interdigitates with the overlying photoreceptors. Among other functions, the epithelial cells engulf and degrade obsolete rod and cone cell segments. The accumulation of lipofuscin appears to be a result of this phagocytic activity. In humans, the quantity of lipofuscin increases with age and the number of melanin granules decreases (Kuwabara and Cogan 1977). The inner transparent layer of the sensory retina comprises nine layers, named from within outward: internal limiting membrane, nerve fiber layer, ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, external limiting membrane, and the layer of rods and cones. The retinal receptors in the dog are predominantly rods. That feature (and others) indicates that the dog is predominantly a nocturnal animal (Prince et al. 1960). To the ophthalmologist, the visible portion of the posterior globe is the ocular fundus, which for descriptive purposes is commonly divided into a dorsal tapetal fundus and a ventral nontapetal fundus (tapetum nigrum). The optic disc is usually at the junction of the two. The optic disc (optic papilla) is the rounded, raised area where the optic nerve leaves the eye. Jenkins (1978) emphasizes the following important features relative to the optic nerve: Because the optic nerve is a fiber tract of the brain, it has no neurilemmal sheath of Schwann and regeneration is not possible; it contains neuroglial elements; has a meningeal investment but no epi-, peri-, or endoneurium; and the individual nerve fibers are all myelinated. Wilcock (1985) points out that, in contrast to most domestic species, the dog’s fibers in the optic papilla are myelinated. Because the optic nerve is in direct contact with the eye and brain via neurons and CSF, it can be affected by diseases of both the eye and brain. Branches of the ciliary and internal ophthalmic arteries and veins lie just along the optic nerve. When they reach the eye, some plunge into globe and others (medial and lateral long posterior ciliary arteries and veins) pass into the superficial layers of the sclera and are externally visible for a distance along the horizontal meridian of the eye. These vessels serve as landmarks in orienting the eye at trimming. The transparent media of the eye include the cornea, aqueous humor, lens, and vitreous body (the retina, excluding the pigment epithelium, is also transparent). The aqueous humor is the clear fluid contained in a cavity bounded anteriorly by the cornea and posteriorly by the lens. The space between the cornea and the anterior surface of the iris is the anterior chamber and the space between the posterior surface of the iris and the lens is the posterior chamber. Aqueous humor is produced in the posterior chamber and drains from the anterior chamber into a meshwork of channels located at the junction of the cornea, sclera, and iris (iris angle, filtration angle). The lens is composed entirely of epithelial cells whose basement membrane is the thick outermost capsule of the lens. The bulk of the lens is formed by layers of epithelial processes that cannot be shed, but are compacted with age to the center of the lens. The adult lens depends entirely on the aqueous humor for delivery of nutrients and removal of wastes. The vitreous body is the transparent gel that fills the inner portion of the eyeball between the lens and the retina. The vitreous shows considerable shrinkage with most fixatives (over 99% of the vitreous is water) and its normal, in-life distribution is not apparent in microscopic sections. Third Eyelid. The dog has a third eyelid, or nictitating membrane, located at the medial angle of the palpebral aperture (medial canthus, nasal canthus). A T-shaped hyaline cartilage forms the skeleton of the third eyelid. The cross of the T supports the free margin of the lid. The lower shaft of the T is surrounded by a mixed lacrimal gland (superficial gland of the third eyelid, nictitans gland). According to Getty (1967), the dog does not have a Harder’s gland (deep gland of the third eyelid). The inner conjunctival covering (bulbar conjunctiva) contains numerous lymphoid nodules. Ear The ear is the organ of hearing and equilibrium. It is composed of three connected divisions, each of which is referred to as an ear: external (outer) ear, middle ear, and internal (inner ear). The

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inner ear is the organ for both hearing and equilibrium. The external and middle ears are sound collecting and conducting apparatuses (Getty 1967). External Ear. This consists of the pinna (auricle) and the external auditory meatus (ear canal). The pinna is composed of a sheet of elastic cartilage (auricular cartilage) covered by skin on both sides. The auricular cartilage is pierced by many foramina that permit the passage of blood vessels. Histological sections that include foramina give the impression that the cartilaginous sheet is not continuous. The function of the pinna is to direct air vibrations into the funnel-like ear canal to the tympanic membrane (ear drum). The tympanic membrane is the partition between the external ear and the middle ear. Middle Ear. This consists of the tympanic cavity, the auditory tube, and a chain of three small bones (auditory ossicles) that connect the tympanic membrane to the oval window of the inner ear. The tympanic cavity is filled with air and communicates with the nasal pharynx by means of the auditory tube (eustachian tube). Inner Ear. This is contained within the temporal bone and consists of the membranous labyrinth containing the organs of hearing and equilibrium and the bony labyrinth surrounding the membranous labyrinth. The inner ear is fluid filled. The bony labyrinth, which lodges the membranous labyrinth, contains a fluid, perilymph, and communicates with the cerebrospinal space by way of the vestibular aqueduct. The membranous labyrinth is filled with a fluid called endolymph. Necropsy and Laboratory Techniques Eye Many lesions of the eyelids, conjunctiva, and cornea will be clinically apparent; however, most lesions within the globe will be hidden to the pathologist without the assistance of an ophthalmoscopic examination. Ideally, the pathologist will have the notes, drawings, or photographs of an ophthalmologist to direct him or her to pathology within the eye. Minute lesions can be difficult to find without directions and lesions involving the lens could be obscured or lost because of the difficulty in obtaining complete, artifact-free sections of the lens. Autolytic changes can be detected in the canine retina within 5 min of death (Saunders and Rubin 1975). Unless there are compelling reasons to do otherwise, the eyes should be the first organs removed at necropsy and both eyes should be in fixative well within the first 5 min of the necropsy. To meet the 5-min deadline, all preliminary procedures (weighing, external examination, clipping of hair) should be done prior to exsanguination. Good exsanguination limits the degree of hemorrhage into the orbit during dissection. The enucleation can be done by a single prosector, but the assistance of a second person is required to have both eyes removed, cleaned, and in fixative within the 5-min time frame. A single incision through the lateral palpebral angle will free the upper and lower eyelids sufficiently to give access to the eye. With an assistant stabilizing the head and stretching the skin to spread the eyelids, the prosector grasps a lateral fold of palpebral conjunctiva with tissue forceps and with curved blunt scissors (one of the authors prefers Metzenbaum scissors) makes an initial incision in the conjunctiva behind the forceps. With gentle traction and well-directed cuts with the scissors, the prosector severs the conjunctiva and extraocular muscles on the lateral, dorsal, and ventral sides of the eye, working toward the cerebral pole where the optic nerve and remaining muscles are cut. The eye is pulled gently forward and remaining extraocular tissue is cut, including the third eyelid. The position of the forceps never need be changed. The globe is placed on a wet sponge to cushion it and the extraocular muscles, fat, and fascia are gently removed close to the sclera, using the convex surface of the scissors. Although the left and right side identity of the eyes can be made using anatomical landmarks, it is easier to maintain the identity of the eyes by placing

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the globes in identified containers or to tie a piece of black thread to a bit of loose extraocular tissue on one of the eyes. The eyes are gently lowered by the thread or forceps into the fixative. At least 5 mm of optic nerve should be left attached to the eye. With proper cleaning, the eye will sink in fixative. The thorough cleaning is required to permit rapid penetration of fixative and to prevent retinal detachment because of the pressure exerted by contracting muscles. For the same reason, the globe should not be grasped by the fingers. Usually, eyes are not weighed. Measuring the diameters of unfixed globes is time consuming and increases the likelihood of retinal detachment. The volume of an eye is easily determined by displacement. Choose a graduated cylinder with an inside diameter just great enough to accept an eye. Partially fill the cylinder with fixative, read the volume, lower the eye into the fixative, and read the new volume. The difference in the volume readings is the volume of the eye. Using an appropriate cylinder, the volume of a beagle eye can be measured to 0.2 ml. After measuring, the eye is poured into the primary container of fixative. There appears to be no ideal fixative for the eye. Ten percent neutral buffered formalin generally gives good results for the cornea and lens but poor results for the retina. Rapidly penetrating fixatives such as Zenker’s, Helly’s, and Bouin’s are generally good for the retina, but require strict attention to a fixation schedule to avoid overfixation. Many additional fixatives have been used. One of the authors has had good results with 2.5% phosphate buffered glutaraldehyde. Dog eyes placed in this fixative can be “cut in” in 1.5 hr to 2.0 hr, allowing a gross examination while the tissues retain much of their original color and the lens is still relatively clear. Fixation is complete in about 72 hr. Davidson’s fixative (Humason 1972) is a simple, easy-to-use fixative that gives good results. Unless there is good reason to do otherwise, the canine eye should be trimmed perpendicular to the horizontal meridian (posterior ciliary vessels) to obtain a midsagittal block that includes the tapetal and nontapetal fundus and optic nerve. A complete sampling of the eye would include a transverse section of the optic nerve. Preparing top-quality eye sections requires special skill and (as with neurohistological technique) should be reserved for the experienced histotechnician with the time and interest to devote to the subject. Ear The middle and inner ears are not routinely examined in toxicology studies; however, it is not difficult to collect and prepare sections that include the tympanic membrane and middle and inner ears. The external ear is removed close to the tympanic membrane. After removal of the brain and disarticulation of the mandible, two transverse cuts are made through the base of the skull just anterior and posterior to the external acoustic meatus. One of these cuts will usually just enter the tympanic cavity of the middle ear. After ample fixation and decalcification (the temporal bone is very dense), the specimens can be trimmed and processed in a routine manner. Pathology Only Glaister (1986) reported ocular disease, but his observations are limited to sores and conjunctivitis without describing these lesions. The conjunctivitis was associated with sawdust bedding and dust in 4% of young beagles. Oghiso et al. (1982) described lymphoid cell infiltration of the third eyelid and gland of the third eyelid. Hottendorf and Hirth (1974) examined both eyes in 85% of their studies but reported no ocular pathology in 630 beagles. Rubin and Saunders (1965) reported Toxocara granuloma in the retina or choroid in three young beagles. Heywood et al. (1976) followed ocular changes in 86 laboratory beagles from 6 months to 8 years of age, at which time the animals were necropsied. Prominent posterior lens sutures were the only ophthalmological change seen in beagles under 3 years of age. From 3 to 8 years of age, ophthalmological changes included corneal opacities, asteroid bodies, and tapetal pigmentation. For the lens they reported prominent posterior sutures; nuclear opacities; and anterior, posterior, and peripheral capsular opacities. Histological findings at 8 years of age included keratitis, cystoid

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degeneration of the retina, thinning and absence of tapetal cells, pigment cells in rod and cone layer, scarring of the retina, and calcified body between the pigment epithelial layer and choroid. Schiavo and Field (1974) examined 532 beagles of various ages ophthalmoscopically and biomicroscopically and reported the following in dogs between 6 and 12 months of age: superficial keratitis in 35 (6.6%); deep keratitis, posterior polar opacities, posterior cortical opacities in 54 (10.1 %); prominent posterior lens sutures, lenticular sheen, vacuoles in lens cortex, persistent hyaloid vessel remnants, zones of discontinuity (lens), vitreous floaters or filaments, atapetal fundi, tigroid fundi, tapetal aberrations/pigment clumps, peripapillary reflectivity, and increased tapetal reflectivity. Lenticular sheen is a yellowish reflection from the lens media and is generally expected as a senile change. It was observed in 120 (22.6%) of beagles sampled. The hyaloid artery is responsible for development of the vitreous and for nourishment of the fetal lens. Normally, the vessel regresses shortly after birth, but sometimes remains to be seen in the adult. The incidence of persistent hyaloid vessels was 142 (26.7%). Zones of discontinuity reflect stratification of lens fibers and are thought of as a presenile change. They occurred in 32 (6%) of beagles sampled. Vitreous floaters are asteroid bodies (small calcium bodies). Vitreous filaments are fibrous strands from vitreous hemorrhage or remnants of the posterior vascular capsule. The incidence of vitreous floaters and filaments was 53 (10%). Tigroid fundi describe the appearance of choroidal vessels through areas of nonpigmented pigment epithelium. Tapetal aberrations include areas of hyperreflectivity and old hemorrhage or scars. These aberrations occurred in 25 beagles (4.7%). Bellhorn (1974) reports similar ocular findings in 8- to 10-month-old beagles plus prolapse of the third eyelid. The abundance of ophthalmoscopic observations and the paucity of histological findings emphasize the comparative sensitivities of the two modes of examination. The discrepancy is easily understood, for the ophthalmologist has the entire living cornea, iris, lens, and fundus to examine. On the other hand, the pathologist is limited to one or two thin sections of an organ that is both difficult to fix and difficult to section. Peripheral retinal cystoid degeneration describes single or multiple microcysts within the retina at or near the ora serrata. This is a common change in older beagles, but it also occurs in young animals. At 8 years, the incidence rate was 85%. The lesion has no apparent functional significance. Focal retinal dysplasia manifested as retinal folds, retinal rosettes, focal absence of retinal cells, and blending of nuclear layers. Protrusion of the third eyelid can be seen in young beagles. Histological findings include inflammation of the bulbar conjunctiva and stroma of the superficial gland. The ducts of the gland might be dilated and contain leukocytes. The conjunctival lymphoid tissue is usually hyperplastic. There is no mention of ear pathology in the articles reviewed. Cutaneous lesions of the external ear were discussed with the integumentary system.

METABOLISM Among the nonrodent species, the dog has been the best characterized with regard to xenobiotic metabolism. This is probably because the dog has been extensively used in biomedical research for more than 100 years. Its relatively large size also provides the advantage of allowing repeat serial sampling of large amounts of biological fluids for time-course analyses. Data on the dog are a staple in numerous review articles and book chapters published on species comparisons elsewhere (see chapter 1). The focus here is not only on species differences, but also on other items (e.g., inducibility) that have not necessarily been covered in detail elsewhere. Insofar as the beagle dog is the most common breed and the most common nonrodent model used in toxicity testing, this chapter focuses on data from beagles, and presents data from other breeds where available. At the onset, it should be noted that dog is generally believed to have a higher intestinal pH than man, leading to some differences in oral absorption of more water-soluble drugs (Dressman 1986; Lui et al. 1986). Although otherwise similar motility patterns and pH profiles prevail in the

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two species for the most part, there are some differences that could affect the time profile and extent of drug absorption. These include slower gastric emptying in the fed state, faster small intestine transit, and higher and more variable intestinal pH in dogs compared with humans. An attempt is made to identify drug and dosage-form properties that would lead to differences in drug absorption in the two species (e.g., drug physiochemical properties, dosage-form size, and pH dependency of dosage-form release characteristics). As reported by Gregus et al. (1983), the dog has a somewhat smaller liver to body weight ratio than the rat (2.3% vs. 4.0%) and somewhat less microsomal protein (20.5 vs. 25.7 mg/g liver). Identified P450 CYP isozymes are listed in table 8.16. Some of the more common parameters of hepatic xenobiotic metabolism in the dog are summarized in table 8.17. Concentrations of cytochrome P-450 range from about 0.30 to 0.80 nmol/mg protein. Using HPLC and other techniques, Amacher and Smith (1987) characterized the cytochrome P-450 isozymic “fingerprint” of naive beagle dogs. Their results suggested that cytochrome P-450 exists as three distinct isozymic groups in female dogs, and two main groups and two to three subgroups in male dogs. Further, they identified sex-related differences in chromatographic behavior between the major isozymic groups. This is an interesting finding because few, if any, sex-related differences in the activity of the microsomal mixed function oxidase (MMFO) have been identified in dogs. If table 8.17 is compared Table 8.16 Identified P450 CYP Isoforms Active in the Dog CYP CYP CYP CYP CYP CYP CYP CYP CYP CYP CYP CYP CYP

1A1 1A2 2A6 1B11 2C19 2C21 2C41 2D6 2D15 2E11 3A4 3A12 3A26

(Low Km; Shou et al. 2003) (Chauret et al. 1997) (Chauret et al. 1997) (Low Km; Shou et al. 2003) (Chauret et al. 1997) (Low Km; Shou et al. 2003) (Chauret et al. 1997) (High Km; Shou et al. 2003)

to similar endpoints for man or other model species (such a table being presented in other chapters), it is readily seen that the dog is frequently not a good metabolic model for man and is poorly comparable to the rat and mouse. McKillop (1985), using SDS-PAGE techniques, identified three major cytochrome P-450 isozymes in uninduced adult male dogs. Phenobarbital increased the levels of two of these major isozymes, but primarily caused an increase in another (fourth) isozyme. β-Naphthaflavone caused increases in three other isozymes that are only present in very small amounts in naive animals. Hence, as a generality, the beagle dog has at least seven isozymic forms of cytochrome P-450, including a cytochrome P-448 with some homology with that of the rat, but that are differentiated on the basis of molecular weight, substrate specificity, and responses to inducers. In general, the dog has less ability than the rat to form hydroxylated aromatic metabolites via the MMFO. For example, in the metabolism of amphetamine, the dog produces very little 4-hydroxyamphetamine (as reviewed by Williams 1972). Cook et al. (1982) reported that the dog, as opposed to the rat, produces no phenoxyl metabolites of disopyramide. Aniline-hydroxylating activity in dogs tends to be less than that of rats. Gregus et al. (1983) have reported benzo(a)pyrene-hydroxylating activity in the dog is much less than that of the rat. There are exceptions; for example, the dog has a much higher rate (15 times) of metabolism with 2,2',4,4',5,5-hexachlorobiphenyl than the rat (Duignan et al. 1987). Some care must be taken in interpreting data on aniline hydroxylase in the dog, as this species produces both ortho- and para-aminophenol from aniline at a ratio of 1/2 (p/o) (Williams 1972). In the

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Table 8.17 CYP Specific Metabolic Activities in Beagle Dogs Activity

Beagle Dog

References

7-Ethoxyresorufin O-dealkylation 7-Methoxyresorufin O-dealkylation Caffeine 3-demethylation Benzphetamine N-demethylation 7-Benzoxyresorufin O-dealkylation 7-Pentoxyresorufin O-dealkylation Coumarin 7-hydroxylation 7-Ethoxy-4-trifluoromethylcoumarin deethylation Ethoxycoumarin O-dealkylation Tolbutamide methyl-hydroxylation Chlorzoxazone 6-hydroxylation Mephenytoin N-demethylation

1A1/2

Kastner and Neubert, (1994)

2B11 2B11

Ohmori et al. (1993)

4-Nitrophenol hydroxylation N-Nitrosodimethylamine N-demethylation Androstenedione 15α-hydroxylation Androstenedione 16α/β-hydroxylation Dextromethorphan O-demethylation Dextromethorphan N-demthylation Testosterone→ Androstenedione* Testosterone 2α-hydroxylation Testosterone 2β-hydroxylation Testosterone 6β-hydroxylation Testosterone 7α-hydroxylation Testosterone 15α-hydroxylation Testosterone 15β-hydroxylation Testosterone 16α-hydroxylation Testosterone 16β-hydroxylation Lauric acid 11-hydroxylation Lauric acid 12-hydroxylation

R enantiomers twice as fast as S

Yasumori et al. (1993)

2B 2D15


3A12


2B11, 2C21 3A12, 2B1146

Ohmori et al. (1993) Ohmori et al. (1993)

rat, this ratio is 6:1. As the common colormetric method (formation of a quinolinidine complex with phenol) of determining aniline hydroxylation is specific for p-aminophenol, the activities reported for aniline hydroxylase in the dog are probably low. With many other typical substrates (dealkylation rather than aromatic hydroxylation), MMFO activity in the dog is often comparable to that of the rat. Microsomal aminopyrine metabolism, for example, is about the same in dogs as in rats (Lan et al. 1983). Gregus et al. (1983) reported that the rat had about twice the activity with benzamphetamine, but four times the activity than with ethylmorphine than dogs. In contrast, dogs have a higher baseline level than rats for 7-ethoxycoumarin (primarily a substrate for P-450-dependent MMFO) and 7-ethoxyresorufin (primarily a substrate for P-448 dependent MMFO) deethylating activities (Gregus et al. 1983; McKillop 1985), but greater degrees of induction (with either phenobarbital or β-naphthaflavone) occur in the rat than in the dog (McKillop 1985). Thus, in comparing the rat and the dog (probably the two most common species in toxicity testing), one should not assume that the rat has the more rapid rates of MMFO activity but does have a more extensive range of activities. Nelson et al. (1996) and Zuber et al. (2002) report that the dog CYP1A, 2E1, 3A12, and 3A26 actvities are not well correlated with those in humans. CYP2D15 is well correlated with human CYP2D6 activity. The dog is also the only mammalian species able to metabolize polycylic aromatic hydrocarbons through its CYP2BN enzyme. There is considerable evidence that the MMFO of rats has stereospecificity. For example, Heimark and Trager (1985) compared the microsomal metabolism of R and S warfarin. The overall rate of oxidation (total product) was much greater with the R entaniomer. There is some evidence for MMFO stereospecificity in the dog. Cook et al. (1982) examined the disposition of (R) and (S) disopyramide in vivo in dogs. The (R) entaniomer had significantly longer half-life than the

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(S). In addition, a much higher percentage of dose of S-disopyramide was excreted in the urine as the major metabolite. In general, one should expect stereoselective metabolism of racemic mixtures in the dog. Dog 1A and 2E1 are somewhat different than humans and are not typically good models for humans. Only mammalian species are able to metabolize polycyclic aromatic hydrocarbons through CYP2B11 (Zuber et al. 2002). Celecoxib, a cyclooxygenase–2 inhibitor had its pharmacokinetics in dogs evaluated as part of its nonclinical development program prior to FDA approval. Celecoxib is extensively metabolized by dogs to a hydroxymethyl metabolite, with subsequent oxidation to the carboxylic acid analog. Of the major CYP enzymes, CYP 2D15 was extensively involved in polymorphism of metabolism in dogs, probably due primarily to other CYP subfamilies than 2D (Paulson et al. 1999). Urea synthesis of both fresh and thinned liver slices is higher than those of rat, cynomolgus monkeys, rhesus monkeys, hamsters, minipigs and rabbits. However, the dog has lower testosterone metabolism than any other species (Kanter et al. 1997). MMFO is inducible in the dog. McKillop (1985) examined the inducing effect of phenobarbital and β-naphthaflavone in beagle dogs. Phenobarbital (in saline) was administered intraperitoneally for 7 days: 20 mg/kg for 2 days, 10 mg/kg for 2 days, and 20 mg/kg for the final 3 days. β-Naphthaflavone in arachis oil was given intraperitoneally for 6 or 7 days, 10 mg/kg. The phenobarbital treatment increased cytochrome P-450 by approximately 250%, whereas the β-naphthaflavone caused approximately a 100% increase, with a shift from cytochrome P-450 to cytochrome P-448. In contrast, β-naphthaflavone caused a greater increase (175%) in NADPH: cytochrome C reductase than phenobarbital (41%). This latter change contrasts with that of the rat, in which typical P-448 inducers do not cause increases in the reductase activity. The increases were accompanied by the expected increases in enzyme activity: aldrin epoxidase was increased by phenobarbital, 7-ethoxyresorufin deethylase was increased by 6-naphthaflavone, and 7-ethoxycoumarin was increased by both. Duignan et al. (1987) used a stepwise regimen (to avoid excessive sedation) to induce beagle dogs with phenobarbital (Na+ salt): 10 mg/kg (po) for 2 days, followed by 30 mg/kg for 4 days, then 30 mg/kg for a final 8 days. This regimen more than doubled the microsomal concentration of cytochrome P-450 and significantly increased the activities toward 7-ethoxycoumarin, warfarin, and androstenedione. There were changes in region and site specificity that indicate that induction in the dog, as in the rat, is accompanied by shifts in isozymic character of cytochrome P-450. Aldridge and Niems (1979) examined the effects of phenobarbital and β−naphthaflavone on the metabolism of caffeine in vivo in the dog. Both phenobarbital (10 mg/kg/day po for 7 days) and β-naphthaflavone (20 mg/kg/day intraperitoneally for 3 days) decreased the half-life of caffeine, but only β-naphthaflavone caused a qualitative shift in the spectrum of urinary metabolites. Gascon-Barre et al. (1986) studied the effects of phenobarbital induction of the MMFO on vitamin D metabolism in mongrel dogs. Dogs were given approximately 80 mg/kg/day for 30 days and induction was monitored by following changes in vivo [14C]aminopyrine metabolism 14C-CO2 production). The paper does not mention complications due to the sedative effects of phenobarbital. As might have been expected in an outbred population, some dogs were more inducible than others. In fact, in two dogs, no induction occurred at all. Not surprisingly, induction resulted in increased hepatic catabolism of vitamin D3. The important points to be stressed here are that variable responses of dogs to inducing agents can be a potential problem and that in vivo clearance of aminopyrine could provide a noninvasive probe for screening of “good” versus “poor” responders to inducing agents of the phenobarbital class. The inducing effects of drugs and chemicals other than phenobarbital and β-naphthaflavone (typical experimental tools) have also been studied in the dog. Lan et al. (1983) compared and contrasted the inducing effect of hexahydroindazole (10–250 mg/kg/ day for a month) in three species. Increases in relative liver weights occurred in all three species, but increases in microsomal protein only in rats and monkeys (and not the dog). Increases in cytochrome P-450 and aminopyrine

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metabolism occurred in all three species, but the largest increases (compared to concurrent controls) were observed in dogs. Abramson et al. (1986) and Abramson and Lutz (1986a, 1986b) have extensively studied the relationship between enzyme induction, in vivo antipyrine metabolism and increases in alpha-1 acid glycoprotein. Although the relationship between the latter two parameters is not a simple one, it can be used to monitor the extent of induction by phenobarbital-type agents in the dog. Using these techniques, they demonstrated that phenytoin (Abramson and Lutz 1986a) and rifampicin (Abramson and Lutz 1986b) are effective inducing agents in the dog, whereas medroxyprogesterone is not (Abramson et al. 1986). The latter finding is of interest because medroxyprogesterone has been reported to be an inducing agent in rats. This underscores again the point that there are species-related differences in responses to inducing agents. Epoxide hydrolase is an important enzyme in the metabolism of reactive arene oxides, but until 1980 little work had been done to characterize this enzyme in the dog. Pacifici et al. (1981) reported the activity of hepatic microsomal epoxide hydrolase (with styrene oxide) in the dog to be 9.7 ± 2.0 nmol/min/mg protein, which was intermediate between that of the mouse (1.9 nmol/min/mg) and the baboon (31.3 nmol/min/mg). Gregus et al. (1983) confirmed that the dog has relatively high epoxide hydrolase activity (approx 15 nmol/min/mg protein) compared to many other commonly used laboratory species, including the rat. Little other work has been completed to distinguish, either on structural or substrate specificity basis, epoxide hydrolase of the dog from that of other species. Given its high activity, however, it can be expected to play an important role in xenobiotic metabolism in the dog. As in other species, conjugative metabolism (other than mercapturic acid formation) has been studied in the dog longer than oxidative metabolism. As reviewed by Hirom et al. (1977), the majority of the conjugative reactions were described in Germany during the latter part of the 19th century. The dog, like most mammals, excretes phenols as sulfates and glucuronides. In the dog, the ratio of sulfate to glucuronide (at an aromatic hydroxyl group) varies with substrate. For example, with phenolphthalein, the dog will excrete 18% as the glucuronide and 82% as the sulfate (as reviewed by Hirom et al. 1977), whereas with acetaminophen, 75% will be excreted as the glucuronide and 10% to 20% as the sulfate (Hjelle and Grauer 1986). With aryl acetates, the dog has a high tendency to form conjugates with amino acids. For example, with benzoic acid, the dog will excrete 82% as the glycine conjugate (hippuric acid) and only 18% as the glucuronide. Relatively little work has focused on the biochemical and molecular characterization of the enzymes involved in conjugation in the dog. Gregus et al. (1983) examined the in vitro activity of PAPS-sulfotransferase of the dog against four substrates. Activity was noted with all four, the highest with 2-naphthol and the lowest with taurolithocholate. When compared to other species, the sulfotransferase activity of the dog tended to be lower than most. Gregus et al. (1983) examined the activity of UDP-glucuronosyl transferase against a wide variety of substrates. The dog had somewhat higher activities than the rat with 1-naphthol, p-nitrophenol, estrone, morphine, and digitoxigenin-monodigitoxoside, whereas the opposite was true with diethylstilbestrol and bilirubin. Hence, in terms of relative activity there is little to distinguish the UDP-glucuronosyl transferases of the dog from those of other species. There is evidence, however, of species differences in stereoselectivity. Wilson and Thompson (1984) examined the stereoselectivity of dog hepatic microsomal UDP-glucuronosyl transferase by examining differences in activity toward (R)- and (S)-propranolol. When racemic mixtures were studied, the (S) entaniomer was the preferred substrate, with 3 to 4 times more (S)-propanolol glucuronide produced than that of the (R) entaniomer. Additional experiments with separate entaniomers demonstrated that the Km and Vmax of the dog microsomal UDP-glucuronosyl transferase are much higher with the (S) than the (R) entaniomer. In humans, plasma levels of (R)-propranolol have been shown to be higher than those of (S)-propranolol (Silber et al. 1982), whereas the opposite has been shown in dogs (Walle and Walle 1979), and this difference might be due to the differences in stereospecificity of glucuronidation in the dog versus human (Von Bahr et al. 1982). Stereospecificity of UDP-glucuronosyl

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transferase with substrates other than propranolol has been reported for other species (Sisenwine et al. 1982). Thus, stereospecificity of UDP-glucuronosyl transferase is not unique to the dog or propranolol. Schmoldt and colleagues (1987) examined the in vitro glucuronidation rates of dog liver microsomes toward various cardiac glycosides. In contrast to most other species examined, dog UDP-glucuronosyl transferase is capable of conjugating digitoxin; otherwise glucuronidation rates between rats and dogs were similar for all other cardiac glycosides examined. In fact, neither species has detectable activity toward digoxin. As both dogs and humans rapidly eliminate (in vivo) administered doses of digoxin, these results suggest that there might be greater similarity between canine and human UDPglucuronosyl transferase than between the human and rat enzyme with regard to substrate specificity. UDP-glucuronosyl transferase exists as a family of different isozymes in rats (Knapp et al. 1988). By examining a variety of model substrates and various inhibitors, Schmoldt et al. (1987) concluded that although there might be more than one canine isozyme, a single isozyme in the dog was responsible for glucuronidation of all cardiac glycosodise. The dog has long been recognized to have less active N-acetyl transferase activity than other species (Williams 1972). This was more recently confirmed by Gregus et al. (1983), who demonstrated that the dog had almost imperceptible activity with five different substrates. This is a qualitative species difference that could result in toxicologically important species-related differences in metabolism. For example, arylamines require acetylation to be activated to mutagens and carcinogens. In an interesting paper, Neis et al. (1985) compared the cytochrome P-450 content, N-acetyltransferase activity, and mutagenic activation activity of canine and human isolated hepatocytes. The mutagenic activation of five different arylamines was examined, and it was found that human hepatocytes had much greater mutagenic activation activity; this difference was attributed to the N-acetyltransferase present in human cells. Paroxon, an inhibitor of acetyl transferase, decreased mutagenic activation by human hepatocytes. They noted that dog hepatocytes were approximately the same size as human hepatocytes, but those of the dog had higher concentrations of cytochrome P-450, 210 ± 10 versus 94 ± 2 pmol/10E6 cells. N-acetylation is thus involved in the activation of arylamines in humans, and therefore the dog might not be the appropriate model in which to study the toxicity (relative to humans) of arylamines. Arylamines are not only substrates of the MMFO, but they can also be substrates for the flavin-based mixed function oxidase (FMFO). The FMFO of the dog has not been isolated and well characterized like that of the pig or the rat, but differential arylamine metabolism and the use of specific inhibitors has provided a probe for studying the involvement of this enzyme in dog xenobiotic metabolism. 2,[(2,4-Dichloro-6phenyl)phenoxyl-ethalamine (DPEA) is a specific cytochrome P-450 inhibitor and methimazole is a competitive inhibitor of FMFO. Using these, Hammons et al. (1985) have reported that the FMFO metabolized 2-acetylaminofluorene in pigs and humans, but not in dogs and rats. This group also studied the in vitro metabolism of 1-naphthylamine (1-NA) and 2-naphthylamine (2-NA) in rats, dogs, and humans. N-hydroxylation is a major pathway in all three species for 2-NA, but not for 1-NA, and this reaction is exclusively mediated by the MMFO. Ring hydroxylation, however, also occurred inall three species, and might be partially mediated by the FMFO as well as the MMFO because it was not completely inhibited by DPEA. For both chemicals, the dog has a higher rate of metabolism than the rat. Interestingly, there were greater individual human-to-human differences in the microsomal metabolism of these chemicals than the differences between rats and dogs. Like all known and studied mammals, dogs have hepatic cytosolic glutathione S-transferase (GSH-T). Grover and Sims (1964) described this enzyme in dogs as early as 1964, and were the first to note the high activity the dog GSH-T has with 1,2-dichloro-4-nitrobenzene. Gregus et al. (1983) examined glutathione S-transferase activity against six different model 1-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene; otherwise rats had much higher activity than dogs with all other substrates examined.

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Weiner (1986) confirmed in mongrel dogs the observation as to GSH-T activity toward 1,2-dichloro-4-nitrobenzene, and further compared the activities of rat and dog cytosolic preparations against seven other substrates and found that activity in the dog ranged from 2.5% (4-nitropyridine N-oxide) to 95% (ethacrynic acid) that of the rat. Overall, the highest activity in dogs (and rats, for that matter) was obtained with 1-chloro-2,4-dinitrobenzene. Weiner further characterized GSH-T in various cytosolic protein fractions. He found that dog GSH-T exists as four different isozymes composed of three different classes of subunits. In general, this type of isozymic pattern for this enzyme has been seen in other species. The dog enzymes are also not very different with regard to sensitivity to inhibitory ligands, such as bomcresol green or 8-anilino-1-naphthalene (Weiner 1986). Therefore, except for the expected molecular and quantitative activity differences, GSH-T in the dog is not startlingly different from that of other species. Weiner did observe that dog GSH-T has some activity in the denitrification of organic nitrates, such as isosorbide-2,5-dinitrate, and that this activity was responsible for the rapid in vivo conversion of 2,3-di-O-nitro-adenosine-5'-(N-ethyl-carboxamide) to the mono-nitro chemical in the dog (Wiener et al. 1983). Whether this is a pathway unique to the dog remains to be established. In general, little in-depth work on extrahepatic metabolism in the dog has been reported, the exception being work done at the Lovelace Inhalation Toxicology Research Institute on the respiratory tract. Hadley and Dahl (1983) studied the cytochrome P-450-dependent mono-oxygenase activity in the nasal membrane of several species. The dog tended to have the lowest amount of cytochrome P-450 and the lowest MMFO activities. Bond et al. (1988) characterized the cellular and regional distribution of xenobiotic metabolizing enzymes in the respiratory airways of beagles and found detectable cytochrome P-450-dependent activity throughout the airway, from the alar fold to the peripheral lung, but there were regional and substrate differences in specific activities. For example, the ethmoid rubinate had high activities for both benzo(a)pyrene and ethoxycoumarin metabolism, whereas higher nasal regions (alar fold, nasoturbinate, and maxilloturbinate) had much higher activity with ethoxycoumarin. In general, epoxide hydrolase and glutathione S-transferase activities were present along the entire airway, but tended to be higher in the nasal region and pulmonary airways compared to the major airways. UDP-glucuronosyl transferase tended to be evenly distributed. These detoxification enzymes were present in much greater activity than the MMFO-cytochrome P-450-dependent activation system. This work provides an excellent basis for using the dog in the study of site-specific chemical carcinogenesis of the respiratory tract. For example, Petridou-Fischer and colleagues (1987) studied the in vivo disposition of nasally instilled dihydrosafrole in the dog, and recovered metabolites from the nasopharyngeal mucus, thus confirming the potential importance of the MMFO activities in the upper respiratory tract in the metabolic activation of potential carcinogens. Other aspects of extrahepatic metabolism in the dog have been partially investigated as part of studies of various species-related differences in target organ toxicity. Garst et al. (1985) examined the pulmonary metabolism of perilla ketone by a variety of species, and concluded that the insensitivity of the dog lung to perilla ketone toxicity resulted from the relatively low amount of cytochrome P-450 with low MMFO activity toward perilla ketone. Poupko et al. (1983) reported that the dog bladder microsomes had very low activity with both 1- and 2-naphthylamine. Menard and colleagues (1979) examined both testicular and adrenal cytochrome P-450 in the dog, guinea pig, and rat. The dog and rat had comparable concentrations of adrenal cytochrome P-450 (1.1–1.2 nmol/mg microsome protein), whereas the guinea pig had roughly twice this concentration. In contrast, the dog had the highest concentration of testicular cytochrome P-450 (0.170 nmol/mg microsomal protein) and the rat the lowest (0.067 nmol/mg microsomal protein). Administration of spironolactone, a 7-{alpha}-thiosteroid, led to the destruction (40%–60%) of the cytochrome P-450s of both tissues in the dog. In contrast, spironolactone had no effect on adrenal cytochrome P-450 in the rat, but caused large decreases (88%) in testicular cytochrome P-450.

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Pacifici et al. (1981) reported on species and tissue difference in epoxide hydrolase and glutathione S-transferase and found the epoxide hydrolase activity of the dog kidney to be comparable to that of the guinea pig and rabbit, but less than that of the hamster. Glutathione S-transferase activity of the kidney of the dog (with styrene oxide, the same substrate used to determine epoxide hydrolase activity) tended to be much lower than that of the guinea pig and rat, but higher than those of the human and monkey. There are species differences in extrahepatic cytochrome P-450 of which the investigator should be aware, but it does not appear that the dog offers advantages over any other species in the study of specific routes of extrahepatic metabolism. Very little work has been reported on developmental or age-related changes in drug metabolism in the dog. Tavoloni (1985) examined the age-related development of the cytochrome P-450 MMFO and UDP-glucuronosyl transferase in the beagle dog. Activities were lowest at birth. They increased thereafter, tending to plateau after 6 weeks of age. Increases in the MMFO, but not UDP-glucuronosyl transferase, were induced by phenobarbital. Baarnhielm et al. (1986) compared and contrasted the in vivo and in vitro metabolism of felodipine in the rat, dog, and human. Felodipine is a calcium channel blocker that is very lipophilic, well absorbed from the GI tract, highly protein bound, and extensively metabolized. Comparative in vitro rates of microsomal metabolism of felodipine were rat > dog > human. There was an excellent correlation between the V{max}S and the concentrations of cytochrome P-450. The same rank order was observed for in vivo plasma clearance. It would appear that with chemicals that are well absorbed and rapidly metabolized, interspecies extrapolations are possible and fairly predictive. In their studies on the disposition of etodolac, Cayen and colleagues (1981) observed that felodipine also was well absorbed, partially metabolized (different metabolites in dog and rat), and undergoes extensive intrahepatic circulation. There were differences in the serum half-life (human > dog > rat) and clearance (dog > human > rat), but there was greater similarity between the dog and human. In general, the dog appears to be a better pharmacokinetic model (more predictive of behavior in humans) for humans than the rat or other rodents for chemicals that are well absorbed and have a high hepatic clearance.

REFERENCES Abramson, F., and Lutz, M. (1986a). The effects of phenytoin dosage on the induction of alpha-1-acid glycoprotein and antipyrine clearance in the dog. Eur. J. Drug Metab. Pharmacokin. 11, 135–143. Abramson, F., and Lutz, M. (1986b). The kinetics of induction by rifampicin of alpha-1-acid glycoprotein and antipyrine clearance in the dog. Drug Metab. Dispos. 14, 46–51. Abramson, F., Moore, C., and Hill, M. (1986). Medroxyprogesterone acetate does not induce antipyrine clearance and only weekly increases alpha-1-acid glycoprotein in beagle dogs. Res. Commun. Chem. Pathol. Pharmacol. 53, 65–78. Adam, W. S., Calhoun, M. L., Smith, E. M., and Stinson, A. W. (1970). Microscopic anatomy of the dog: A photographic atlas. Springfield, IL: Thomas. Adams, D. R., and Hotchiss, D. K. (1983). The canine nasal mucosa. ZbI. Vet. Med. C. Anat. Histol. Embryol. 12, 109–125. Aiello, S. E. (1998). The Merck veterinary manual (8th ed.). Whitehouse Station, NJ: Merck & Co. Al-Bagdadi, F. A., Titkemeyer, C. W., and Lovell, J. E. (1977). Hair follicle cycle and shedding in male beagle dogs. Am. J. Vet. Res. 38, 611–616. Aldridge, A., and Neims, A. H. (1979). The effects of phenobarbital and β−naphthaflavone on the elimination kinetics and metabolite pattern of caffeine in the beagle dog. Drug Metab. Dispos. 7, 378–382. Amacher, D., and Smith, D. (1987). Differences in the constructive forms of hepatic cytochrome P-450 in male and female adult beagle dogs. J. Chromatogr. 419, 61–73. Amann, R. P. (1982). Use of animal models for detecting specific alterations in reproduction. Fund. Appl. Toxicol. 2, 13–26. Andersen, A. C. (1970). The beagle as an experimental dog. Ames: Iowa State University Press.

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CHAPTER

9

Primates Toxicology:

Mark D. Walker, Joyce K. Nelson, John C. Bernal Charles River Laboratories, Preclinical Services

Pathology:

Gary B. Baskin Charles River Laboratories, Preclinical Services

Metabolism: Shayne C. Gad Gad Consulting Services

CONTENTS Toxicology......................................................................................................................................665 Husbandry.............................................................................................................................666 Institutional Policy and Regulatory Issues ..............................................................667 Facilities....................................................................................................................667 The Physical Plant ....................................................................................667 Building Materials ....................................................................................668 Ventilation, Temperature, and Humidity Control.....................................668 Power and Lighting...................................................................................668 Drainage ....................................................................................................669 Storage Areas ............................................................................................669 External Environmental Influences...........................................................669 Sanitation Facilities...................................................................................669 Animal Rooms ..........................................................................................670 Support Areas............................................................................................670 Special Areas.............................................................................................670 Biohazard Areas ........................................................................................671 Caging and Equipment.............................................................................................671 Individual Housing....................................................................................671 Social or Pair Housing..............................................................................672 Equipment Ancillary to the Cage .............................................................673 Systems of Removing Waste ....................................................................673 Cages and Housing for the Great Apes....................................................673 Environmental Enrichment and Special Concerns ...................................673

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Nutrition and Water ..................................................................................................673 Physical Form and Presentation ...............................................................674 Available Diets and Analysis ....................................................................674 Food Restriction........................................................................................674 Water .........................................................................................................674 Prevention of Disease and Injury.............................................................................675 Procurement, Quarantine, and Conditioning ............................................675 Occupational Health Program...................................................................677 Common Diseases ....................................................................................................677 Respiratory Diseases .................................................................................677 Enteric Diseases ........................................................................................677 Tuberculosis ..............................................................................................678 Viral Diseases............................................................................................678 Licensing and Records .............................................................................................678 Licensing ...................................................................................................678 Records......................................................................................................678 Study Design ........................................................................................................................679 General Considerations ............................................................................................679 Selection of Primate Species for Toxicology Studies .............................................679 Humane Endpoints ...................................................................................................679 Dose Levels ..............................................................................................................680 Dosing Techniques ...............................................................................................................680 Dose Volumes ...........................................................................................................680 Oral Administration..................................................................................................681 Techniques for Oral Administration .........................................................681 Equipment .................................................................................................681 Test Article Preparation ............................................................................682 Dose Administration .................................................................................682 Capsule ......................................................................................................683 Other Oral Dose Administrations .............................................................683 Intravenous Administration ......................................................................................684 Peripheral Venous Administration (Bolus) ...............................................684 Alert-Capture Techniques .........................................................................684 Tube Restraint Technique .........................................................................685 Intravenous Infusion Techniques ..............................................................686 Vascular Access Port Administration .......................................................................687 Intramuscular Injection.............................................................................................689 Subcutaneous Injection.............................................................................................689 Miscellaneous Routes of Exposure..........................................................................690 Intranasal ...................................................................................................690 Intraperitoneal ...........................................................................................690 Infrequent Routes of Dose Administration...............................................690 Data and Sample Collection Techniques .............................................................................691 Clinical Observations ...............................................................................................691 Physical Examinations .............................................................................................692 Body Weights ...........................................................................................................692 Physiological Measurements ....................................................................................693 Body Temperature (Rectal).......................................................................695 Blood Pressure ..........................................................................................695 Heart Rate .................................................................................................695 Respiration Rate........................................................................................695 Neurological Evaluation ...........................................................................695

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Ophthalmologic Examinations .................................................................................696 Electrocardiograms ...................................................................................................697 Blood Collection and Normative Clinical Pathology Data .....................................697 Urine Collection .......................................................................................................699 Pharmacokinetic and Toxicokinetic Evaluations .....................................................700 Invasive Cardiovascular Procedures .........................................................700 Necropsy ...............................................................................................................................700 Specialty Toxicology ............................................................................................................701 Neurotoxicology .......................................................................................................701 Developmental and Reproductive Toxicology .........................................................701 Safety Pharmacology (Cardiovascular Safety Assessment) ....................................703 Ocular Toxicology ....................................................................................................704 Immunotoxicology....................................................................................................704 Morphologic Evaluation Methods ............................................................704 Functional Evaluation Methods ................................................................705 Humoral Immune Response .....................................................................705 Cellular Immune Response.......................................................................705 Innate Immune Response..........................................................................706 Pathology........................................................................................................................................706 Nonneoplastic Spontaneous Diseases ..................................................................................707 Background Changes................................................................................................707 Bacterial Diseases.....................................................................................................707 Shigellosis, or Bacillary Dysentery ..........................................................707 Campylobacteriosis ...................................................................................708 Salmonellosis ............................................................................................708 Bacterial Pneumonia .................................................................................708 Tuberculosis ..............................................................................................709 Viral Diseases............................................................................................709 Simian Retroviruses ..................................................................................712 Parasitism..................................................................................................................714 Strongyloidiasis.........................................................................................714 Oesophagostomiasis ..................................................................................714 Pulmonary Nematodiasis ..........................................................................715 Filariasis ....................................................................................................715 Gastrodiscoides hominis ...........................................................................715 Pulmonary Acariasis .................................................................................715 Balantidium coli........................................................................................715 Other Diseases..........................................................................................................716 Neoplasia ..............................................................................................................................716 Metabolism.....................................................................................................................................716 References ......................................................................................................................................723 Acknowledgments ..........................................................................................................................730

TOXICOLOGY The phylogenetic and physiological similarity between humans and nonhuman primates has resulted in an increased demand for certain species in safety and efficacy assessments of new drugs and biologics. Only a few of almost 200 primate species are utilized in toxicology studies. The most commonly used species are Old World monkeys (cynomolgus monkey, rhesus monkey, and baboon), New World monkeys (squirrel monkey and marmosets), and the great apes (chimpanzees).

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Because of the genotypic and phenotypic resemblance to humans, nonhuman primates have been used in the study of induced or naturally occurring human diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis, diabetes mellitus, and atherosclerosis. Our understanding of the human brain, vision, aging, reproductive function, and behavior has been enhanced by studies in primates. Safety (and sometimes efficacy) evaluations of drugs, vaccines, and biotechnology products are conducted in nonhuman primates prior to approval for general use by the public. A failure to investigate the potential teratogenic effects of thalidomide in a primate model prior to exposing pregnant women resulted in tragic consequences in the 1950s and early 1960s (Somers 1963). Unfortunately, the teratogenicity of thalidomide could not be demonstrated in rodents prior to human exposure, but was subsequently shown to cause fetal abnormalities in primates (Hendrickx 1973; Hendrickx et al. 1966). The development of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase inhibitors (statins) has been a significant advancement for the treatment of hypercholesterolemia (and related hyperlipidemias). The withdrawal of a popular drug in this class, cerivastatin, highlighted the importance of preclinical toxicology in rodent and nonrodent species, as the development of life-threatening rhabdomyolysis in humans ultimately was associated with differences in animal model (and human) pharmacokinetics among drugs in the class, as well as multiple drug therapy and renal insufficiency in the target population (Evans and Rees 2002). In spite of the obvious predictive value of primates for human responses, they must be utilized conservatively, and models in lower species should be used when appropriate. Nonhuman primates used in biomedical research are purpose-bred or ethically obtained from large feral populations. The animals are imported or domestically bred. Special handling procedures resulting from the developing body of knowledge about the zoonotic potential of known (and emerging) infectious diseases to humans create unique challenges and increase the cost associated with the use of nonhuman primates. Nevertheless, the humane use of nonhuman primates as a physiological, pharmacological, and toxicological research model is critical for safety assessment of new drugs and biotechnology products, increasing the need for prudent use of primate inventories. The intent of this section is to define the utilization of nonhuman primates in toxicology studies and to describe the basic husbandry and technical procedures used in a primate facility. It is not intended to be an exhaustive review of the voluminous primatology literature. (See King et al. 1988, for an excellent summary of the role of nonhuman primates in research.) Husbandry Husbandry includes all aspects of housing and adequate care. Primates encompass a variety of species ranging from lower forms such as the tree shrews to the great apes. Much in the literature has generalized the nonhuman primate as one animal, when in reality the primate can be as small as several grams (e.g., shrews) to as large as more than 100 kg (e.g., great apes). The primary genus utilized in toxicological studies has been the Asian macaque, with lesser emphasis on the New World monkey and the chimpanzee. The primary focus of this section is on the macaque with specific reference when necessary to New World primates and apes. Federal regulations enacted through the Animal Welfare Act, Parts 1, 2, and 3, further define the need to provide optimal caging, adequate veterinary care, protocol development through scientific and Animal Care Committee review, and programs geared toward a variety of diverse procedures in assuring the physical and psychological well-being of the animals through environmental enrichment programs. In addition to welfare regulations concerning housing and care, the nonhuman primate requires special enrichment programs to assure social and mental stimulation. Social housing of nonhuman primates, ranging from periodic commingling to group housing, has become standard in toxicology research. Special caging and compatibility assessments are needed for successful social interactions with the animals. By matching the commingled animals by dose group and scheduling the interactions to avoid interference with study procedures, successful social housing can be accomplished with almost any study. Nonhuman primates are highly intelligent,

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and successful environmental enrichment requires a variety of toys, puzzles, and other devices rotated frequently enough to maintain the interest of the animals. Decreased stress and more normal physiologic responses are the rewards of a carefully designed socialization and environmental enrichment program. Institutional Policy and Regulatory Issues Institutional animal facilities and programs should be operated in accordance with the Animal Welfare Act (9 CFR, Parts 1, 2, and 3) and as described in the Guide for the Care and Use of Laboratory Animals (National Research Council 1996) and other applicable federal, state, and local laws, regulations, and policies. Nothing in the regulations intends to restrict the investigator’s freedom to conduct animal experiments in accordance with scientific or humane principles. Any program of husbandry must be developed considering the proper care and humane treatment of the animals used in research or testing. Aspects of conservation, alternatives to animal use, elimination of experimental duplication, and strong scientific principles must be paramount in experimental design. The U.S. Department of Agriculture (USDA) Animal Welfare Regulations (Federal Register 1989) and the Public Health Service through the Office for Protection from Research Risks of the National Institutes of Health (NIH/OPRR) mandate the appointing of an Institutional Animal Care and Use Committee (IACUC) to review the humane principles of research protocols and the total animal care and use program of each research institution. Included in this review are semiannual facility and program reviews; protocol review concerning euthanasia, appropriate care, and related issues; and provision of an annual written report to the responsible administrative official on the status of the institutional animal care and use program. Special consideration of restraint needs and surgical procedures come under the purview of the IACUC and must be scientifically justified. Facilities The Physical Plant A key to a successful toxicological research program is the design of the animal facilities. A good design assures efficiency in animal care and personnel movements and provides managers with the facilities necessary for an economic and sound animal care program. A basic design premise is to accommodate projects involving both small and large numbers of primates. Briefly, the following functional areas are essential to assure that diverse projects, different primate species, dosing and collection requirements, and husbandry and sanitation needs can be met (National Research Council 1996): 1. Separation of primate housing from administrative or human occupancy areas. Areas with animal procedure rooms (as distinguished from laboratories) can be adjacent to basic animal housing areas. 2. Special areas such as quarantine or receiving require isolation from primary study rooms or laboratories to assure project contiguity and disease-free studies. 3. Separate areas for surgery, necropsy, postsurgical care, clinical pathology, radiography, diet preparation, or animal treatment. 4. Office areas for administration and facility support areas, separate from animal areas. 5. Support areas for personnel, such as a break room, lockers, showers, and toilets. 6. Feed and supply storage and receiving areas. 7. Caging storage, washing facilities, and incinerator or waste disposal. 8. Central supply and receiving.

The size and complexity of these areas depends on the size of the facility. A facility less than 1,000 square feet has much different requirements from a facility housing numerous primates with

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areas of thousands of square feet. Efficiency and economy in utilization of research workers’ time must be carefully balanced with the need to separate animal and human facilities. Careful consideration of personnel and animal traffic patterns must be utilized in the design of animal facilities. Security has become a key issue in animal facility design. Barriers, entry locks, separate corridors, and separate floors all can enhance security. Building Materials Economy, maintenance, and sanitation must be considered in the selection of materials for the research facility. Key features of an animal room are durability, aesthetics, and ease of sanitation requirements while providing a humane and comfortable housing area free of pathogens and vermin hiding places. Construction Criteria. The design of primate housing areas is unique. Basic aspects to consider are the following: 1. Corridor widths of adequate size to permit easy movement of caging to wash area and bumper guards to prevent wall or corner damage. 2. Animal room doors with viewing windows, swinging toward the corridor only when an anteroom is present. Preferable construction is solid or metal doors to eliminate vermin hiding places. The doors accessing animal areas should have self-closures. 3. Floors of durable, sanitary, nonslip construction, such as epoxy or sealed concrete. 4. Walls of sealed impervious paints or coatings using coved and sealed junctions at floor and ceiling. 5. Ceilings of nonpermeable construction or sealed with washable coatings, and light fixtures of waterproof sealed construction.

Ventilation, Temperature, and Humidity Control Environmental control is an essential component of facility design in a toxicology laboratory. Nonhuman primates have specific requirements for ventilation, temperature, and humidity. Therefore, environmental consideration must always be given to the specific species being housed. For example, New World primates are specifically affected by low humidity (less than 50%), which can contribute to upper respiratory afflictions (National Research Council 1996). Ventilation at 10 to 15 air changes per hour is essential to reduce environmental aerosol contamination and provide odor and ammonia control. Recirculation of air is not advised unless it has been treated to remove particulate or toxic gaseous contaminants (Besch 1980). Temperature control systems are required to assure appropriate ranges of temperature from 64°F to 84°F (18°C–29°C; National Research Council 1996). The two most important considerations in the physical environment of primates are humidity and temperature, as they closely correlate to metabolism and behavior. Humidity is recommended at between 30% and 70% for Old World species. The toxicological laboratory must be more sensitive to temperature or humidity variations because such finite measurements as cellular parameters and hormonal indexes can be affected by environmental variances (Gortan and Besch 1974). Power and Lighting Steady and uninterrupted power with backup emergency generators is essential to assure critical environmental controls are maintained. Lighting of sufficient intensity is necessary to provide good husbandry and sanitation practices, observation of animals, security, and safe working conditions. A biphasic or a variable intensity lighting system might be necessary in certain facilities to provide

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adequate observations, yet provide soft diffused lighting for the majority of the day. Illumination of 75 to 125 foot-candles (Bellhorn 1980) has been recommended for observation periods. Time-controlled systems are essential in a research animal facility to assure regular circadian rhythms. Lights should be sealed and preferably fluorescent for energy efficiency. Some facilities have gone to the daylight ultraviolet bulb to simulate natural lighting; either is acceptable and appropriate for the subject species. Drainage Adequate waste control is essential to assure that contamination, odor, and waste stoppages of drains do not occur. Research colonies are at increased risk of disease transmission from sewage overflow. All waste fixtures should be of adequate size to permit full flow of waste biscuits, feces, hair, and similar materials. Drain sizes from 4 to 6 in. are recommended (National Research Council 1996) with appropriate floor sloping to facilitate the removal of water and waste materials. Cages should be provided with flush pans and troughs to direct and keep waste off of the common pathways. Facilities using dry paper or bedding systems should have adequate disposal systems for these materials. Lockable drain covers or appropriate-sized grids might be necessary to prevent improper materials from being deposited into (or obstructing) the drain system. Storage Areas A variety of storage areas are necessary to permit effective husbandry procedures. These include clean food and bedding storage; temperature-controlled food storage to prevent vitamin and nutrient deterioration; refuse storage areas for dry bedding or paper; and equipment storage for clean or dirty caging, disinfectants, and personnel items (masks, gloves, etc.). Consideration should be given to the myriad of equipment (precision scales, ECG machine, blood pressure monitors, infusion pumps) required to support the research effort. Most of this equipment must be stored in a dedicated area that remains clean and dry. External Environmental Influences The animal facility should be free of unusual external influences, as adrenal and immune cellular function can be affected by sudden or unusual noise. Cage washing areas should be separate and apart from animal rooms. Soundproofing materials should also be utilized to prevent unwelcome noise in the animal facility (Fletcher 1976; Peterson 1980). Large primates can add significant noise pollution by shaking their cages. These animals should be housed an appropriate distance from more sensitive studies and animal species. Special consideration is required for primate behavioral or hormonal evaluations. External influences, such as turning on a light or sudden personnel intrusion during nighttime periods, can adversely impact these studies. Fire alarm tests should be done with muffled alarms to prevent severe animal distress. Sanitation Facilities Critical to the husbandry of any facility are areas for washing and sterilizing animal cages, racks, water bottles, and similar accessories. The USDA/Animal Welfare Regulations mandate the sanitation and washing of cages at a minimum of every 2 weeks. Temperatures not lower than 180°F are recommended with use of appropriate disinfectants (National Research Council 1996). Key factors to consider in sanitation facilities are the following:

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1. 2. 3. 4. 5. 6.

Location related to traffic, animal rooms, elevators, and waste disposal. Soundproofing. Utilities such as hot and cold water, steam, drainage, and power. Proximity to storage and appropriate storage area to meet clean and dirty cage demands. Ventilation and employee safety. Access and corridor width.

The size of the facility dictates whether manual washing using brushes and portable units is satisfactory or whether large automated washers are necessary. Animal Rooms Animal rooms for toxicological experiments should be variably sized to house either large or small numbers of animals and to provide project segregation during (at a minimum) the dosing phase. Usually this is best accomplished by having a variety of sizes of animal rooms. Laminar flow and bubble-isolette room systems have been designed to permit multiple studies in fairly large rooms. Generally, animal rooms should have an anteroom provided with a sink and area for outer garment storage (e.g., disposable coveralls, masks, gloves, and dedicated footwear). The animal room proper should be constructed with all of the parameters noted earlier to permit an effective sanitary and humane environment (see figure 9.1). Recent concerns about psychological enrichment might dictate play areas, group housing areas, or special cage constraints to assure animal wellbeing and to provide the opportunity for exercise. Social needs of the nonhuman primate influence animal room design, research, and husbandry management techniques. All programs using primates should have primate environmental enrichment and some level of socialization incorporated into the facility design. Support Areas Personnel support areas, including appropriate break areas, locker rooms, and shower areas, are essential to any husbandry program. These areas should be carefully designed and managed for traffic flow to prevent risks to animals and personnel from contamination and disease. Special Areas Most facilities will have special needs for aseptic surgery, involving pre- and postoperative care, surgeon scrub areas, and operating rooms; pathology and necropsy rooms; clinical pathology; radiology support; treatment and procedure areas; and so on. The size and complexity of the facility

Figure 9.1

Nonhuman primate animal room. Floors and walls are made of nonporous materials that are readily sanitized. Watering systems are typically automatic.

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will dictate the size of each of these areas, but all are necessary parts in the support of the overall facility, husbandry, and management procedures. Biohazard Areas The study of drugs for AIDS, hepatitis, and similar diseases dictates the need for special facilities to work with hazardous agents whether chemical, biological, or physical. Each project will dictate the need for the level of containment. The Centers for Disease Control (CDC) has listed four levels for dealing with potential biological hazards (from BSL-1 to BSL-4), each with specific criteria. Special hoods, filters, room negative pressures, procedures, and personnel practices are involved. The reader is referred to the Center for Disease Control (1999) publication Biosafety in Microbiological and Biomedical Laboratories for reference to this vital area. Caging and Equipment The well-being and health of the nonhuman primates is critical to the success of a research project. The caging or housing system must be designed to assure that the research objectives can be met through minimizing experimental variables and assuring the “normality” of the animal via the maintenance of health and well-being. The Institute for Laboratory Animal Research (ILAR) Guide (National Research Council 1996) provides the following factors to consider in any housing system: 1. Provide adequate space to assure freedom of movement and normal postural adjustments with a resting place. 2. Comfortable environment. 3. Escape-proof caging. 4. Easy access to food and water. 5. Adequate ventilation. 6. Meets the biological needs of the animal. 7. Keeps animals dry and clean. 8. Reduces unnecessary physical restraint. 9. Protects animals from known hazards. 10. Provides options for environmental enrichment (grooming bars, commingling capability).

Individual Housing Cages for nonhuman primates must meet the space recommendations noted in the Guide for the Care and Use of Laboratory Animals (National Research Council 1996). Table 9.1 lists the current space requirements provided in the Guide. Institutions are encouraged to provide alternatives to individual caging. Infants and juveniles can be housed in group cages, for example. If adults are to be housed in groups, it is essential that only compatible animals be kept together. Newly grouped animals must be closely monitored to detect injuries due to fighting. Space in group cages should be enriched with structures such as resting perches and shelters. The minimum height of pens and runs used to house nonhuman primates should be 6 ft (1.8 m). For chimpanzees and brachiating species (orangutans, gibbons, spider monkeys, and woolly monkeys), the minimum cage height should be such that the animals can, when fully extended, swing from the cage ceiling without having their feet touch the floor. Historically, cages have been constructed of a variety of materials. Caging must be constructed of sturdy, durable materials and be designed to reduce the possibility of contamination to adjacent cage units. All surfaces should be smooth and free from sharp edges or broken wires. A minimum of ledges, corners, or angles is recommended to prevent dirt or fecal retention. The squeeze device should be easily operable to avoid primate injury. The cage should facilitate animal observations

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Table 9.1 Individual Housing Animals

Weight (kg)

Nonhuman primatesb Group 1 >1 Group 2 1–3 Group 3 3–10 Group 4 10–15 Group 5 15–25 Group 6 > 25 a b‘

Type of Housing Cage Cage Cage Cage Cage Cage

Floor Area/Animal ft2 m2 1.6 3.0 4.3 6.0 8.0 25.1

0.15 0.28 0.40 0.56 0.74 2.33

in. 20 30 30 32 36 84

Heighta cm 50.80 76.20 76.20 81.28 91.44 213.36

From the resting floor to the cage top. The designated groups are based on approximate sizes of various nonhuman primate species used in biomedical research. Examples of species included in each group are as follows: Group 1: marmosets, tamarins, and infants of various species; Group 2: capuchins, squirrel monkeys, and similar species; Group 3: macaques and African species; Group 4: male macaques and large African species; Group 5: baboons and nonbrachiating species larger than 15 kg; Group 6: great apes and brachiating species.

and provide feeding access and appropriate watering devices. Cage designs have included wallmounted, floor, rack (figure 9.2), and permanent installations, all of which are satisfactory depending on species and project needs. The cage requirement for an adult chimpanzee is much different than for the New World monkey, mostly a function of space and strength of design. Social or Pair Housing The benefits of social housing primates in toxicological studies are numerous. Psychological well-being programs should have written procedures for compatibility assessment, sexual maturity, gender separation, and cage security. Nonhuman primates are highly social, and interaction with other animals best satisfies this need and reduces stress. Pair housing when study procedures allow or periodic commingling of compatible animals within the same dose groups has effectively been used during toxicology studies. Special caging has been designed with sliding doors to intermittently connect cages to allow access between cages or grooming bars to allow tactile interaction if full cage access cannot be allowed. Pair housing can also be used during periods of nonobservation or during nighttime hours for some studies. Animals can be housed socially in larger groups when not on study using larger pens at both indoor and outdoor facilities.

Figure 9.2

Hanging cage rack system for mature macaques. Pair housing is accomplished through commingling doors on the side of adjacent cages.

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Group caging has its own set of requirements that by necessity must consider group dynamics, behavioral needs, usable surface areas, volume versus square footage, escape areas, and management practices. Equipment Ancillary to the Cage Equipment necessary to move the cage, handle the animal, and maintain safety of the handler and animal is essential to the husbandry operation. This equipment can include cage lifts, transfer cages, and restraint apparatus (e.g., pole and collar, leather gloves, capture gun, nets). Systems of Removing Waste The two primary methods of waste handling are dry and wet methods. Dry methods involve the use of bedding or plastic or paper liners under the cage. This system permits aerosol-free waste removal and cleaning of animal rooms, yet has the disadvantage of excessive waste disposal and high labor. This procedure for waste removal is sometimes employed where there are heightened biosafety considerations (biological radioactive waste). The alternative system is the wet procedure using water under high pressure to move the waste down the trough to the floor drains. The advantage of this system is fast cleanup, but care must be taken to prevent aerosolization and potential contamination of adjacent animals and personnel. A modification of this method using a lowpressure manual or automatic flush system is another approach. The use of wall-mounted proportionators allowing dual disinfectant and rinse flushing reduces contamination possibilities. Cages and Housing for the Great Apes The most important considerations for housing the ape, specifically the chimpanzee, are strength, environmental and behavioral enrichment, space for adequate movement and exercise, and design simplicity. Group housing of chimps might become the rule. New rules for individual housing could require at least two times the current space recommendations for adult chimpanzees of 25 ft2. This would certainly have a direct impact on housing requirements and protocol design. Environmental Enrichment and Special Concerns Congress has coined the term “psychological enrichment” as a housing requirement for nonhuman primates. Essentially this has been meant to include environmental enrichment, social housing, behavioral well-being, and similar terms. Any primate research facility must take measures to assure that programs are in place utilizing and developing techniques to enrich the environment of primates on or off study. The USDA requires a written program for environmental enrichment. A variety of toys, puzzles, mirrors, and so on can be purchased or constructed for use in environmental enrichment. Care must be taken to assure that the equipment can be sanitized no parts of the equipment are hazardous. Use of occasional treats with human interaction or forage boards or as part of a feeder puzzle can be used. Such treats should not be of a quantity to adversely affect intake of a balanced diet, and certain research studies might limit the type or nature of the treats. Nutrition and Water Fortunately, there are adequate diets available commercially for nonhuman primates most commonly used in research. Most important, concern must be shown with the feeding techniques, feeding frequency, and feeding receptacles used with primates. Careful observation of feeding behavior is essential. An empty feed cup does not signify that a primate is eating because most of

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the biscuits might be on the floor. Nor does a half-empty feed cup indicate an animal is not eating adequately, as each animal’s metabolic rate is different. Regular monitoring of body weights and body condition are the best measures for determining the adequacy of the diet. Physical Form and Presentation The majority of commercial primate feed is produced through an extrusion process resulting, after baking, in a very hard product. A young primate might find the product to be unpalatable unless softened by soaking. (The liquid used for moistening must be provided to the primate, as the vitamin C sprayed on the biscuit will be rinsed off into the liquid if discarded.) Some toxicology studies require oral dosing using a specific vehicle. Careful attention to amount of vehicle, particularly on twice or three times a day dosing regimens, is required to avoid “filling” the primate up with resultant reductions in food intake and potential nutritional deficiency. Careful attention should be given to dates of manufacture and rotation of feed to prevent deterioration of vitamin C. There are now diets containing stabilized forms of vitamin C, which allows the shelf life of commercial primate diets to be extended up to 180 days. Check with the feed manufacturer to make sure they will guarantee this shelf life and set up a rotation schedule appropriate to the manufacturer’s recommendations. The food must be kept palatable to the primate. Feed presented on the bottom of the cage without a feed cup can be contaminated by urine or feces, causing disease or an iatrogenic anorexia. Available Diets and Analysis Commercial diets are usually adequate and can be followed with pre- or poststudy analysis. Certified diets are also available and are required by GLP regulations. Analysis of noncertified commercial and noncommercial diets should be made to avoid potential contamination. Special diets for special studies, such as low-sodium or high-fat diets might be available commercially or might require custom preparation. Food Restriction It is common to fast the primate overnight prior to collection of clinical chemistry samples that might be affected by feeding or for preparation for anesthesia and surgery. The animals should not be fasted for excessive periods to prevent hypoglycemia. Some level of food restriction might be necessary to facilitate dose level when bioavailability is affected by the presence of food in the stomach or proximal intestine. Longer term food restriction during studies requires careful analysis to assure that animal well-being is not compromised (when balanced against potential scientific gains). Such modification to routine feeding practices must be justified to the IACUC. Water Clean, fresh water should be available at all times and should meet or exceed the appropriate regulatory standards for drinking water. Modern husbandry systems provide water through filtered automatic water systems. Clean water bottles are also an acceptable method to provide water but require more monitoring and maintenance to assure sanitation and proper function of the bottle. Water can be a source of potential contaminants that can interfere with a variety of study results. Careful analysis for chemical and microbiological contaminants should be conducted routinely. Chemical testing includes analysis for contaminants (i.e., pesticides, heavy metals, and specific organic compounds). Special testing can be conducted if required for certain test articles or studies.

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Prevention of Disease and Injury Procurement, Quarantine, and Conditioning The assurance that the animal model used is of high quality and healthy is essential to any research program. This is particularly critical for nonhuman primates, which for the most part are obtained from breeding facilities in China, Indonesia, or the Philippines. Relatively few primates are bred in the United States at this time. To prevent introduction of contagious conditions or zoonotic hazards into the animal colony, new animals should be quarantined a set period of time. The CDC requires a 31-day quarantine for imported primates, but a 7- to 14-day quarantine is more appropriate for animals introduced from domestic sources. The primary reason for the quarantine length is to detect potentially latent diseases and prevent the introduction of these agents into a larger (and likely susceptible) laboratory animal colony. Procurement Sources. Most nonhuman primates used for research today are foreign or domestic purpose-bred animals. Although some primates are feral caught, this represents a small proportion of the animals used in preclinical research. Nonhuman primates that are island-bred or raised in a controlled environment are typically of a superior health status, and they readily adapt to the laboratory environment. Cesarean-delivered or specific pathogen-free monkeys might be required on occasion, based on the study objectives and the known properties of the test article. Quarantine and Conditioning Procedures. Because most of the monkeys used for research are transported from breeding centers or quarantine facilities in other countries to the laboratories, quarantine and conditioning procedures are vital to assuring the health of the animals and protecting the laboratory colony from infectious diseases. A quarantine and conditioning program that pertains to the macaque is described as an example. It should be understood, however, that the following procedural descriptions might not be applicable to all institutions that utilize nonhuman primates because of differences in research needs, staffing, and facilities. The quarantine facility should consist of standard animal rooms equipped with squeeze-back cages. Prior to receipt of newly arrived monkeys, each cage should be washed in a cage washer and sanitized, and the room should be washed, disinfected, and rinsed thoroughly. It is recommended that iodophores and phenolics should be used for their tuberculocidal properties. The monkeys are delivered in crates containing individually segregated animals. Shipment lots can contain up to 120 animals. Immediately after receipt into the institutional quarantine facility, the monkeys are transferred from their shipping containers to quarantine cages directly and without handling. Drinking water and fruits are offered to assist in adaptation. The animals are fed a commercial 15% to 20% protein monkey diet supplemented with fruit on the afternoon of arrival and are allowed to acclimatize for a few days. After 3 to 5 days acclimatization, the monkeys are sedated with ketamine hydrochloride (10 mg/kg, intramuscularly) and examined. Stress to the animals and hazards associated with physical restraint are contraindicated; therefore, mild chemical restraint is better for the animal’s welfare and the safety of personnel. An electronic thermometer with an acrylic, rectal probe is used to obtain temperatures. The probe is shielded with a disposable plastic cover, which is changed and discarded between animals. Elevated temperature in the sedated primate is a more accurate disease indicator than rectal temperature measured in the physically restrained animal. If indicated by clinical signs of diarrhea or blood in the stool, fecal cultures can be obtained prior to temperature measurement. The animals are then examined for respiratory problems, suspicious enlargements, oral herpetic lesions, lacerations, dermatitis, and ectoparasitism. Minor health problems, including skin wounds and dermatitis, are treated at this time and supportive therapy is administered to monkeys with diarrhea or dehydration. Consideration of severe disease problems in individual monkeys that might require extensive workup is reserved until

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all animals have been examined. Additional fecal cultures and hematological evaluations are performed as indicated. During physical examination, monkeys are tested for tuberculosis (TB) using 0.1 mL of veterinary mammalian tuberculin, full strength, administered intradermally in the eyelid (National Academy of Sciences [NAS] 1986). Each monkey is given an injection of Ivermectin the first week of the conditioning program. Additional parenteral anthelmintics (e.g., Droncit for tapeworms and levamisole for roundworms) are administered the third week of the conditioning program. A second injection of levamisole is administered the fifth week of the conditioning program. Prophylactic injections of amoxicillin (20–40 mg/kg subcutaneously) or enrofloxacin (5 mg/kg, intramuscularly) might be indicated for respiratory or GI diseases, depending on the suspected infectious agent. Vitamins C and B complex (0.25–0.50 mL, intramuscularly) can also be given to improve the overall nutritional status of the animal. Monkeys are tattooed consecutively, typically using a combination of letters and numbers per institution policy. Information such as weight, abnormalities, and clinical signs is entered on the animal’s individual record. The quarantine period procedures are based on requirements of the CDC. In summary, monkeys are quarantined for at least 31 days to diagnose and eradicate TB. A total of three consecutive negative TB tests on each animal (after receipt) in the quarantine room must be achieved prior to releasing the group from quarantine. All monkeys that test positive for TB (reactors) are examined, possibly radiographed, euthanized, and necropsied. Tissues (typically the mediastinal lymph nodes) are evaluated microscopically (acid fast stain) for the presence of the TB organism. If deemed to be positive for TB, the quarantine procedures for the remaining animals need to be reevaluated to determine if depopulation or a repeat of the quarantine period is appropriate. All monkeys in the facility are observed daily by a technician or veterinarian. If needed, the veterinarian prescribes treatments and institutes diagnostic protocols for sick or debilitated animals. Urgency of treatment is paramount in any primate facility. For example, a debilitated animal exhibiting anorexia, nasal discharge, or diarrhea requires prompt attention. Treatment is initiated immediately to alleviate clinical signs. Diagnostic tests will only be performed if absolutely necessary during the CDC quarantine period. Diarrhea is one of the most common clinical signs in monkeys, particularly in new or stressful environments. Diarrhea is initially treated with pink bismuth (5 mL/kg/day, per os). Rectal cultures are collected when blood or mucus is seen in the feces, and a broad-spectrum antibiotic is given immediately. This original treatment is continued or changed depending on the results of culture and sensitivity testing. Other antidiarrheal agents that have been used successfully are amino pentamide hydrogen sulfate (0.1 mg/4.5 kg/day, intramuscularly), kanamycin sulfate (10 mg/kg/day, intramuscularly), and sulfa combinations. Because specific animal groups usually harbor bacteria with similar antibiotic sensitivities, one drug can usually be employed for treatment. Antibiotics are continued for at least 5 days after cessation of clinical signs. Fluid therapy (lactated Ringer’s solution intravenously or subcutaneously or a commercially available oral electrolyte solution) is often used for monkeys with diarrhea or dysentery. Pneumonia is a problem associated with the stress of shipment. Supportive therapy, including fluids, nutritional support, a temperature-controlled environment, and administration of antibiotics (e.g., penicillin or gentamicin) is recommended to treat pneumonia caused by secondary bacterial invaders. Severe epizootics of pneumonia have occurred in rhesus or cynomolgus monkeys that were associated with measles (rubeola) outbreaks in the first few weeks of quarantine (Potkay et al. 1971). Vaccination with modified live measles vaccine is recommended after the third TB test has been evaluated. Other disease problems include amebiasis, dermatophytosis, and miscellaneous traumatic injuries. Most of these entities manifest clinically during the first 14 to 21 days after arrival. Thereafter, the health status of most animals stabilizes considerably. Blood is obtained following the quarantine period from selected animals to further evaluate their health status and for reference serology. Fecal examinations for parasites are conducted.

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Specific antihelmintics, antiprotozoal agents, and antibiotics are prescribed as indicated to individual monkeys or to groups. The preceding description of quarantine and conditioning procedures is applicable to cynomolgus and other macaques imported from purpose breeding farms (NAS 1986; Renquist 1975). Domestically bred macaques that have been tested and found to be free of infectious diseases communicable to humans (e.g., shigellosis herpes B virus infection and TB) do not require such rigid quarantine measures. It is necessary, however, to provide a sufficient period of time for such monkeys to become adapted to a laboratory cage environment, as the majority are born and raised in large harem-type cages or corrals. During the adaptation period, valuable baseline data (hematological, serological, microbiological, and physiological) can be compiled for individual monkeys. Occupational Health Program Transmission of disease between primates and humans has been described. Recently, cases of herpes B, latent in the macaque but fatal in humans, have been incriminated in several deaths of animal handlers (CDC 1987a, 1987b). An occupational health program is essential, including preemployment screens for TB and physical examination with complete medical history. Banked reference serum samples and continuous monitoring are necessary with evaluation by the physician to assure employee health. The reader is referred to the NAS (1986) standards for specific information on evaluation criteria (CDC 1987a, 1987b). There are three central components to a sound occupational health program in a primate facility. The first component is the staff. They must be well trained in the biohazards of working with primates. The institution must provide adequate personal protective equipment (PPE). Procedures and policies requiring the use of PPE should be incorporated into the training program. The second component is to require written programs and procedures to be followed in the event that there is an exposure (scratch, bite, bodily fluid) from a primate or primate-soiled equipment. These procedures can include postexposure disinfecting of the wound or area, as well as biological samples from the animal and human for surveillance and medical care where appropriate. The third component is the need to educate the medical community. Occupational health providers, emergency room staff, and medical specialists (e.g., virologists, trauma surgeons) should be a part of your medical support structure in the event that an exposure results in human disease. This will facilitate better care for your employees. Common Diseases The diseases and descriptions noted herein are those most common to the primate toxicology laboratory. A more comprehensive overview of the pathology associated with infectious diseases in presented in the pathology portion of this chapter. Respiratory Diseases Pneumonia is seen in primate colonies and is most often due to changes in environment related to stress of shipment, or sudden changes in humidity or temperature. In macaques, this is often a complication of rubeola (measles) and specific bacterial pathogens (e.g., Branhamella cattarhalis or streptococcus). Appropriate antibiotics and measles vaccination is recommended (Good and May 1971). Enteric Diseases Diarrhea and dysentery are probably the most common diseases in nonhuman primates. The major causative organisms are gram-negative enteric bacteria (e.g., Salmonella, Shigella, Campylobacter,

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Yersinia, and E-coli; refer to Renquist 1987a; Weil et al. 1971). Unfortunately, latent infection without clinical signs is common and can be exacerbated by the rigor of the experimental protocol. Clearing protocols might be necessary to reduce the latent carrier state; however, these antibiotic regimens are often costly and should not be performed without culture and sensitivity testing. When evidence of clinical disease is seen, appropriate therapy is often the best approach. Tuberculosis The bane of the research laboratory is TB caused by a variety of mycobacterial organisms (Renquist 1987a). Appropriate tuberculin testing coupled with rigorous quarantine procedures can decrease the likelihood that it will enter the colony. A diligent intradermal mammalian tuberculin (MOT) TB testing program, a health surveillance program to include radiology where appropriate, and specialized assays such as the Gamma interferon test can be employed to achieve a comprehensive approach to TB prevention. Viral Diseases Measles (rubeola) is a common viral disease in humans and nonhuman primates. Certainly, however, other viral pathogens (e.g., poxviruses, hepatitis, simian hemorrhagic fever, and rabies) have also caused problems in a variety of research facilities. Most recently, serious concerns have been caused by herpes B (Renquist 1987b). Unfortunately, the incidence of herpes B antibody approaches 60% in most colonies. Herpes B is latent in macaques, causing a perioral blister-like lesion that is typically not associated with other abnormalities. When an individual is scratched or bitten by a positive primate, fatal consequences can result. The CDC has developed a set of guidelines for herpes B exposure and Ebola-like disease that should be carefully followed for both liability and employee health reasons (CDC 1987b, 1990). Simian retroviruses (SRVs) have been associated with morbidity that has confounded the results of toxicological studies. SRV is prevalent in many macaque populations, and therefore should be part of a prestudy viral testing protocol. It is prudent to eliminate SRV-positive animals from assignment to long-term and pivotal toxicological studies. Licensing and Records Licensing The Animal Welfare Act (Public Law 89-544), as amended most recently in 1991 (Federal Register 1991), requires the registration and inspection of primate facilities. The USDA Animal Plant Health Inspection Service is involved with enforcement. Direct importers of primates must also be registered with the CDC. Other regulations and agencies covering primates and their care and use are the Department of the Interior, Federal Wildlife Permit Regulations, Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), and the National Academy of Sciences, Institute of Laboratory Animal Resources. Records Accurate recordkeeping is required by law and is essential from the source to the completion of a project. This involves source country, date of receipt, importer, age, sex, body weight, health histories, and tattoo numbers. This is particularly critical for animals for which a federal wildlife permit is required or when animals are on the threatened or endangered CITES list (e.g., chimpanzees).

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Study Design General Considerations The design of a preclinical toxicology study protocol (also referred to as a study plan) is dependent on the intended therapeutic indication of the investigational drug, the human (or animal) population for which it is intended (Hobson and Fuller 1987), or the potential for environmental exposure for population risk assessment. Guidance documents issued by worldwide regulatory agencies, including the U.S. Food and Drug Administration (FDA), the U.S. Environmental Protection Agency (EPA), the European Union Organisation for Economic Cooperation and Development (OECD), the Japanese Guidelines for Nonclinical Studies of Drugs Manual, and the International Conference on Harmonization (ICH) Tripartite Guidelines, typically provide information to assist the researcher in the development of appropriate study designs intended for safety assessment. Study duration ranges from singledose acute studies to chronic multiple-dose studies that could exceed 1 year of test article exposure. The number of animals required for each study design varies substantially, and is directly impacted by the objectives of the study, duration of exposure, toxicologic endpoints, and potential need for assessment of posttreatment recovery. Acute (single-dose) and short-term repeated-dose toxicity studies are typically used to determine appropriate dose levels for subsequent studies of longer duration. Drugs that are intended for the reproductively active population sometimes require a threesegment testing plan (not to be confused with the standard three-segment reproductive evaluation in rodents; Hendrickx and Binkerd 1990; Hendrickx and Cukierski 1987). In segment I, menstrual cycles and hormone levels (estrogen, progesterone, luteinizing hormone, and follicle-stimulating hormone) are monitored for 90 to 180 days, depending on protocol design, and spermatogenesis and testosterone levels are evaluated in males during a minimum 60-day treatment period. In segment II studies, pregnant females are treated during the period of organogenesis (gestation days 21–89) and a cesarean section is usually performed on day 100 for assessment of fetal abnormalities. The late gestational effects of a test material are examined in segment III studies. Pregnant females are administered the drug from gestation days 90 to 150 for evaluation of any abnormal neonatal neurological or behavioral responses. Selection of Primate Species for Toxicology Studies As discussed earlier in this chapter, there are many different species of primates used in research and determining the best nonhuman primate species for a particular research study is challenging. In toxicological studies in which many compounds are novel to primates, animal availability and industry trends have had a significant impact on the choice of species used for these studies. From the early research into polio vaccine development (during the 1940s) and continuing until approximately 20 years ago, the rhesus macaque was the monkey of choice for most research programs. Today the cynomolgus macaque is the most commonly used monkey in toxicological studies, although the rhesus monkey still remains a suitable surrogate from a scientific standpoint. The investigator should be aware that there could be more than one species suitable for toxicity studies. When choosing a primate for a toxicological program, it is often preferable to use one particular species from the same country of origin throughout the drug development program to minimize variability in the clinical parameters evaluated. Other considerations when choosing primates for studies include study design, dose administration techniques, health specifications (e.g., specific pathogen-free animals) weight of the animal, degree of maturity, and gender. Humane Endpoints Humane endpoints are the physical, clinical, or other in-life observations that mark or describe adverse toxicity that results in an animal welfare concern. Humane endpoints in primates can be a

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valuable tool to quantitatively communicate morbidity to investigators, study directors, sponsors, and the IACUC. Humane endpoints are intended to describe or quantify the course and progression of adverse toxicities; they should be determined by active communication among all members of the study team. It is critical to any humane endpoint determination that individuals performing the assessment are qualified in the evaluation of these parameters and consistent methodology is employed. Close evaluation of the in-life data could serve as an early predictive indicator of present or impending adverse effects. If excessive pain, distress, or an unnecessary level of illness will result if the procedures of a study continue, humane endpoints are determined for the study to effectively modify the procedures or humanely euthanize the animals (if necessary). Dose Levels Dose levels tested are usually based on a proposed human dose or exposure. The lowest dosage in a safety evaluation study is usually greater than the expected human dose and should define the highest no-effect level. The highest dose is some multiple (e.g., 10–100 times) of the proposed clinical human dose and should induce toxicity. The middose is an intermediate level usually with minimal toxicity to provide characterization of a dose response to the test article. Dosing Techniques Dosing procedures used in research protocols will vary depending on the experimental objectives or the biochemical characteristics of the compound. The dosing technique utilized in safety studies with nonhuman primates approximates as closely as possible the expected route and frequency of drug exposure in humans. Typically, pharmaceuticals are administered via nasogastric intubation, orally by gavage, or via capsule or masking in dietary supplements. Biotechnology-derived materials are given either intravenously (bolus or IV infusion), intramuscularly, or subcutaneously. Other less common routes of dose administration are intranasal, intraperitoneal, intravitreal, or site-directed administration (i.e., intrajejunal, hepatic artery, or intra-articular). On occasion test article formulations are administered using alternate oral routes such as in fruit slices, sugar cubes, or a fruit juice drink. Methods of dosing can also be dictated by volume and characteristics of test article, duration of administration, and intended therapeutic use. Dose Volumes Guidelines for maximum dose volume administration will vary from facility to facility, and there is a paucity of information available in the literature. In general, the dosing volumes for nonhuman primates are described in table 9.2. Table 9.2 Dosing Volumes for Nonhuman Primates Route

Maximum Volume

Comments

Capsule Nasogastric/oral IV bolus (≤ 3 min) IV infusion (≤ 1hr) IV infusion (1–6 hr) IV infusion (6–24 hr) IV infusion (> 24 hr)

Single “0” capsule 10 mL/kg 10 mL/kg 15 mL/kg 10 mL/kg/hr 5 mL/kg/hr 4 mL/kg/hr

Capsule size limitation due to anatomic limitations Per dose; twice daily dosing possible, minimum 4 hr apart Also described as “slow push” Maximum volume might be exceeded depending on formulation Animal should be closely monitored for urine output Single dose; monitor urine output Continuous infusion, chronic administration possible

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Oral Administration Oral administration of drugs in safety evaluation studies has generally been conducted in squirrel monkeys or young macaques, and in limited numbers of immature baboons. The reasons for the size limitations in nonhuman primate models are the expense of special handling equipment and the size and aggressive nature of mature monkeys and great apes. Also, an adequate amount of historical data is available only in a limited number of primate species. Procedures must be employed that minimize stress to the animal, yet protect personnel and animals from injury. Infectious agents that might be carried by some of the primate models (i.e., herpes B virus) could be transmitted to animal handlers with potentially fatal consequences. The fecal or oral transmission of bacterial pathogens (salmonella, shigella, campylobacter, and E. coli species, to name a few) can potentially occur, leading to disease in the animal handler. As a result, the zoonotic potential of these (and other) infectious agents must be considered for all handling and dosing procedures. However, with proper training of personnel in the handling of nonhuman primates, oral toxicity studies can be successfully conducted and the potential risks minimized. Techniques for Oral Administration Techniques for oral administration of a test article include oral gavage, nasogastric gavage, capsule (using a modified oral gavage tube), or (when palatable) on a piece of fruit or in a drink such as fruit juice. Equipment The nasogastric route of administration is the most common and preferable means of gavage administration for macaques. Commercially available infant feeding tubes are best suited for young adult to adult cynomolgus and rhesus monkeys. When oral gavage is preferred (either due to the size of the animal, restraint method used, or test article properties) gavage tubes are available and range in size from 8 to 12 French (ID), and are utilized for the oral gavage of liquid formulations. When specialized tubing is required (e.g., for capsules), tygon or polyethylene tubing of appropriate size is utilized. The end of the tube is beveled, then blunted by heat to prevent esophageal damage or stomach injury. Capsule administration is not recommended for studies in nonhuman primates of longer duration (greater than 14 days of daily administration), due to the increasing chance of injury to the animal over time. For both nasogastric and orogastric administration, a syringe is attached to the tube for delivery of the test article and the flushing solution. An 8 French feeding tube is generally used for nasogastric intubation (figure 9.3) when small volumes or low-viscosity liquids are presented or for stomach tubing of small primates (e.g., squirrel monkeys or infant macaques). Although large monkeys can be manually restrained for dosing procedures, the risk of injury to personnel is high, as the head of the animal must be held securely by animal technicians. A chair restraint system can be utilized for larger monkeys. This method restrains the animal’s head and limbs during the dose administration procedure, minimizing the risk to technical personnel. In addition, this restraint system can be less stressful to the animal than manual restraint, if the animal is properly acclimated to the procedure. When using this system an aluminum or hard plastic collar is placed around the neck of an anesthetized animal and remains in place for the duration of the study. Aluminum poles with snap-type hooks are inserted into the cage and attached to the collars. The animal is led from the cage and placed in a chair (figure 9.3) and the collar secured to the chair. In this manner, minimal handling of the animal’s head is required for dosing. It is important that proper positioning of the animal is achieved to reduce the risk of test article aspiration in the event vomition occurs. After dosing, the animal can be led back to the cage and released. Animals typically adapt to this handling procedure within a short period of time (typically 1–2 weeks).

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Figure 9.3


Nasogastric dose administration.

Chimpanzees are utilized on a very limited basis for acute nonterminal toxicity studies, where the characteristics of the test article preclude the use of other nonhuman primate species. Animals of this size are usually maintained anesthetized for serial bleeds over a maximum of 6 hr, after which they can be immobilized with titrated doses of ketamine HCl for subsequent collections. Some chimpanzees can be trained for alert bleeding, but only experienced personnel should be allowed to handle the animals under these circumstances. Test Article Preparation The test article formulation is typically prepared on the day of dosing, although there might be instances where the known stability of material allows for preparation at an earlier date. A number of vehicles are used for test article formulation, depending on the aqueous solubility of the compound. True solutions (where aqueous solubility is high) commonly use distilled water, buffered saline, or aqueous admixtures that increase stability and solubility. Polyethylene glycol (PEG400 or similar) is commonly used when the aqueous solubility of the test article is poor; however, dose volumes should not exceed 1 mL/kg due to the increased incidence of diarrhea with this vehicle. Suspensions of the test article are commonly prepared in carboxymethylcellulose (typically 1%), and this material is well tolerated in all nonhuman primates. Capsules can be prepared prior to the day of use, if a powder; if liquid, it is necessary to fill the capsule shortly before administration. Control animals should be administered a concentration and volume of the vehicle equivalent to that received by the high-dose animals. Dose Administration For oral or IV administration, the individual doses are drawn up into graduated syringes in the formulation laboratory or in the animal room. If prepared in the formulation laboratory, syringes are labeled with the animal number, the study, and group color code. A research technician not involved in the dose preparation verifies this information. Syringes are placed in a rack, also color coded, and

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transported to the animal room. If dose withdrawal occurs in the animal room, the source container is appropriately identified with the study and group information. The test article formulation, dose volume for each animal, and dose administration is verified and documented by the technical staff. Depending on the test article, monkeys can be fasted overnight prior to a morning dosing. Manual restraint methods include cageside dosing, removal from the cage and immobilization by the technician, tube restraint, and chair restraint. Cageside dosing is achieved by use of the squeezeback mechanism of the animal’s cage. Once the animal is appropriately restrained, the dose is administered. One technician can accomplish this procedure. For dose administration outside of the cage, the animal is caught (using protective gloves) after partial closing of the squeeze-back mechanism. The animal is removed from the cage and restrained (typically cradled on the side of the animal handler). A second technician administers the dose, which is confirmed by another individual in the room. For tube restraint (most appropriate for IV dose administration and femoral venous blood collection), the same method of extracting the animal from the cage is used, and the animal is placed (headfirst) into an appropriately sized clear Lucite tube. The animal’s legs are restrained and the dose is administered into the saphenous vein. For chair restraint dosing, two handlers, each with a pole, are required to capture an animal and place it in the primate chair. The door to the animal’s cage is opened slightly, a pole inserted, and the pole attached to a ring on the collar. The animal is restrained within the cage, the door is opened, and the other pole is attached to the ring on the opposite side of the collar. The animal is then led out of the cage and into the chair for restraint. The third technician, meanwhile, has the animal’s dosing syringe and tube (nasogastric or oral) available for dose administration. A technician, wearing appropriate gloves and arm covers, restrains the animal’s head. The tube is inserted and slight negative pressure is applied to the syringe to check for air bubbles, indicating a possible lung intubation. The dose is administered, a flushing syringe containing a small amount of water or vehicle is attached, and the remaining contents of the tube are flushed into the stomach. The tube is removed and the animal is observed for a moment to check for vomiting. The poles are reattached to the collar and the animal is returned to the cage. Risks to the animals are minimal if the proper procedure is followed; however, if the dose is inadvertently placed in the lung rather than the stomach chances for animal survival are minimal. Caustic or irritating compounds or large gavage volumes might induce an emetic response that, if aspirated in the lungs, can result in the death of the animal. Consideration must be given to reducing the dose or reformulating the test article to reduce the possibility of aspiration. Capsule Plain gelatin or enteric-coated capsules are administered when testing a potential new drug delivery system or when a slower absorption rate is required. The technique for dosing with capsules is similar to oral dosing with a liquid. The plain or coated capsule is slightly wedged into the end of an 8- to 14-in. length of tygon tubing of slightly larger inside diameter than the diameter of the capsule. A syringe, usually containing 10 to 15 mL of tap water, is attached to the end of the tube. The capsule-plugged end of the tube is intubated and the capsule flushed into the stomach. The administration of multiple capsules simultaneously is possible, but this is generally used only in single-dose studies. Several capsules can, in this way, be administered to one animal without repeated intubations. A stylet can also be used to expel capsules out of the tube once properly placed in the stomach. In general, several hundred milligrams of test article can be administered in this manner. Prestudy acclimation to the dosing procedures will minimize the possibility of dosing errors and animal injury. Other Oral Dose Administrations Nonhuman primates have a propensity for playing with their food and expelling portions of their dietary rations from their cage or breaking it up and dropping it through the cage floor, thus

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making it essentially impossible to quantify dietary consumption (or assure a specific dose of test article, if added to the diet). On a limited basis, small quantities of test article have been placed on sugar cubes, in small pieces of fruit, or dissolved in a commercially available fruit drink. Animal technicians present the test-article-treated food item and observe to verify that the animal consumes it. Liquid can be administered from a syringe such that the animal voluntarily drinks the dosing solution. Expected problems encountered with these methods include animals removing portions of the sugar cube or fruit from their mouth and refusal to complete the dosing, or losing some of the fruit drink through spillage and refusal to swallow all of their dose solution, making quantification of the total dose administered difficult. These problems are minimal or nonexistent if the test article or food mixture is highly palatable to the animal. Additionally, the effects of the content of such vehicles on metabolism of the compound must be ascertained as part of the study design. Intravenous Administration The IV route of drug administration is a commonly employed technique in preclinical toxicology studies. This route is preferred for test articles with poor bioavailability or limited GI absorption, and for biotechnology-derived proteins and monoclonal antibodies. The cephalic and saphenous veins are the most accessible for peripheral venipuncture, in both single-dose and repeat-dose studies. Peripheral veins, including the femoral and the jugular, can be catheterized for acute or subchronic IV infusions, although many veins require surgically invasive procedures for catheter placement. In addition, specialized restraint devices are required for protection of the catheters from the animals (e.g., primate chairs, vascular ports, jacket-tether infusion, or backpack-port ambulatory infusion systems). Peripheral Venous Administration (Bolus) The two largest, most accessible superficial veins for IV dose administration are the cephalic and saphenous veins. The cephalic vein is located on the dorsal arm near the bend of the elbow of the nonhuman primate. The saphenous vein can be found on the ventral side of the leg from the knee to the ankle. Toxicology studies requiring a single daily injection for as long as 12 months can be performed successfully if proper techniques are utilized to minimize vascular and perivascular tissue damage. Alert-Capture Techniques Study animals are housed singly in squeeze-back cages for the study duration. Wearing appropriate protective apparel (lab-specific clothing, gloves, mask, arm guards), trained technicians capture the animals and administer the test article. Small and medium-sized squirrel monkeys (weight 5–6 kg) are caught by technicians wearing heavy leather gloves with long gauntlets by reaching into the cage, placing a thumb and forefinger firmly around the animal’s body, and gently removing it from the cage. The animal is restrained on a table, stomach down, by the technician, exposing the back of the leg for a saphenous injection administered by a second technician. Large macaques and small baboons are usually not removed from their cages for treatment. The animal is pulled to the front of the cage by the squeeze mechanism, which is locked in position. The technician carefully maneuvers an arm or leg near the feeding hole in the lower front of the cage. Once the foot or hand is gripped tightly, the squeeze mechanism is allowed to return approximately one-third of the width of the cage, permitting the animal to lean toward the rear of the cage. The limb can now be extended through the hole for accessibility to the vein (figure 9.4). Prior to injection, a portable clipper is used to remove the hair from 2 to 4 in. along the length of

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Figure 9.4

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Intravenous (saphenous) injection of rhesus monkey.

the vein. The exposed area is cleaned with an alcohol pad and pressure is applied to the vein with the thumb while holding the limb with the hand. Pressure is applied proximal to the intended injection site. As blood flow is impeded, the vein becomes visible to the technician. The needle, attached to the dosing syringe, is placed bevel up against the skin. Pressure is exerted until there is a slight give, usually indicating entry into the vein. Slight back pressure on the syringe will draw blood into the hub of the needle, verifying venous placement, at which point the test compound can be administered. Syringe and needle sizes vary with the volume and characteristics of the compound to be injected. Needle sizes usually range from 21 to 25 gauge and syringes from 1 to 35 mL. Injection volumes greater than 10 mL are more successfully administered when a small butterfly catheter is placed into the vein for dosing and the animal is removed from the cage. This can be facilitated by the use of two technicians: one to restrain the limb and ensure that the butterfly is not dislodged and the second to administer the dose through the distal end of the catheter and subsequently flush the remaining dose with appropriate vehicle (e.g., saline). Tube Restraint Technique The tube restraint technique is used for femoral venous blood collection techniques and for IV bolus (or slow push) administration of dosing formulations into the saphenous vein. The tube is constructed of clear Lucite plastic and varies in wall thickness, diameter, and length. A variety of tube sizes should be available to accommodate differences in age, species, and size of monkey restrained. Animals acclimate to the tube restraint technique quickly, as the barrier between the handler and animal not only provides a greater level of safety, but with repeated use it also has a calming effect on the animals. This restraint technique should be considered if frequent blood sampling is required, as it minimizes the potential for inadvertent cageside trauma (i.e., bruising and abrasions) and has been found to decrease errors related to strict time collection requirements.

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Intravenous Infusion Techniques A variety of techniques are utilized to administer dosing solutions via slow IV infusion. The following section provides a brief overview of these techniques; however, a more comprehensive discussion of IV infusion in nonhuman primates can be found in Healing and Smith (2000). Chair Restraint Technique. Studies requiring an IV infusion for periods of time ranging from 30 min to a suggested maximum of 4 hr are conducted using primate restraint chairs. Studies requiring a longer duration of infusion, or repeated daily doses similar to the maximum infusion duration, should be conducted using the jacket-tether or ambulatory infusion model (see later). Animals should normally be adapted to chairs prior to study initiation. Several chairing sessions beginning 2 weeks prior to study start reduces stress related to the chairing procedure. Animals are first anesthetized with ketamine HCl (approximately 10 mg/kg) and an indwelling catheter introduced into the cephalic or saphenous vein (Flynn and Guilloud 1988). The animal is placed in a restraint chair and allowed to recover from the anesthesia prior to dosing. If the cephalic vein is used for dosing, some provision must be made to secure the opposite hand and arm to prevent the catheter from being pulled from the vein by the animal’s free hand. The prepared dose is drawn up in an appropriate-sized syringe and placed on an infusion pump set to deliver the designated dose concentration at a preset rate. An extension tube (typically 10 in. in length) is attached from the syringe to the catheter for delivery of the dosing solution. The volume of the extension tube (and other external components, such as in-line filters) must be factored into the dose preparation calculations to assure sufficient volumes to dose all animals. At the termination of dosing, the catheter is removed, hemostasis is induced, and the animal is removed from the chair and returned to the home cage. Jacket and Tether Infusion Technique. The femoral and jugular veins are ideal for chronic catheterization and the conduct of chronic infusion studies. When the catheter is externalized, the placement of an indwelling catheter requires a surgical procedure, but this technique offers the advantage of limited animal inaccessibility to the catheter. Using the jacket and tether infusion technique, the catheter is channeled subcutaneously to an exit site located on the dorsal thoracic region of the animal and it is protected by a jacket and tether system (Bryant 1980). The dosing formulation is delivered via an infusion pump external to the cage, and doses can be administered over extended periods of time (i.e., several hours or days). These procedures are applicable for New and Old World monkeys but are not feasible for animals as large as adult chimpanzees. Animals are acclimated to the tethers and jackets for 2 to 3 days prior to placement of the catheter. During this period, the jacket is worn by the animal while the tether (flexible steel tube) hangs freely in the cage unattached to the animal. After the acclimatization period, the animal is sedated and taken to the surgery suite. The animal is prepared for surgery and an incision is made in the skin above the vein or artery to be catheterized. The vessel is located and separated from connective tissue by blunt dissection. The vessel is elevated with sutures to impede blood flow and a small cut is made in the vessel with iris scissors. A catheter introducer is inserted into the incision to facilitate introduction of the catheter. The catheter is inserted into the vein or artery for 5 in. to 8 in., depending on the size of the animal. The most desirable placement of the catheter is in the femoral artery or vein so that the catheter tip extends to either the vena cava or aorta, facilitating infusion and blood sampling procedures. The catheter is held in place by ligatures placed around the vessel and on both sides of an elevation (or “donut”) located several inches up the catheter. This prevents the catheter from being pulled from the vein or artery by pressure exerted from normal body movement. As an additional precaution, a stress loop is made in the catheter and anchored with a suture or sutures just under the skin. A trocar is used to tunnel the catheter under the skin to the middle of the back

PRIMATES

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where it is exited. The catheter is then passed through the mounting plate attached to the jacket and through the tether to the appropriate channel on the underside of the swivel. Another length of catheter is attached to the same channel on the top of the swivel and then to an infusion pump. Aseptic techniques in handling the exposed end of the catheter are extremely important to the success of this system. Frequent observations for potential problems with jackets and the tethering system are important if study objectives are to be met. Primates are particularly adept at manipulating this system, and thus require frequent monitoring to correct animal attempts to remove the jacket. Vascular Access Port Administration The vascular access port (VAP) is a subcutaneously implanted device that provides chronic vascular access and eliminates the need for multiple venipunctures in repeat-dose IV bolus or IV infusion studies (Dalton 1985). The VAP is a silicone rubber septum housed in a titanium metal casing that can be anchored with sutures under the skin. The placement of the vascular port is usually dorsolateral thoracic region, from which the catheter (typically polyurethane or silicone) is tunneled under the skin to the femoral vein (or another vein or artery) for catheterization. VAPs have proven to be quite durable and reliable, as long as local and systemic infection is rigorously monitored and treated proactively. Repeat-dose studies in excess of 9 months in duration have been successfully conducted; however, it should be recognized that continuous use of these devices results in an increasing rate of catheter failure after 13 weeks of dosing. Catheter failure is typically preceded by repeated dermal or subcutaneous infections, formation of adhesions, and necrosis of the skin overlying the VAP, as well as infections along the catheter tract characterized by swelling and occasional necrosis or drainage. Although there is no clear explanation for the increased incidence of catheter failure in chronic studies, gross and histopathologic findings from animals chronically instrumented with VAPs indicate that changes occur at the catheterization site. At gross necropsy, tissue adjacent and distal to the tip of the catheter thickens, becomes more brittle, and strictures form as fibrous connective tissue replaces the nonviable regional vasculature over time. Histopathologic findings include fibrosis of the vascular wall, fibrous capsule formation, chronic active inflammation, thrombosis, and hypertrophy of vascular and perivascular tissues. Dosing is performed by injecting the test article into the port and the catheterized vein. Location of the port and septum is easily accomplished by palpation. The area of the skin above the port is prepared using aseptic techniques to minimize the chance of infection. To prevent a coring effect, a 21-gauge Huber point (noncoring) needle is used for port access. If possible, locking solutions containing other materials should be aspirated prior to flushing the port. When flushing the port and administering a dosing solution, the Huber needle is inserted through the septum, and the dose is administered. A saline solution (or saline containing heparin up to 40 IU/mL) is used to flush the remaining test article from the catheter, and the appropriate locking solution is instilled to maintain catheter patency. Ambulatory Intravenous Infusion. Ambulatory infusion employs the use of a VAP for dose administration, and offers some advantages over the jacket and tether technique. Animals are first instrumented with a VAP, and sufficient healing time (approximately 7–10 days) is allowed prior to initiation of acclimation procedures. Each animal is fitted with an appropriately sized infusion jacket, which contains a dorsal opaque (black canvas) pocket. A battery-powered ambulatory infusion pump (CADD Legacy PLUS, or equivalent, SIMS Deltec, Inc., Minneapolis, MN) and self-contained dosing reservoir (50 or 100 mL cassette fill volume), along with a winged infusion set (i.e., infusion line connecting the pump and reservoir to the VAP via Huber-point needle) is contained within the dorsal pocket (figure 9.5 and figure 9.6).

688


Figure 9.5

Cynomolgus monkey fitted with ambulatory infusion jacket. The winged infusion set is used to access the VAP, which is implanted in the dorsolateral thoracic region under the jacket.

Figure 9.6

Cynomolgus monkey moving freely in the home cage using the ambulatory infusion system.

The ambulatory infusion system allows the investigator to implant a number of animals prior to initiation of dosing, thus providing a readily available pool of monkeys for a series of studies. If intermittent infusion is required (i.e., multiple daily doses or a defined dosing intervals per week), this system can result in decreased animal handling due to the programmable nature of the infusion pump. In addition, the jacket and infusion system can be removed between doses (e.g., once or twice weekly), allowing the animal more normal activities and minimizing infusion-system-related injuries (i.e., pressure necrosis to the skin, obscured infections, etc.).

PRIMATES

689

Ambulatory infusion is not appropriate for test articles of limited room temperature stability, or for those formulations that require very small or very large volumes of administration (i.e., less than 5 mL/dose or more than 250 mL/dose for the designated infusion period). In addition, this system is not optimal for monkeys larger than 5 kg, due to handling and safety concerns. Additional information concerning the use of ambulatory infusion in nonhuman primates can be found in the earlier referenced literature, as well as the bibliography for this chapter. Intramuscular Injection Intramuscular administration of a test article formulation provides for a reasonably consistent and rapid absorption and distribution profile, depending on the test article characteristics and the properties of the vehicle and excipient formulation. Biologics delivered in oil are released much slower than those from an aqueous carrier; in addition, test article suspensions, where the particle size is larger than for true solutions, might have a longer time to maximal plasma concentration (Cmax) and a concomitant longer half-life (t1/2). In nonhuman primates, the injection site is usually the outer thigh (quadriceps femoris mm), although the triceps are also utilized in larger animals. Monkeys and small baboons can be dosed alert, whereas large baboons and chimpanzees should be sedated. The cage squeeze mechanism allows the monkey to be immobilized to allow access for injection to a leg or arm. Hair is clipped from the injection site and the site is either cleansed with alcohol or aseptically prepared using a Betadine solution. Using an appropriate-sized needle (we suggest no larger than 21 gauge) and syringe, the skin and muscle are penetrated. A slight vacuum is applied to the syringe to check for possible venous puncture. If blood is drawn into the syringe, the needle should be removed and the process repeated. If no blood appears, the plunger is depressed slowly until the dose is administered. The needle is then removed and pressure applied to the injection site for approximately 30 sec with a gauze pad to complete the dosing procedure. Dosing volume should be limited to less than 0.5 mL/kg to minimize pain and the incidence of skeletal muscle irritation. If larger dosing volumes are required, two injection sites should be utilized. Subcutaneous Injection Subcutaneous administration of a test compound is frequently utilized in toxicity studies when a slower absorption rate is required, or if the characteristics of the formulation (i.e., low pH, largeparticle suspension, large volume, etc.) preclude the use of the intramuscular route. The dose is delivered between the dermal and muscle layers, usually in the dorsal midscapular area. The loose skin at this site allows for volumes up to 5 mL/dose to be administered. Other subcutaneous injection sites to be considered are the inner thigh and limbs for smaller volumes of dosing material (less than 0.5 ml). The relatively large potential subcutaneous space at each dose site should be demarcated with either a tattoo or indelible pen, depending on the duration of dosing and need to evaluate the dose site(s) for adverse reactions. Capture procedures for the animals are as previously described. The injection site is shaved and cleansed with alcohol, or prepared aseptically with a Betadine solution. The skin is grasped between the thumb and forefinger and retracted from the underlying muscle. The skin is penetrated with the needle at approximately a 15° angle to the injection site. As the plunger is depressed, a small bubble should appear as the dosing progresses. There should be no resistance to the injection. If resistance is noted and the bubble is firm, close to the surface, and appears white in color, the injection is being administered intradermally. The needle should be inserted to the deepest point into the subcutaneous space initially, and slowly removed as dosing is completed to assure a broad distribution of the dosing solution. The needle exit site should be digitally compressed for approximately 30 sec after dosing to prevent leakage of the formulation. One animal technician should restrain the animal while another

690


administers the dose to ensure complete delivery of the test compound and prevent possible injury to the animal or handler. Miscellaneous Routes of Exposure Intranasal The development of aerosol drug delivery systems for inhalation therapy has resulted in increased intranasal toxicity assessments. Test articles are administered into one (or both) nostrils with the aid of a calibrated and metered aerosolizer device. Discharge from the reservoir can be accurately calibrated, but total delivery into the nasal cavity is not always successful (or consistent) in an alert animal. Depending on the frequency of dosing and size of the animal, dosing can be accomplished via hand restraint (i.e., one animal handler for restraint, and one technician for dose administration), or via the use of chair restraint (for multiple doses over a short period of time, or for larger monkeys). This technique allows for better control of the head while the delivery tube is placed a short distance into the nostril for dose administration. Intraperitoneal Although not a common route for exposure to a test compound in nonhuman primates, intraperitoneal injections or infusions permit rapid absorption into the portal circulation. In humans, this route would place therapeutic agents in close proximity to tumors of the abdominal organs; thus nonhuman primate testing by this route can occur on a limited acute basis. The jacket and tether system is ideal for continuous intraperitoneal infusions for extended periods of time. This method has been utilized successfully to deliver tumor-specific monoclonal antibodies in safety-evaluation studies. Serial interperitoneal injections (needle or catheter) of nonhuman primates are not recommended because of the high probability of infection as well as the potential for bladder or intestinal puncture. Single injections are usually performed on anesthetized animals. The entire abdomen and inner upper thigh is shaved, cleaned, and sterilized. Using a Betadine swab (or solution) the injection site, which is 1 in. to 2 in. below and 1 in to 2 in. lateral to the navel, is again sterilized. The skin is pulled slightly to the center of the abdomen with the thumb of one hand while inserting the needle at an angle of approximately 45° into the injection site with the other hand. The purpose for pulling the skin to one side and placing the needle or catheter at an angle ensures that when the needle or catheter is removed an interrupted channel into the body cavity is established. This reduces bleeding and decreases the chances for infection. A short catheter (2 in.) or needle reduces the chances of perforating the bladder, intestine, or other organs. The location of the needle or catheter can be checked by injecting 1 to 2 ml of saline followed by gentle vacuum with a syringe. If blood, fecal material, urine, or fluid other than saline is aspirated into the syringe, the procedure must be reattempted on the opposite side. Health-threatening consequences could result from organ perforation; thus the veterinarian should be notified immediately and appropriate antibiotic therapy begun. If an organ is not punctured, thumb pressure on the skin is released, which stabilizes the needle or catheter for injection. As the plunger of the syringe is depressed, attention must be directed at the injection site for possible swelling, which would indicate a subcutaneous or intramuscular dose. If swelling occurs, dosing is stopped and the procedure is repeated. Following completion of the injection, the needle or catheter is removed and pressure is applied to the injection site for approximately 30 sec, followed by observation for any bleeding or swelling at the site. Infrequent Routes of Dose Administration Drug delivery systems designed for special therapeutic applications have been evaluated in safety studies but are not considered routine procedures. Such procedures include osmotic

PRIMATES

691

minipumps or other drug-containing implants; topical administration; vaginal, rectal, or colonic administration via suppositories or liquids; and sublingual dosing. Periocular and intraocular dosing (i.e., administration of dosing formulations around the eye or via intravitreal injection) is accomplished in an anesthetized animal, and is an increasingly common route for the development of targeted therapies for degenerative ocular disease. Many of these unique routes of dose administration require surgical procedures or can only be administered to anesthetized animals or animals physically restrained in primate chairs. Such methods are typically utilized only for acute dosing protocols and might not be acceptable for subchronic or chronic toxicity assessments. Inhalation toxicology is a unique area requiring specialized equipment and facilities not usually found in general toxicology laboratories and is thus beyond the scope of this review. Data and Sample Collection Techniques All GLP primate toxicology studies require approved protocols including a defined listing of in-life and postlife evaluations to adequately characterize and evaluate the toxicologic and pharmacologic effects of the test article. Various worldwide regulatory agencies provide guidance as to the basic elements of regulatory toxicology study designs, but much is left to the individual researcher in defining (and justifying) the parameters evaluated. Commonly used evaluations include daily clinical observations, body weights, blood and urine collection for clinical chemistry, hematology, coagulation, and urinalysis evaluations. Additional evaluations might include physical examinations, measurement of cardiovascular parameters, ophthalmic examinations (direct or indirect), and serum and plasma test article concentrations for toxicokinetic studies. Occasionally more specialized tests or samples such as menstrual cycle status, pregnancy tests, telemetry-based cardiopulmonary monitoring, nerve conduction velocity, electroencephalograms (EEGs), electroretinograms (ERGs), and tissue biopsy are required. After completion of the dosing (i.e., test article exposure) phase, animals are commonly euthanized, gross necropsies are conducted, organ weights are measured, and a protocol-specified list of tissues are collected, preserved, processed, and evaluated for potential histopathologic changes. Most studies of 4 weeks duration or longer include both terminal phase (i.e., immediately after completion of dosing) and recovery phase groups of animals to assess the potential for the reversal of target organ changes and toxicity. Recovery phases range from 2 weeks to a number of months, depending on a variety of factors including the known toxicology, pharmacology, and tissue distribution, and toxicokinetics of the test article. Sample collection from great apes is limited to nonterminal and non-life-threatening techniques. Tissues occasionally become available from moribund animals from these species, which are humanely sacrificed, or from animals dying of natural causes. Both study design and animal inventory planning must consider that primates should only be subjected to one major experimental surgical procedure during their lifetime (Federal Register 1991). A major surgical procedure is defined as one that penetrates a body cavity or is considered invasive in nature (examples include bone surgery, intracranial administration of test article, or medical device implantation). Additional procedures within the same experimental protocol must be approved by the IACUC, include appropriate perioperative measures for analgesia, and be justified by decreasing the overall number of animals used in experimental research. Clinical Observations Because primates have complex and highly individualized behavior patterns, obtaining meaningful observation data depends on the experience of the observer, the knowledge of expected observations for the species in a laboratory setting, and an innate familiarity with individual animal responses. The occasional use of group caging makes this task even more complex for more traditional toxicologic evaluations; however, behavior studies prefer the establishment and maintenance of a well-defined animal hierarchy. Ideally, observations should be made at the same time(s)

692


and by the same observer each day. Many studies include postdose evaluations that coincide with the expected maximum plasma concentrations (Cmax), which allows the researcher to determine if any effects are present the following day (predose). Changes in behavior or appearance are most important to note. An experienced observer should be quick to note the following (Silverman 1988): • • • • • • • • • • • • • • • • •

Lethargy Dehydration Unusual posture or movement within the cage Poor condition of hair coat Brightness or dullness of the eyes Unusual fluid from body orifices Unusual motion within the cage and proprioceptive deficits Self-mutilation Hyperventilation Hyperactivity Hyperreactivity (startle responses) Apparent pain or discomfort Favoring of individual limbs Evidence of changes in appetite or water consumption (if possible) Watery or discolored stool Changes at injection sites (where relevant) Any other unusual behavior or appearance

Appetite should be assessed at least once daily, and is commonly conducted prior to offering the morning food ration. Persistent inappetance can result in serious (and possibly life-threatening) hypoglycemia in most Old and New World species, as these animals have limited stores of glycogen and become ketotic with starvation. Physical Examinations The physical examination findings can be one of the most important overall criteria to determine acceptability of an animal for study use. These evaluations include, at a minimum, the assessment of the overall physical condition of the animal, body temperature, respiration rate, heart rate, thoracic auscultation, abdominal palpation, integumentary assessment, overall behavior and mentation, and other evaluations that are specified in the testing facility standard operating procedures or study protocol. Specialized examinations, which can include endocrinological profiles or more extensive neurological examinations, might be required, depending on the type of toxicology study (Keeling and Wolf 1975). Clinical pathology (serum chemistry, hematology, and coagulation parameters) and fecal parasite evaluations are typically included in the general physical examination. Table 9.3 and table 9.4 detail typical normative clinical hematology and serum chemistry data for nonhuman primates commonly used in toxicity evaluation studies. Body Weights Toxicologists who are not accustomed to reviewing data from primate studies are often surprised by the degree of inter- and intra-animal variability of body weights. It is not uncommon in a study of 2 weeks duration (or less) to encounter weight changes of 10% or more that are totally unrelated to the experimental protocol. These changes can be minimized by weighing and feeding at the same time each day, acclimating the animals to in-life procedures prior to initiation of dosing, and providing individualized care to animals that might not adapt as quickly to the requirements of the

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Table 9.3 Hematology of Nonhuman Primates, Normal Ranges Cynomolgus monkeya (Macaca fascicularis)

Rhesus monkeya (Macaca mulatta)

Baboonc,d (Papio sp.)

Chimpanzeea (Pan Troglodytes)

Test

Units

Squirrel monkeyb (Saimiri sciureus)

White blood cells (WBC) Red blood cells (RBC) Hemoglobin (HGB) Hematocrit (HCT) Mean corpuscular volume (MCV) Mean corpuscular hemoglobin (MCH) Mean corpuscular hemoglobin concentration (MCHC) Platelets (PLT) Reticulocytes Differential Segmented neutrophils (SEGS) Lymphocytes (LYMPH) Monocytes (MONO) Eosinophils (EOS) Basophils (BASO) Coagulation Prothrombin time (PT) Activated partial thromboplastin time (APTT) Fibrin degradation products (FDP) Fibrinogen (FIBRIN)

thsd/mm3 mill/mm3 gm% percent µ3

3.4–14.8 7.1–10.9 12.9–17.0 — 41.4–62.7

4.5–14.0 4.6–6.5 10.0–13.1 30–41 57–68

5.5–15.4 4.0–6.6 10.9–15.5 35.2–47.6 65–79

— — 8.7–13.9 31–43 63–90

7.2–16.8 4.8–5.8 12.0–5.8 36.0–49.5 64–102

µµg

13.9–20.1

18–22

21–27

18–27

20–34

percent

29.2–34.8

30–33

30–34

28–34

27–36

— —

200–550 0.1–1.1

230–650 0–1.9

225–544 0.3–2.3

150–450 0.5–1.5

percent

13.0–79.0

10–50

32–83

23–78

55–80

percent percent percent percent

19.0–82.0 0.0–6.0 0.0–22.0 0.0–4.0

50–80 0–2 2–8 0–2

34–69 0–2 0–6 0–1

14–76 0–3 0–8 0–1

12–45 0–5 0–2 0–1

sec sec

— —

8.9–12.9 16.9–33.9

9.4–13.4 19.5–25.3

11.5–13.0 29.5–38.0

10.0–13.4 18.9–35.6

µg%

—

3.5 Prothrombin time (sec > control) 1–4 a

Grade B 2.0–3.0 3.0-3.5 Easily controlled Minimal Good

Grade C > 3.0 < 3.0 Poorly controlled Advanced Poor

2 Points 1 or 2 Slight 2–3 2.8–3.5 4–10

3 Points 3 or 4 Moderate >3 < 2.8 > 10

5–6 total points = mild dysfunction: 7–9 = moderate dysfunction: > 9 = severe dysfunction.

870


Liver disease can influence absorption and drug disposition by altering the primary parameters: unbound intrinsic clearance, CLu,i; unbound plasma fraction, fU; and liver blood flow, QH. Alteration in those parameters can then produce measurable changes in several absorption and disposition parameters, as shown in table 14.6. Table 14.6 Primary Parameter Alterations and Effect on Pharmacokinetic Parameters Altered Primary Parameter CLu,I fU QH

Pharmacokinetic Parameter Effected CLs → T1/2 (restrictive clearance) ERH → F (nonrestrictive clearance) V → T1/2 CLs → T1/2 CLs → T1/2 (nonrestrictive clearance)

Table 14.7 summarizes the pharmacokinetic changes for a number of (low) restrictively cleared compounds during liver disease (mostly cirrhosis). The healthy control values are listed in parentheses. In most cases the value for CLs decreases and T1/2 increases (due to a decrease in CLU,i). The latter might in part be due to an increase in apparent volume of distribution. In some cases, notably compounds undergoing phase II conjugation metabolism (e.g., lorazepam, oxazepam), there is no change in CLS. In at least one case, tolbutamide, there is an increase in CLS, which is probably a result of an increase in fU. Antipyrine is a frequently used marker for CYP 450 oxidative metabolism because it is completely metabolized, not plasma protein bound, and has a low clearance. For the influence of various liver diseases on antipyrine t1/2, the normal value of about 10 hr increases markedly to more than 25 hr for all liver disease states. Note, however, there is an extremely wide variation in the values (from about 10 hr to more than 50 hr). One reason for this is the lumping together of all liver disease states into one category. The average values for t1/2 depend on the specific disease state, but variation remains wide in all cases. Clearance would be a better parameter to compare. Variability remains very high in both the control and disease groups and the raw data indicate considerable overlap of the clearance values. If the clearance values are normalized for estimates of liver volume, both variability and overlap decrease considerably. Reduced enzyme activity, as measured by CLu,i, would be expected to decrease the hepatic clearance of low or restrictively cleared drugs, as noted previously. In contrast, such changes should have no influence on the clearance of high or nonrestrictively cleared drugs. The clearance of such drugs will be influenced by changes in liver blood flow, QH, which is associated with many liver disease conditions. Table 14.8 summarizes the data for a number of high-clearance drugs. Clearance is reduced in most all cases and this is a reflection of altered blood flow. Note also that bioavailability increases substantially in all cases. This is a reflection of a reduction in CLu,i. A reduction in CLu,i results in a reduction in the hepatic extraction ratio, which in turn results in a decrease in the hepatic first-pass effect. In other words, the absolute oral bioavailability (F) increases. This will be most dramatic for drugs having a very high hepatic extraction ratio. Thus, if ERH is 0.95 in normals and the value changes to 0.90 (less than a 5% decrease) in liver disease, the value for bioavailability (F) increases twofold, from 0.05 to 0.10. (Recall that, F = 1 – ERH). An additional part of this increase in F is due to the existence of portal shunts that permit some of the absorbed dose to go directly from the GI tract into systemic circulation. The propranolol and nicardipine concentrations following oral dosing in cirrhosis patients are much higher than those in normals.

C

Flurosemide

C

Tolbutamide

Volume 59.1 ± 43.1 liters (19.5 ± 4.6) (Vss) 49.9 ± 4 litersa (65.9 ± 4) (Vss) 428 ± 108 ml/kg (321 ± 77) (V) 0.48 ± 0.14 liter/kg (0.33 ± 0.06) (Vss) 1.4 ± 0.6 liters/kg ( 1.1 ± 0.4) (V) 1.74 ± 0.21 liters/kga (1.13 ± 0.28) (V) 533 ml/kga (210) (V) 12 ± 3.5 liters (9.3 ± 3.7) (Vss) 1.14 ± 0.26 liter/kg (1.25 ± 0.24)(V) 1.57 ± 0.64 liters/kg (1.25 ± 0.24) (V.) 2.01 ± 0.82 liters/kg (1.28 ± 0.34) (V) 1.52 ± 0.61 liters/kg (1.2 ± 0.34) (V) 60.9 ± 9.5 liters (61.2 ± 12.2) (V) 51.7 ± 17.2 liters (47.7 ± 16.7)(V) 69 ± 13 liters (70 ± 8) (V) 115 ± 32 liters (106 ± 35) 0.563 ± 0.08 liter/kg (0.482 ± 0.08) (V) 0.15 ± 0.03 liter/kg (0.15 ± 0.03) (V) 0.19 ± 0.04 liter/kg (0.21 ± 0.02)(V)

Clearance 280 ± 136 ml/min (342 ± 80) 59.2 ± 8.4 ml/mina (168.6 ± 9) 7.6 ± 1.08 ml/kg/hr (13.8 ± 1.2) 7.7 ± 2.1 ml/mina (15.4 ± 4.4) 463 ± 145 ml/min (511 ± 93) 13.8 ± 2.4 ml/min (26.6 ± 4.1) 192 ml/min (194) 120 ± 36 ml/min (142 ± 42) 1.88 ± 0.70 ml/min/kga (3.32 ± 0.99) 1.26 ± 0.49 ml/min/kga (3.32 ± 0.99) 0.81 ± 0.48 ml/min/kg (0.75 ± 0.23) 0.74 ± 0.34 ml/min/kg (0.75 ± 0.23) 155.5 ± 70.4 ml/min (136.0 ± 46.3) 137.4 ± 51.4 ml/min (113.5 ± 30.7) 278 ± 79 ml/min (256 ± 56) 476 ± 139 ml/min (543 ± 126) 18.8 ± 11.3 ml/hr/kga (63.0 ± 28.5) 26 ± 5.4 ml/hr/kga (18 ± 2.8) 6.1 ± 0.9 liters/hr (6.1 ± 0.7)

T1/2 1.90 ± 0.56 hra (1.31 ± 0.15) 10.45 ± 1.14 hra (4.6 ± 0.3) 40.1 ± 5.1 hra (16.5 ± 3.6) 62.7 ± 27.3 hra (23.8 ± 11.6) 2.9 ± 1.1 hr (2.3 ± 0.7) 105.6 ± 15.2 hra (46.6 ± 14.2) 2.2 hr (0.79) 129 ± 75 min (74 ± 18) 509 ± 174 mina (340 ± 140) 1.017 ± 450 mina (340 ± 110) 31.9 ± 9.6 hra (22.1 ± 5.4) 25.0 ± 6.4 hr (22.1 ± 5.4) 5.8 ± 1.1 hr (5.6 ± 0.8) 5.3 ± 0.7 hr (5.1 ± 1.3) 3.0 ± 1.0 hr (3.3 ± 1.0) 166 ± 41 min (124 ± 16) 28.8 ± 14.3 hra (6.0 ± 2.1) 4.0 ± 0.9 hra (5.9 ± 1.4) 23 ± 5 hr (25 ± 3)

Note: C = cirrhosis; L = type of liver disease not cited; AVH = acute viral hepatitis; CAH = chronic active hepatitis.

AVH

C

Theophylline

Warfarin

C

CAH

AVH

C

AVH

C (compensated) C (uncompensated) C

Rantidine

Prednisolone

Oxazepam

Lorazepam

Hexobarbital

C

Diazepam

C

L

C

C

Cimetidine

Chlordiazopoxide

C C

Disease

The Influence of Liver Disease on the Pharmacokinetics of (Low) Restrictively Cleared Drugs (Williams 1984)

Chloramphenicol

Ampicillin

Drug

Table 14.7

SUSCEPTIBILITY FACTORS 871

0.6 0.87

Propranolol

Verapamil C

C

C

C

C

C

+140d

+42

+278

+65

+81

+91

+1,000

Bioavailability (% Change)

263 ± 28 liters (232 ± 53) (Vss) 5.56 ± 1.8 !iters/kg (5.94 ± 2.65) (V) 4.0 ± 0.3 liters/kg (3.2 ± 0.2) (V) 23 ± 1 3 liters/kg (2.9 ± 2 4) (Vss) 356 ± 94 liters (415 ± 107) (Varea) 380 ± 41 litersc (290 ± 17) (Varea) 481 ± 141 litersc (296 ± 67) (Vss) 9.17 liters/kg (6.15) (Vss)

526 ± 31 litersc (805 ± 91) (Varea) 2.22 ± 0.94 liters/kg (1.70 ± 0.21) (Varea) 310 ± 180 liters/kg (2.00 ± 0.5) (Vss)

Volumea T1/2 8.7 ± 4.0 hr (6.6 ± 2.4)b 170 ± 24 min (187 ± 26) 343 ± 234 minc (108 ± 70) 160 min (90) 12.5 ± 4.5 hr (7.7 ± 2.0) 359 ± 77 mind (213 ± 25) 6.99 ± 2.74 hrc (3.37 ± 0.82) 7.2 ± 1.2 hr (4.2 ± 1.1) 2.2 ± 1 3 hr (25 ± 1.5) 396 ± 115 minc (230 ± 28) 11.2 ± 12 hr (4.0 ± 0.3) 815 ± 516 minc (170 ± 72) 840 min (220)

Clearance

5.2 ± 2.1 ml/min/kgc (9.2 ± 0.8) 13.0 ± 3.9 ml/minc (20.0 ± 3.9) 814 ± 144 ml/minc (1,002 ± 304) 523 ± 158 ml/minc (900 ± 316) 649 ± 228 ml/minc (1,261 ± 527) 0.61 ± 0 13 liter/ min (0.01 ± 0.11) 1,153 ± 345 ml/minc (1,233 ± 427) 675 ± 296 ml/min (1.246 ± 236) 580 ± 140 ml/min (860 ± 90) 0.545 ± 0.181 liter/minc (1.571 ± 0.405) 1.22 liter/min (1.26)

12.8 ± 4.8 ml/min/kgc (18.1 ± 29)

Note: C = cirrhosis; AVH = acute viral hepatitis a Vss, volume of distribution at steady state; V = volume of distribution (one compartment); Varea clearance × 0.693/T1/2. b Numbers in parentheses indicate values observed in healthy controls. c Statistically significant differences between patients and healthy controls. d Because of a sixfold variability, caution is suggested in interpretation of this change in bioavailability.

0.8

0.6–0.8

Morphine

Pentazocine

0.15

Metoprolol

AVH

C

0.5

AVH

C

Meperidine

0.7

Lidocaine

C

C

0.7

Labetalol

C

Disease

Lorcainide

0.9

Extraction Ratio

Chlormethiazole

Drug

Table 14.8 The Influence of Liver Disease on the Pharmacokinetics of (High) Nonrestrictively Cleared Drugs (Williams 1984)

872 ANIMAL MODELS IN TOXICOLOGY

SUSCEPTIBILITY FACTORS

873

The increased apparent volume noted for a number of compounds in table 14.9 is a reflection of the altered (reduced) plasma protein binding of those compounds. The increase in the unbound fraction, fU, is the result of decreased plasma protein concentrations (especially albumin) or the accumulation of endogenous compounds that would normally be eliminated by hepatic metabolism. The latter compounds might compete with drug for protein binding sites. Table 14.9 lists the percentage increase in the unbound fraction to plasma proteins for a variety of drugs. Binding either decreases or does not change, as noted in the table. Table 14.9 Increase in the Unbound Fraction to Plasma Proteins in Liver Disease (Williams 1984)

Drug Highly extracted drugs Udocaine Meperidine Morphine Propranolol Poorly extracted drugs Amobarbital Azapropazone Diazepam Diazepam Phenylbutazone Phenylbutazone Phenytoin Phenytoin Quinidine Tolbutamide

Diseases

Percentage Increase in Fraction Unbound

AVH AVH AVH/C AVH/C

No change No change 15 38

AVH/C CAH/C C C C AVH/C AVH C C AVH

38 477 210 65 400 500 33 40 300 28

Note: AVH = acute viral hepatitis; C = cirrhosis; CAH = chronic active hepatitis.

Hepatic metabolism and aging is very difficult to discuss as there do not appear to be any general rules. The most likely reason for this, unlike renal function, is that there are a host of factors that influence the efficiency of drug metabolism (e.g., genetics, nutrition, drugs, disease, live size, etc.). There is huge variation among the population for hepatic clearance at any given age. It is not unusual to find a tenfold range of clearances among otherwise normal, healthy subjects at a given age. On that basis it is difficult, if not impossible, to tease out the effect of age per se on hepatic clearance (Rowland and Tozer 1995) The general impression is that hepatic drug clearance either decreases or remains unchanged with age at least for phase I metabolic processes. Although not well proven, it appears that age has less of an effect on those compounds that undergo phase II metabolism (i.e., conjugation). Impaired GI Absorption GI absorption has been claimed for many years to be impaired in the elderly. There is simply no good evidence for this and, in fact, the statement has been made because of poor experimental design and incorrect interpretation of data. There are definitely many changes in the gut that could effect absorption efficiency, including the following: • • • •

GI fluid pH GI fluid contents Gastric emptying rate Intestinal transit rate

874


• • • • •

GI blood flow GI surface area and “membrane” characteristics Nutritional intake and eating habits Age-related drug ingestion altering physiology or affecting absorption of other drugs Age-related GI disease

There is not very good information about a number of these factors. There is a greater incidence of achlorhdria (lack of acid secretion in the stomach) that could have some implications in terms of drug dissolution and drug stability, but at present this condition does not appear to be an important clinical issue. Gastric emptying rate and intestinal transit rate are expected to decrease with age; there is the general thought that gut activity slows with aging. This is difficult to conclude, however, and the data shown in table 14.10, which are the only data available, are directly conflicting. In one case there is no change and in the other the elderly empty more slowly than younger subjects. Even less is known about intestinal transit rates but, once again, the prevailing thought is that they decrease with age. Table 14.10

Two Studies That Have Examined the Influence of Age on Gastric Emptying Half-Time (in Minutes, Inversely Related to Emptying Rate) n

M

Age, Years Range

26 77

23–31 72–86

n

M

Age, Years Range

10 10

31 76

24–51 71–88

M

Liquid Meal Range

50 123

Liquid Phase M SEM 68 94

7 ns 13 ns

21–132 67–4,541

M

p < .001

Solid Phase SEM

104 105

10 ns 17 ns

Note: n = number of subjects; SEM = standard error of the mean; ns = not significantly different

One of the most interesting stories concerns the use of d-xylose for the estimation of GI absorption. That carbohydrate is often used to assess the presence of malabsorption syndromes, especially in the case of sprue. It has also been applied to the elderly and pediatric populations. Unfortunately, the data have been totally misinterpreted and this has led to the general statement about impaired absorption with aging. The basis of the method is quite simple: Ingest a 5- or 25g dose of d-xylose and collect urine for 5 hr. The amount excreted is then compared to a normal range. In reviewing the literature one can plot, from a number of different studies, the percentage dose recovered in 5 hr as a function of age. Note the lines decrease, suggesting impaired absorption with age. However, it is very difficult to explain line A, which is obtained following an IV dose of d-xylose; it also declines with age! The only explanation for this is not a decrease in absorption (after all, absorption is complete, 100%, after IV dosing) but a reduction in elimination. The latter makes sense, when we realize that, as discussed later, renal function declines with age and that we are therefore obtaining an incomplete urine collection. The general conclusion with regard to GI absorption is that the rate of absorption might decline with age but there are no differences in the extent of absorption as a function of age. The only exceptions to the latter rule are drugs that have a high hepatic extraction ratio and undergo substantial first-pass metabolism. For such drugs, absorption might increase with age as a result of reduced hepatic clearance. There are virtually no data about the influence of age on drug absorption by other routes of administration such as intramuscular, transdermal, pulmonary, and so on. Distribution might be altered as a result of changes in plasma protein concentration, changes in anatomy, and blood flow differences. Changes in plasma protein binding can result from this


875

observation, but it might also occur due to the use of many drugs by the elderly and the consequent interaction in displacing one compound by one or more other drugs. Another major change that occurs with aging is the relative body content of water and adipose tissue. In both males and females the relative percentage of weight that is adipose tissue increases with age, whereas water content (or lean body mass) declines with age. Another important issue in drug distribution onvolves blood flow. Age per se does not appear to alter cardiac output. However, among the population in which there is substantial coronary artery disease, there appears to be an inverse relationship with age. One needs to first define the question to provide a correct answer. The conclusions with regard to drug distribution and aging are as follows: • Plasma protein binding either decreases or does not change with age. • Clearance could change (increase) as a result of altered binding (low-clearance drugs). • Apparent volume of distribution will increase for lipid-soluble drugs and decreases for watersoluble drugs. • Half-life could change as a result of a change in apparent volume. • Blood flow decreases in most elderly subjects and this can alter the clearance of nonrestrictively cleared drugs.

Pregnancy There are numerous and substantial changes that occur during pregnancy and, although there is not a great deal of quantitative information with regard to drug disposition and response, several of these factors can be discussed. There is little quantitative information with regard to the effect of pregnancy on the efficiency of the GI absorption process or, for that matter, absorption by other routes of administration. Several changes in the GI tract do occur, however, including a reduction in gastric acid secretion, an increase in mucous secretion, and a slowing in gastric emptying and intestinal motility. Any of these changes can affect the drug dissolution and absorption processes, at least in theory, but as yet we do not have the data to indicate the existence of such alterations in absorption. Increased peripheral blood flow might also suggest more rapid absorption following intramuscular or subcutaneous and, perhaps, transdermal dosing. There are considerable anatomical changes that occur during pregnancy and might affect drug distribution. This increase in weight in terms of fluid volumes, blood, and fat is illustrated in table 14.11. Table 14.11

Change in Blood Distribution During Pregnancy

Parameter Blood volume (mL) RBC volume (cells/mm3) Hematocrit (%)

Late Pregnancy

Nonpregnant State

Increase (%)

4,820 1,790 37.0

3,250 1,355 41.7

48 32

Note: RBC = red blood cell. Source: Pritchard (1965).

The increased volumes of both water and fat will likely increase the apparent distribution space for water-soluble and lipid-soluble drugs. An additional consideration, however, is the distribution space afforded by the fetus (assuming the drug can traverse the placenta) and breast milk (assuming the drug can undergo mammillary transfer). The other major factor that can affect distribution volume as well as clearance (and, therefore, half-life) is plasma protein binding. As with other factors, one must consider the time course of any possible change over the normal duration of pregnancy. Serum albumin concentration declines and, for many of the drugs studies to date, plasma protein binding also decreases, resulting in a greater fraction of unbound drug.

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There are substantial changes in hemodynamic functions such as cardiac output and blood flow to different body regions, as shown in table 14.12. Table 14.12

Hemodynamilc Parameters Throughout Pregnancy Position

1st Trimester

2nd Trimester

3rd Trimester

Postpartum

L 5 L S L S L S L S L S

77 ± 2 76 ± 2 75 ± 3 82 ± 5 3.53 ± 0.21 3.76 ± 0.24 302 ± 2 301 ± 3 98 ± 2 106 ± 2 53 ± 2 57 ± 2

85 ± 2 84 ± 2 86 ± 4 85 ± 4 4.32 ± 0.22 4.19 ± 0.21 290 ± 5 286 ± 4 91 ± 2 102 ± 2 49 ± 2 60 ± 1

88 ± 2 92 ± 2 97 ± 5 87 ± 5 4.85 ± 0.27 4.54 ± 0.28 281 ± 4 260 ± 4 95 ± 2 106 ± 2 50 ± 2 65 ± 2

69 ± 2 70 ± 2 79 ± 3 79 ± 3 3.30 ± 0.17 3.33 ± 0.21 310 ± 5 307 ± 5 97 ± 2 110 ± 2 57 ± 2 65 ± 1

Heart rate (beats/min) Stroke volume ml/min) Cardiac output 1/mm/m2 Left ventricular election time (msec) Systolic blood pressure (mmHg) Diastolic blood pressure (mmHg) Note: L = lateral: S = supine.

The implications of these changes in terms of drug distribution have not been clearly delineated but the findings suggest that there would be an increase in distribution volume during pregnancy that then returns to normal sometime following birth of the child. There is a very dramatic increase in renal function as judged by estimates of glomerular filtration (e.g., creatinine and inulin clearances). This is illustrated in table 14.13. Table 14.13

Changes in Kidney Function in Pregnancy

Time

Renal Plasma Flow (ml/min)

Glomerular Filtration Rate (ml/min)

13.0 20.8 38.0 20.0 80.0

804.67 749.13 589.00 491.00 549.00

161.33 157.11 146.00 100.00 97.00

weeks pregnancy weeks pregnancy weeks pregnancy weeks postpartum wk postpartum

This increase in renal function has its direct counterpart in the renal clearance of drugs that undergo excretion by the kidney. Ampicillin, for example, is primarily excreted unchanged by the kidney and has a greater clearance in the same women during pregnancy (613 ml/min) compared to the value after giving birth (394 ml/min). Elimination half-life is also shorter and the apparent volume of distribution is larger. Findings similar to these have been made for other water-soluble antibiotics (e.g., cephalosporins) and digoxin, among other renally excreted drugs. Therefore, it is very likely that larger than usual doses of such drugs will need to be given to pregnant women to achieve the desired steady-state plasma concentration and response. As noted earlier in the discussion of age, it is far more difficult to address the issue of drug metabolism because of the large number of variables that affect hepatic metabolism and, once again, there are only a limited number of studies available. Two studies examined the difference in disposition of the drug metoprolol in the same women during and after pregnancy. These results are summarized in table 14.14. These data suggest that there is an increase in hepatic metabolic activity during pregnancy, at least for the enzyme system responsible for metoprolol metabolism. General conclusions concerning the metabolism of other drugs during pregnancy are far from being unequivocal. Metabolic clearance


Table 14.14

877

Metoprolol Absorption and Disposition Parameters in Women During and Following Pregnancy

Parameter Oral dose (100 mg) CLo (ml/min/kg) T1/2 (hr) IV dose (10 mg) CLs (L/min) V (L/Kg) T1/2 (hr) Oral dose (100 mg) CLo (L/min) F

Pregnant

Nonpregnant

362 1.27

82 1.70

1.38 6.87 5.38

0.65 3.85 5.36

9.56 0.21

1.71 0.42

Note: Studies done in 5 women during third trimester of pregnancy and 3 to 6 months after giving birth.

is affected by several factors, some of which have already been noted. First, intrinsic hepatic or metabolic clearance might change in response to, for example, hormonal alterations during pregnancy. In fact, it appears that many if not all of the changes in metabolic efficiency are related to hormonal activity, as will be noted later when we consider the influence of oral contraceptives on drug metabolism. Furthermore, the effects of hormones on drug metabolism are not always clearcut but often appear contradictory. The latter point probably reflects the fact that there are other factors, currently not well recognized or understood, that have an impact on the overall metabolic disposition of drugs. For example, an increase in testosterone concentrations increases hepatic microsomal enzyme activity, thereby enhancing the rate of hepatic metabolism of some drugs. In contrast, progesterone and estradiol can act as inhibitors of certain enzymatic processes and thus reduce the rate of metabolism. The other factors that play a role in hepatic clearance are plasma protein binding and hepatic blood flow. The former has already been discussed and, assuming a general decrease in binding during pregnancy, one would expect an increase in hepatic clearance. Liver blood flow, on the other hand, does not appear to be dramatically affected during pregnancy and, therefore, should have a minimal effect on drug metabolism. The preceding issues have important ramifications in terms of appropriate drug dosing during pregnancy to maintain the desired therapeutic response and minimize adverse, toxic effects. One can make a good argument for therapeutic drug monitoring for those drugs with a narrow therapeutic range along with careful, continued monitoring of the response to the drug. Making the foregoing issues more complicated are concerns for the fetus, which is exposed to the drugs that the mother is taking. To minimize adverse effects on the fetus, it might be necessary to select a drug that undergoes minimal placental transfer or exerts little effect on the fetus. An additional complication arising after birth is the mammillary transfer of drug and subsequent exposure of the breastfeeding infant. We have long known that there are certain cycles or rhythms that biological systems undergo that can have a dramatic affect on various aspects of the system. The term given to this area of study is chronobiology and it has received considerable interest in recent years. The menstrual cycle is a good example of chronobiology, yet its implications are poorly understood with regard to drug disposition and pharmacologic response. It has been only in very recent years that we have begun to learn of the impact that menstruation might have on the outcome of therapy. It is not clear whether or not GI absorption is altered during the menstrual cycle as this process has not been thoroughly studied. One well-designed study that examined the oral absorption of d-xylose indicates no significant differences during the follicular (days 4 and 5), ovulatory (days 16 and 17) or luteal (days 23 and 24) phases.

878


A 20% increase in creatinine clearance has been reported between the beginning and end of the menstrual cycle, which is consistent with the approximate 24% increase in d-xylose renal clearance. In contrast to these findings, however, no changes were noted in creatinine clearance and tobramycin pharmacokinetics during the corresponding phases of the menstrual cycle in another study. As a result, and because there is a lack of other basic studies, one cannot reach a general conclusion concerning the changes in renal function during the menstrual cycle. Unfortunately, but not surprisingly, similar disparities exist with regard to consideration of drug metabolism during the menstrual cycle. To date there have been at least two different studies that have examined the pharmacokinetics of the model compound antipyrine during different times of the menstrual cycle. The results of both of these studies suggest that there is no time-dependent alteration in antipyrine metabolism during the menstrual cycle. One positive finding is the results of a study that examined methaqualone pharmacokinetics during days 1 and 15 of the cycle and these results are shown in table 14.15. There is a very dramatic difference in the values for clearance and half-life and, although the mechanism(s) for this alteration is not currently known, the investigators suggest a hormonal action. Table 14.15

Pharmacokinetic Parameters of Methaqualone During the Menstrual Cycle

Parameter

Day of Cycle Day 1 Day 15

Half-life (hr) Clearance (mL/min.kg) Volume (L/kg)

16.3 1.72 2.12

11.6 3.20 2.84

Influence of Gender on Xenobiotic Absorption, Disposition, and Toxicity It has become quite clear in recent years that women’s health and health issues have not been adequately studied. As a result, we understand far less about the diagnosis of illness and its treatment in women than in men. For the same diagnosis, women are less likely than men to receive important diagnostic or therapeutic modalities (e.g., renal transplantation, cardiac catheterization). The issue, however, is not so straightforward and is complicated by several factors such as age associated with heart disease (earlier age in men than in women) and the dangers of certain therapeutic maneuvers (e.g., catheterization) at those ages. It is important to recognize that the medical treatment for women is based on a male model: The results of medical research on men are generalized to women without sufficient evidence of applicability to women. This statement is best supported by consideration of the large-scale clinical research studies that have been conducted in this country over the past several decades: • The Physicians Health Study examined the use of aspirin for coronary artery disease (22,071 male, 0 female). • The Multiple Risk Factor Intervention Trial examined the modification of risk factors to prevent heart disease (15,000 male, 0 female). • The Veterans Administration Cooperative Study showed the benefits of coronary surgery in angina patients. • The Baltimore Longitudinal Study of Aging, which has been ongoing since 1958, began to include women only since 1978.

Consider the following facts: • Women will constitute the larger population and will be the most susceptible to disease in the future (see table 14.16 and table 14.17). • Overall, women have worse health than men. • Certain health problems are more prevalent in women than in men. • Certain health problems are unique to women or affect women differently than they do men.


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Table 14.16

Life Expectancy: 1989

Total population White Black Hispanic Native Americana Asian Pacific Islanders a a

Men

Women

71.8 72.7 64.8 69.6

78.6 79.2 73.5 77.1

No information. Table 14.17

Percentage of Women Within the Aging Population

Year

65+

85+

1900 1980 1990 2020

49.5% 59.7 59.7 60.0

55.6% 69.6 72.0 73.0

The Assistant Secretary of Health established a Public Health Task Force on Women’s Health Issues in 1983. One result was the publication of Women’s Health: Report of the Public Health Service Task Force on Women's Health Issues (Vol. 1 1985; Vol. 2 1987). One of the most important recommendations of this Task Force was the recommendation that “biomedical and behavioral research should be expanded to ensure emphasis on conditions and diseases unique to, or more prevalent in, women in all age groups.” A consequence of these activities was the creation of the Office of Research on Women’s Health (ORWH) within the Office of the Director of the National Institutes of Health (NIH). Currently, consideration is being given to establishing a permanent office for the ORWH within NIH. The objectives of the ORWH are the following: • Any research supported by NIH must adequately address issues related to women’s health. • Women must be appropriately represented in any clinical research, especially clinical trials. • An increase should be fostered in the enrollment of women in biomedical research, especially in decision-making roles within clinical medicine and the research environment.

A public hearing was held in June 1991 for the purpose of determining the major needs for research into women’s health and the testimony given indicated that the following issues should receive attention (among other issues): • • • • • • • • • •

Cancer prevention (especially breast cancer) Cardiovascular disease Osteoporosis Autoimmune diseases affecting women Hormonal cycles and how these might affect absorption, disposition, action, and elimination of drugs Sexually transmitted diseases Work site safety Domestic violence AIDS Pre- and postnatal care

The ORWH has begun the largest clinical project of its kind ever undertaken in the United States, the Women’s Health Initiative. The purposes of this project are to do the following:

880


• Decrease prevalence of cardiovascular disease, cancer (especially breast cancer), and osteoporosis. • Develop recommendations on diet, hormone replacement therapy, diet supplements, and exercise. • Evaluate effectiveness of various strategies for motivating older women to adopt health-enhancing behaviors.

The project is expected to involve 150,000 women at 45 centers for up to 14 years and cost about $625 million. The health issues to be addressed will be divided according to age and processes: • Consideration of age • Birth to young adulthood (birth–15 years) • Young adulthood to perimenopausal years (15–44 years) • Perimenopausal to mature years (45–64 years) • Mature years (65+ years) • Consideration of process • Reproductive biology • Early developmental biology • Aging processes • Cardiovascular function and diseases– • Malignancy • Immune function and infectious diseases

Certain conditions are more prevalent among women (see table 14.18) than in men and these include the following. Table 14.18

Leading Causes of Death in Women

Condition

Number

Heart disease Cancer Cerebrovascular Pneumonia/influenza Chronic obstructive pulmonary disease Accidents Diabetes Atherosclerosis Septicemia Nephritis

379,754 226,960 90,758 40,828 33,191 31,279 23,393 13,759 11,793 11,512

Source: National Center for Health Statistics (1998).

• Cardiovascular disease • Stroke accounts for a greater percentage of deaths among women than in men at all stages of life. • Half of all women, but only 31 % of men, who have heart attacks die within 1 year. • About 90% of all heart disease deaths among women occur after menopause. • One in 9 women 45 to 64 years old have some clinical cardiovascular disease, increasing to 1 in 3 at age 65 and older. • Mental disorders • Rate of affective disorders (depression, etc.) is 7%, about twice the rate in men. • In elderly women depression affects about 3.6% versus 1% in men. • Alzheimer’s disease • Higher incidence among women • Osteoporosis • Affects more than 24 million Americans, primarily women (see table 14.19). • Hip fractures are the most serious consequence of osteoporosis (about 250,000/year). • Osteoporosis causes 1.3 million bone fractures per year. • 500,000 vertebrae fractures occur per year, and about one-third of women over age 65 will suffer at least one vertebral fracture.


881

Table 14.19

Incidence of Osteoporosis in Women

Age Group

% Incidence

45–49 50–54 55–59 60–64 65–69 75+

17.9 39.2 57.0 65.6 73.5 89.0

• Sexually transmitted diseases • 6 million women per year are affected (one-half are teenagers). • Fifteen to 20 million women have chronic genital herpes or human papillomavirus. • Women are the fastest growing population with AIDS. • Immunologic diseases • Autoimmune thyroid diseases have a 15:1 ratio of women to men. • Rheumatoid arthritis has a 3:1 ratio of women to men. • Systemic lupus erythematosus occurs nine times more often in women than men. • Systemic sclerosis affects women four times as often as men. • Diabetes mellitus and multiple sclerosis occur more often in women.

Certain health problems are unique to women or affect women differently than they do men. • Cancer is the second leading cause of death and the leading cause of premature death in women. • Lung cancer has exceeded breast cancer as the leading cause of death due to cancer (51,000 vs. 45,000 in 1991). • One in 9 women will develop breast cancer versus 1 in 20 in the 1960s. • Two and one-half million women acquire chlamydial genital infections annually. • One million women are treated for pelvic inflammatory disease annually. • Incidence of involuntary infertility and ectopic pregnancies have quadrupled in past decade due to sexually transmitted disease. • Due to perinatal transmission, AIDS is the leading cause of death among Hispanic children and the second leading cause of death among Black children.

The FDA restricts the inclusion of women of childbearing age in early clinical trials except for studies involved with life-threatening diseases. These restrictions are currently being reconsidered. Gender-related differences in drug disposition in nonhuman mammals, especially in metabolism, have been known for some time. There are also several impressive differences in the magnitude of response to selected drugs in male and female rats (e.g., anesthetics and barbiturates). The prevailing thought up to just a few years ago was that these differences did not apply to humans. The gender-related differences in drug metabolism in animals have been shown to depend on the specific drug, metabolizing enzyme system, age, and animal species among other variables. The differences in drug metabolism between the genders appear to be a result of hormonal differences between the sexes. The lack of unequivocal information concerning differences in drug disposition between males and females has been the result of limited studies, small numbers of participants in each study, and the influence of confounding variables. With regard to confounding variables there is often no control for or consideration given to factors such as differences in age, smoking status, use of other drugs (especially oral contraceptives, caffeine, and ethanol), time during the menstrual cycle, and diet. The latter factors might exert a profound influence on the results of any study attempting to examine and compare the disposition of a drug as a function of gender. Several reviews on this topic have appeared in the literature. Few if any studies have indicated substantial gender-related differences in the rate or extent of drug absorption, which might prove to be clinically important. The detection of such differences

882


requires intensive sampling that is seldom applied in the typical clinical study. Furthermore, the incorrect analysis of data could lead to an erroneous conclusion (e.g., comparisons of blood concentrations only, which do not take into account differences in drug clearance). There is one important and interesting exception to this general statement that might have a counterpart for certain other drugs. Women have greater blood ethanol concentrations compared to men following oral doses of ethanol and this has been ascribed to differences in body build (i.e., smaller percentage of body weight that is fat-free in women and into which ethanol distributes). Blood concentrations are similar, however, following IV dosing. Ethanol is metabolized in the stomach by the enzyme alcohol deydrogenase before it is absorbed into the systemic blood circulation. The latter is an example of GI or gastric first-pass metabolism. It appears that women have a much lower gastric alcohol dehydrogenase enzyme activity than men, which results in less metabolism in the stomach and, consequently, a greater part of the ethanol dose being absorbed into the blood stream. Women, therefore, will have a greater blood ethanol concentration than men at comparable oral doses. The approximate absolute oral bioavailability of ethanol in women was 91% compared to 61% for men. There are examples of other drugs that undergo some form of GI metabolism prior to absorption that might be prone to the same effect noted for ethanol. For example, L-DOPA is inactivated in the stomach by a decarboxylase enzyme. Does the activity of this enzyme vary between the genders? Another potential gender-related difference in oral bioavailability could involve hepatic first-pass metabolism. The concept is identical to that noted earlier for gastric metabolism, with the only difference being the site of metabolism. What determines the significance of the hepatic first-pass effect is the hepatic clearance value of the drug; the greater the clearance, the greater the first-pass effect and the lower the systemic oral bioavailability. Therefore, if there are gender-related differences in the hepatic clearance of high-clearance drugs, one would expect there to be a corresponding difference in GI absorption or bioavailability. Although there is currently little information on which to make a conclusion, there are suggestions that hepatic clearance of certain drugs differs between the genders (discussed later ). Drug distribution throughout the body depends on several factors, including plasma protein and tissue binding and body build with regard to relative amounts of adipose and fat-free tissues. These factors, in turn, determine the apparent volume of distribution of a drug that affects the elimination half-life (i.e., half-life increases as the apparent volume of distribution increases, assuming that elimination clearance of the drug remains the same). Plasma protein binding might also affect the clearance of a drug, depending on the nature of the clearing process. The current literature with regard to plasma protein binding differences between the genders is conflicting and inconclusive. Some studies suggest a lower binding in women compared to men for certain highly bound drugs and other studies suggest no differences for the same drugs. The reasons for this disparity include the inclusion of few subjects and the lack of control for a number of variables that might alter binding (e.g., plasma protein concentration, age, health and nutritional status, other drugs, smoking, etc.). As a result no definitive conclusion can be reached, but the general impression is that differences, if they exist, are not dramatic. There are substantial differences between the genders, however, with regard to body build that are further magnified with age (as discussed later). The major difference here is the fact that women have a greater percentage of body weight that is adipose tissue compared to men; conversely, women have a smaller percentage of body weight that is fat-free or that contains water. Therefore, a lipid-soluble drug (e.g., thiopental) will have a larger apparent volume of distribution (i.e., it will occupy a larger space) in women compared to men of the same age on a body weight basis (e.g., volume/kg). In contrast, a water-soluble drug (e.g., ethanol) will have a smaller volume of distribution in women compared to men of the same age. These differences can become important when administering a loading dose of a drug and in terms of the elimination half-life. This is the most difficult topic to discuss rigorously because there is virtually no consensus about how gender affects drug metabolism in general, although specific drug examples can be


883

discussed. The reason for this dilemma is the fact that there have been far too few adequate research studies that have employed a sufficient number of participants and the fact that there are a host of variables, in addition to gender, that can affect drug metabolism. A brief listing of those factors would include consideration of: genetics, age, health status, nutritional status, smoking status, and the use of other drugs (including ethanol, caffeine, and drugs of abuse). Furthermore, when considering gender per se, one must also control for the time of the menstrual cycle and the possibility of pregnancy in addition to the use of oral contraceptives. Clearly, it is very difficult to factor out gender differences as being responsible for any observed differences in metabolism when there are so many other variables. Exquisite care must be given to the control of all aspects of such a comparative study to obtain statistically valid conclusions. Furthermore, it would be far more instructive to obtain estimates of metabolic (or hepatic) clearance of a drug compared to elimination half-life, because the former is an adequate measure of the inherent ability of the liver to metabolize drug, whereas the latter reflects clearance as well as volume. Unfortunately, not all investigations recognize this difference and they report half-life more often than clearance. The relationship among these three parameters is presented in this equation. T1/2 =

0.693 × Volume Clearance

Differences in clearance as a function of gender must also consider the possibility of differences in plasma protein binding that could affect the clearance of certain drugs (those with low clearance values). A further complication in comparisons of clearance values is whether or not the values are adjusted for body weight, as this normalized value is usually greater in women by virtue of their weighing less than men. At this time it is almost counterproductive to list the results of the several studies that have attempted to compare metabolic drug efficiency in men and women, because the results of one study are often diametrically opposite the findings from another. This is the case, for example, for many benzodiazepine derivatives that undergo oxidative (phase I) biotransformation (e.g., diazepam, chlordiazepoxide, etc.) and the frequently used model or test compound, antipyrine. One recent study examined the role of gender in propranolol kinetics subsequent to an earlier report that suggested that females have higher concentrations of the drug compared to males after long-term oral dosing following a myocardial infarct. The investigators report that the oral clearance of propranolol is greater in males (leading to a greater first-pass effect, as discussed earlier). Females, therefore, have greater plasma concentrations of the drug compared to males, supporting the preliminary observation already noted. Complicating this finding, however, is the fact that whereas metabolic clearance was greater for two pathways (a side-chain oxidation and glucuronidation), it was not different for at least one other pathway (ring hydroxylation). Further complicating this issue are the recently discovered gender- and age-specific enantiomer-selective metabolic differences. The considerable recent interest in drug enantiomers (on the basis of different pharmacologic and pharmacokinetic behaviors) has lead to a greater ability to assay these different stereoisomers in biological fluid with the resulting greater understanding of what factors influence the disposition of those forms. One recent study, for example, indicates that there is an age-dependent gender effect as well as a gender-dependent age effect in the metabolism of the R-enantiomer of mephobarbital. In contrast, little difference exists in the metabolism of the S-enantiomer.. Although we have been aware of the age dependence in the metabolism of enantiomers (e.g., hexobarbital), this is the first example of a gender dependence. Undoubtedly, other examples will be forthcoming. In contrast to the difficulty in reaching any unequivocal conclusions about gender-related differences in phase I metabolic processes, one investigator concludes that there is a more consistent

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gender-related trend in the metabolic clearance of those benzodiazepine derivatives that undergo conjugation reactions (phase II). A comparison among several of those drugs is shown in table 14.20. Table 14.20

Drug

Clearance of Selected Benzodiazepine Derivatives Undergoing Phase II Conjugation Reactions in Normal, Young Males and Females Clearance, ml/min Male Female

Lorazepam Oxazepam Temazepam a

77 88 97

55 50a 68a

Significantly different from male value.

These data, however, remain far too limited to allow us to reach any valid general conclusions concerning consistent differences between the sexes for the metabolism of those drugs that undergo phase II metabolism. For that reason recommendations cannot be made with regard to the need to alter dosing regimens as a function of gender. It is quite clear that there is a need for additional studies to clarify the influence of gender on drug metabolism. As alluded to earlier and noted later, the effect of gender is further complicated by consideration of age. In addition to metabolism, urinary excretion is another major route of drug elimination from the body. Unlike metabolic processes, there are fewer variables that affect renal excretion of drugs (e.g., urine flow, urine pH). As a result, the renal clearance of a drug is reasonably consistent among people with similar kidney function. Renal clearance is often estimated by measurement of creatinine clearance or, more often, by serum creatinine concentration. The latter, however, can be quite misleading because factors such as age and body build affect the relationship between concentration and clearance. The following useful relationship between CLCr and Scr has been determined from many male subjects: CL CR (ml / min)MALE =

(ideal body weight, kg )(144-aage, yr ) 72 × SCR (mg%)

The corresponding equation for females requires multiplication by a factor of 0.86. The latter correction is a result of the fact that creatinine is an end product of muscle metabolism and females have smaller muscle mass compared to males. This relationship is rewritten here. Therefore, at equal body weight, age, and serum creatinine concentration the male will have a greater creatinine clearance and, presumably, more efficient kidney function and a greater ability to excrete drugs. To the best of this writer’s knowledge, surprisingly, there has not been a systematic investigation of gender-related differences in renal function and renal excretion of drugs. CL CR (ml / min)FEMALE =

0.86(ideal body weight, kg )(144-age, yr) 72 × SCR (mg%)

Figure 14.1 is a plot of the percentage function remaining for several body functions versus age. Note that this is a linear scale indicating that the decline in function occurs at a constant rate (also note that there is no precipitous drop at age 65 years). The reasons for considering the elderly as a special segment of the population and some of the complications in conducting gerontologic research are outlined in table 14.21.


885

100

% Function remaining

90

Maximal breathing capacity

80

Renal plasma flow Vital capacity

70

Glomerular filtration rate 60

Cardiac index

50 40 30 20

30

40

50

60

70

80

90

Age (years) Figure 14.1

Plot of the percentage function remaining for a variety of body functions versus age. (Mayersohn 1992).

Table 14.21

Characteristics of the Elderly That Warrant Their Being Considered as a Special Segment of the General Population

Consideration

Characteristics

Population

The elderly (i.e., those older than age 65) currently represent about 12% of the U.S. population; this percentage increased to about 16% by year 2005. The elderly experience a greater incidence of disease, physical impairments, and physiological disorders than do younger adults. The elderly occupy a greater share of hospital beds (≈ 33%) and long-term care facilities than do younger adults. The elderly consume more drugs (≈ 25% of total use) per capita than do younger adults. The elderly experience a greater incidence of adverse drug effects and drug interactions than do younger adults. Complication Chronological versus biological age Continuum over years versus arbitrary definition of elderly Longitudinal design (age changes) versus cross-sectional design (age differences) Chronic or acute illness versus good health; institutionalization versus living at home Acute or chronic drug therapy versus no drug use Good versus poor nutrition Smoking versus not smoking, prior environmental exposure of elderly when young versus current exposure in young

Health Institutionalization Drug use Drug effects Factor Age: Definition Age: Comparisons Age: Changes Health status Drug therapy Nutritional status Environment

Source: Mayersohn (1992).

Microbiological Definition Animals, like humans, suffer from infectious diseases ranging from those causing only mild symptoms to acute disease outbreaks with high mortality. Although some of these diseases can be partially controlled by medication, both the disease and the medication could seriously interfere with experimental work. Fortunately, most of the infectious diseases of laboratory animals can be controlled simultaneously by the use of SPF techniques (Bleby 1976).

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Stocks of SPF animals were initially developed by hysterectomy of the pregnant females just prior to parturition using aseptic surgical techniques. The young were resuscitated in a building designed to prevent the entry of disease-causing organisms, or in closed isolators, and then handreared using a sterile milk substitute. This procedure immediately eliminates a wide range of pathogenic organisms that are normally transmitted from mother to offspring because young within the uterus are usually microbially sterile. Such SPF animals are normally free of all parasites, many viruses, and most pathogenic bacteria. Once established, such colonies breed well and can be used to supply high-quality breeding stock that does not carry the risk of introducing disease into an existing colony. Commercial breeders supply SPF mice, rats, guinea pigs, rabbits, and cats. Although some other species have been derived into SPF conditions, they are not freely available at present. Such SPF animals have (or offer) the several advantages. A colony of animals carrying infectious pathogenic organisms is likely to be more variable than a group of SPF animals, and therefore more animals will be needed to achieve the same degree of statistical precision. For example, it has been shown that underweight mice carried five times as many parasitic nematodes as their normal-weight littermates (Eaton 1972). Although in this case cause and effect cannot be separated, it seems reasonable to assume that such an uneven parasite burden will lead to increased variability among the experimental animals. This means that for the same statistical precision, more “parasitized” animals might be needed than if the animal were free of such parasites. In conventional animals, there is the danger that the effects of disease might be mistaken for the actions of the experimental treatment. Vitamin A deficiency, for example, has been recorded as causing pneumonia and lung abscesses in rats, when in fact all that the deficiency is doing is increasing the severity of the infectious chronic respiratory disease found in all non-SPF rats (Lindsey et al. 1971). Pneumonia does not occur in SPF rats that are vitamin A deficient. The activation of latent disease through the experimental treatment can be extremely misleading as the control animals not subjected to the same degree of stress could be unaffected (Baker et al. 1971). In some cases, mild infections might mask the results of an experimental treatment. For example, rats are widely used in inhalation toxicology, yet the lesions of chronic respiratory disease could completely obscure the effects of the experimental treatment. In one case, chlorine gas caused lung lesions, but as the animals grew older the differences between the treated and control groups were completely obscured by the chronic respiratory disease, so that the two groups became histologically indistinguishable (Elmes and Bell 1963). Many long-term studies require a substantial number of animals to reach old age. As fewer SPF animals die as a result of infectious disease, fewer animals need to be started in each experimental group (Lindsey et al. 1971). Thus, for long-term studies SPF animals might be substantially more economical than conventional ones. A sidelight of this is that the use of SPF animals has advanced survivability to the point that consideration has been given to lengthening some study types (e.g., carcinogenicity). Genetic Definition Laboratory animals should also be genetically defined. Behavior; response to drugs; size, weight, and shape of many organs; numbers and types of spontaneous tumors; and response to antigens depends not only on the species, but also on the strain of animal (Festing 1979b). Inbred strains of mice, rats, hamsters, and guinea pigs produced as a result of at least 20 generations of brother–sister mating are readily available. They are much better experimental subjects than the more widely used outbred “white” mice and rats for most studies. Strong (1942) wrote that: It is the conviction of many geneticists that the use of the inbred mouse in cancer research has made possible many contributions of a fundamental nature that would not have been otherwise. Perhaps it would not be out of place to make the suggestion that within the near future all research on mice should


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be carried out on inbred animals or on hybrid mice of known (genetically controlled) origin where the degree of biological variability has been carefully controlled.

Gruneberg (1952) even went so far as to state that, “The introduction of inbred strains into biology is probably comparable with that of the analytical balance into chemistry.” The main characteristics of genetically defined inbred strains are as follows: 1. All individuals of a strain are genetically identical (isogenic). The genetic uniformity of inbred strains means that each strain can be genetically typed for characteristics such as their blood group in the knowledge that all animals within that strain will be the same. Such data are essential in many immunological and cancer research studies, and cannot be gathered in outbred stocks, where each individual is genetically unique. Isogenicity also leads to phenotypic uniformity for all highly inherited characters, and this means that the statistical precision of an experiment using these animals is increased. 2. Inbred strains are genetically stable. Once a strain has been developed it stays genetically constant for many years. Noninbred strains might change as a result of selective forces, but such forces cannot act on inbred strains, which can only change as a result of the accumulation of mutations, a slow process. This stability means that background data on strain characteristics remains constant for long periods—allowing for the use of such information in planning experiments. 3. Inbred strains are internationally distributed. This means that experiments conducted on some of the more common inbred strains, which are maintained in laboratories throughout the world, can easily be confirmed at laboratories in entirely different parts of the world. Moreover, if many laboratories are working with the same strain, background data on the strain are accumulated much faster. 4. Each strain has a unique set of characteristics that could be of value in research. In some cases, a strain might have a disease that in some way models a similar condition in humans. The best known of such models are the strains with a high incidence of a particular type of cancer. The inbred mouse strain C3H develops a very high incidence of breast tumor, strains AKR and C58 develop leukemia, SJL develops reticulum-cell sarcoma (Hodgkin’s disease), and some sublines of strain 129 develop teratomas. Other strains develop autoimmune anemia (NZB), amyloidosis (YBR and SJL), congenital cleft palate (A and CL), hypertension and heart defects (BALB/c and DBA/2 mice, and SHR and GH rats), obesity and diabetes (NZO, PBB, and KK mice), and even a preference for alcohol when given a free choice of 10% alcohol or plain water (strain C57BL mice).

Each of these strains can be studied to obtain a better understanding of the disease in the mouse or rat. Once it is understood in the animal, it will be easier to understand in the human, even though it is unlikely that the conditions are exactly comparable in animals and humans. In fact, it is clear from study of a disease such as hypertension in the rat that the cause of the hypertension in SHR and GH is entirely different (Simpson et al. 1973), emphasizing that diseases of this sort in humans can have several different causes. Obviously, in such cases, some animal models might mimic a human disease relatively closely, whereas in other cases there is little resemblance. Table 14.22 lists some examples of the models of disease that can be found among inbred strains of mice. Inbred stains do not need to model any human disease to be of value in search. Strains can usually be found to differ for almost every characteristic studied, including many aspects of behavior, response to a wide range of drugs and chemicals, response to antigens, response to infectious agents, incidence of spontaneous diseases, and even anatomical features. These differences can be of great value in research in a number of different ways. At the most trivial level, if a scientist is studying a response to some treatment effect, it is often possible for him or her to find, by surveying a number of inbred strains, a strain that is highly sensitive to his or her experimental treatment. In some cases this will mean that fewer animals are needed to achieve the same degree of statistical precision in future experiments. In other cases, the more sensitive strain might show the effect sooner than resistant strains, and this could reduce the time and facilities needed to complete the experiment.

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Table 14.22

Examples of Disease Models and Characteristics of Interest in Inbred Strains of Mice

Character

Strain(s)

Alcohol (10%) preference Aggression/fighting Audiogenic seizures Autoimmune anaekia Amyloidosis Cleft palate Chediak-Higashi syndrome Hypertension and/or heart defects Hyperprolinaemia and prolinuria Obesity and/or diabetes Osteoarthropathy of knee joints Polydipsia Resistance to myxovirus infection Tumors Leukemia Reticulum-cell sarcoma (Hodgkin’s disease) Lung tumors Hepatomas Mammary tumors Ovarian teratomas Induced plasmacytomas Testicular texatomas Complete absence of spontaneous tumors Whisker eating

C57BL, C57BL, C57BR/cd SJL, NZW DBA/2 NZB YBR, SJL CL, A SB BALB/c, DBA/1, DBA/2 PRO NZO, PBB, KK, AY STR/1 SWR, SWV A2G AKR, C58, PL, RF SJL A C3Hf C2H, C2HA-Avy, GRS/A, RIII LT BALB/c, NZB B129/terSV X/Gf A2G

Source: From Festing (1978).

At a slightly more sophisticated level, a comparison of sensitive and resistant strains could give extremely valuable information about the mechanism of some treatment effect. For example, if two strains differ in sensitivity to a drug, it would be of great interest to know whether this is because of differences in absorption, metabolism, excretion, or target organ sensitivity. Such a study could give information that would be extremely useful in evaluating the likely effect of the drug in humans. Preferably, such studies should be carried out on two or more sensitive and two or more resistant strains to show whether the results are uniquely strain dependent, or can be generalized. Any two inbred strains will normally differ from each other at several thousand different genetic loci. However, sets of inbred strains that differ from one another at only one or a few loci have been developed to study in greater detail those loci that are of particular importance in biomedical research. These are known as sets of congenic strains, and most of them have been developed to study the major histocompatibility complex (MHC). This complex locus is responsible for a range of immunological reactions, including immune responses and graft rejection. Obviously, if two strains can be developed that are genetically identical apart from the MHC, it becomes possible to study the MHC in detail simply by comparing the two strains. Such strains can be developed either as a result of a fortuitous mutation within an inbred strain, or by deliberate breeding using conventional genetic backcrossing techniques. Several hundred strains of this type have been developed, and they are not widely used in immunology and cancer research. They have undoubtedly given much insight into the biology of the mouse MHC, which in many respects is very similar to the MHC in humans. There are more than 500 known mutants and variants in the mouse, and a further 100 in the rat, although in the rat many of these have now been lost. Some of these mutants appear to mimic similar mutants in several species, including humans, and can therefore be regarded as “models” of human disease. Other mutants lack an organ such as the thymus, spleen, tail, or eyes, or they suffer from some hormone deficiency or a developmental defect. Such mutants can be extremely valuable for certain types of research even though they might not resemble any human condition. A list of some of these mutants, classified into


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models of disease, a genetic alterations and deficiencies, and biochemical and immunological polymorphisms is given in table 14.23. There are, for example, a number of types of genetically determined obesity and diabetes that have been extremely useful as models of similar conditions in humans Table 14.23

Examples of Mouse Mutants of Medical Interest and of Biochemical and Immunological Polymorphisms

Models of Disease Anemia

Chediak-Higashi syndrome Diabetes and obesity

Inborn errors of metabolism Kidney disease Muscular dystrophy Neuromuscular mutants

Genetic alterations or deficiencies Embryonic defects Hair absent

Hair and thymus absent Growth hormone absent Resistance to androgen Sex reversal Spleen absent

Mouse Mutants of Biomedical Interest Gene Name sla Sl W bg Ay Avy db dbab ob his pro kd dy dy2j jp med qk Swl Tr

Sex-linked anemia Steel Dominant spotting Beige Yellow Viable yellow Diabetes Adipose Obese Histidinaemia Prolinemia Kidney disease Dystrophia-muscularis Dystrophia-muscularis-2J Jimpy Motor and plate disease Quaking Sprawling Trembling

t-allets hr hrrh N nu dw Tfm Sxr Dh

Tailless alleles Hairless Rhino Naked Nude Dwarf Testicular feminization Sex reversal Dominant hemimelia

Biomedical and immunological polymorphisms Polymorphism

Gene Locus

Aromatic hydrocarbon Pancreatic β-D-Galactosidase activity Liver catalase Erythrocyte antigens Esterases (serum and kidney) Friend virus susceptibility G-6-PD regulators Hemoglobin alpha chain Hemolytic complement Histocompatibility Immunoglobulin Macrophage antigen-1 Major urinary protein Phosphoglucomutase Sex-limited protein Thymus cell antigen-1 Thymus leukemia antigen

Ahh Amy-2 Bgs Ce-1 Ea-1 to Ea-7 Es-1 to Ds-7 Fv-1, Fv-2 Gdr-1, Gdr-2 Hba He H-1 to H-38 Ig-1 to Ig-4 Mph-1 MUP-1 Pgm-1, Pgm-2 Slp Thy-1 Tla

Source: From Festing (1978).

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(Festing 1979a). Such animals help to show the immense complexity of the regulation of body fat via the hormonal control of a range of metabolic interactions, each of which can be controlled by regulatory mechanisms that interact with one another. Thus, the finding of a particular biochemical abnormality is no guarantee that it is the cause of the observed obesity. It is much more likely that it is a secondary effect of the primary genetic defect. However, although many of these models of obesity might have no exact counterpart in humans, they can still be useful in screening drugs with a potential for reducing obesity (Cawthorne 1979). One of the most important mutants causing genetic alterations or deficiencies is the athymic nude mutation in the mouse. A similar mutation has now been described in the rat (Festing 1978). The thymus is essential for the full development of the immune system, and homozygous nude mice or rats are deficient in the cell-mediated type of immune response. They are of value in fundamental studies of immune mechanisms as well as in applied cancer research. This is because, lacking the cell-mediated immune response, they are unable to reject transplanted foreign tissue, including transplants of human tumors. Such transplanted human tumors usually grow, but they retain all the characteristics of human tissue. Therefore, it is possible not only to study human tumors when they are growing in an animal, but it is also possible to study the effect of drugs on such tumors. This is obviously of more value than having to rely simply on the study of animal tumors in animals. The interest in mutant “knockout,” and transgenic animals has blossomed since the first edition of this book. This probably follows the successful development of the nude mouse as a research model. Environmental And Nutritional Condition The need to house the defined laboratory animal in defined and stabilized environmental conditions, with a nutritionally adequate and controlled diet, is now becoming recognized. Both diet and environment can drastically alter the physiology of the animal and its response to drugs and other experimental treatments. Moreover, animals obtained from a commercial breeder could well take 2 or more weeks to acclimatize to their new environment. During this period their physiological responses might be unpredictable, depending on the difference between the two environments (Grant et al. 1971). Susceptibility Factors With all the effort (and reasons behind it) that goes into obtaining a “defined” test animal with a relatively narrow range of variation in responses, what then are the components or factors that lead some animals to be more sensitive to the toxicity of agents than others? Consideration of the problem shows that susceptibility factors fall into two large groups—intrinsic and external. The intrinsic factors include sex, stress, age, disease, physiological state (all of which were discussed in detail in the previous chapter), species variations, and strain and animal variations (biological variation). External or environmental factors, meanwhile, include temperature, humidity, light, and time of day. Summary If the human population we are concerned about is such that one or more of these susceptibility factors is present in a substantial portion of the members, steps should be taken to design studies so that such individuals are adequately represented by an appropriate model in the test animal population. Barring that, or in the face of having existing data on studies performed in a standard manner, consideration should be given to these factors when attempting to predict outcome of exposures in people.


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SPECIES PECULIARITIES There are a number of quirks associated with various common species of laboratory animals used in toxicology. Many of these are not well presented in the toxicology literature, although Oser (1981) has done his best to overview problems specific to the rat and Gralla (1986) has published a review of eight species-specific responses to toxicants (a modified form of which is presented in table 14.24). Most of these peculiarities hold at least the potential to impact study design and interpretation. The ones presented here are those that the authors believe should be considered in model selection for acute studies. Table 14.24

Species-Specific Toxic Effects

Type of Toxicity

Structure

Sensitive Species

Mechanism of Toxicity

Ocular Ocular Stimulated basal metabolism Porphyria

Retina Retina Thyroid

Dog Any with pigmented retinas Dog

Zinc chelation Melanin binding Competition or binding

Liver

Estrogen-enhanced sensitivity

Kidney Kidney and bladder Fetus

Human rat, guinea pig, mouse, and rabbit Rats (male) Rats and mice Rats and mice

Heart

Rabbits

Tubular necrosis Urolithiasis Teratogenesis; fetal mortality Cardiovascular

Androgen-enhanced sensitivitya Uricase inhibition Uricase inhibition Sensitivity to microvascular constriction

a

More sensitive than humans for many agents (e.g., caprolactam and halogenated solvents). Source: Adapted from Gralla (1986) with modification.

Species Variation Although anyone who has had to work in biological research with intact animals should be aware of the existence of wide variability between species, examples specific to toxicology should be pointed out along with a comparison of species sensitivities for a number of specific agents. The rodenticide zinc phosphide is dependent on the release of phosphine by hydrochloric acid in the stomach for its activation and efficacy (Johnson and Voss 1952). As a result, dogs and cats are considerably less sensitive than rats and rabbits, as the former species secrete gastric hydrochloric acid intermittently, whereas the latter secrete it almost continuously. That this case is not a rare one can be quickly established by examining some data sets in which we have comparative oral lethality data on several species (including humans), such as those presented in table 14.25 Table 14.25

Comparative Human Acute Lethal Doses and Animals LD50s (mg/kg via Oral Route)

Chemical Aminopyrine Aniline Amytal Boric acid Caffeine Carbofuran Carbon tetrachloride Cycloheximide Lindane Fenoflurazole

Human LDLO

Mouse

220 360 43 640 192 11 43

358 300 345 3,450 620 2 12,800 133

840

Note: LDLO = lowest observed lethal dose.

1,600

LD50 Values Rat Rabbit 685 440 560 2,660 192 5 2,800 3 125 283

160

Dog 150 195

575 224

140 19

6,380 130 28

120 50

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There are numerous additional examples of such differences in pharmacology (Tedeschi and Tedeschi 1968). However, just as important as these variations in general patterns or effect are the species-specific responses that are associated with the commonly employed animal models. Rat The rat is commonly accepted as the best animal model in toxicology, the closest to our ideal (Oser 1981). Table 14.26 presents a list of some of the commonly known advantages and disadvantages of the rat as a model for humans. Table 14.26

Advantages and Disadvantages of the Rat as an Experimental Model for Humans

Advantages

Disadvantages

Commonly used Small size Minimal housing space Prolific Short gestation Short lactation Omnivorous Dry diet acceptable Dosing by multiple routes Inexpensive Low maintenance cost Docile Intelligence

Anatomic Lack of gallbladder Yolk-sac placenta Multiple mammae over body surface No emetic reflex Fur covered Thinner stratum corneum No bronchial glands Physiological Estrus and menstrual cycles Multiparous Hematology Obligatory nose breather Concentrated urine Limited hypersensitivity response Metabolic Purines to allantoin Clinical chemistry Enzymatic biotransformation High β-glucuronidase activity Nutritional Mineral requirements Vitamin requirements Ascorbic acid biosynthesis Histidine biosynthesis Behavior Nocturnal Coprophagy Cannibalism Maintenance requirements Temperature and humidity control Noise control

Calabrese (1983) has published a good comparative review of the rat as a model for humans across a wide range of toxicological and biological parameters, and should be consulted for details on these. Mice Mice share many of the same advantages and disadvantages of the rats as a model for humans, such as an inability to vomit (i.e., no emetic response). Additionally, their small size and high metabolic rate cause the extent of many toxic effects to be exaggerated as homeostatic mechanisms are “overshot.”


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Guinea Pigs The systemic immune response in the guinea pig is exaggerated. As a result, although for many immune parameters it is the best common model for humans, the animal is subject to exaggerated respiratory and cardiovascular expressions of immunologically evoked events. Rabbits There are no SPF rabbits currently commercially available. Rather, the animals tend not to be as homogeneous or as high quality as the other common laboratory animal species. Indeed, they tend to harbor a wider range of subclinical infections that show a seasonal variation in their degree of expression (animals with a visible disease problem are more common in the spring and fall), and the stress of experimentation can cause these subclinical infections to be expressed. Also, the alterations in dermal vascular flow that accompany the changes in phases of hair growth cause marked alterations in percutaneous absorption and in so doing might alter many dermally related responses to chemicals. Dog The dog is currently the first-choice nonrodent model for toxicity studies. It is generally very cooperative. The major physiological peculiarity it has that affects toxicity testing is the ease with which it is provoked to vomit. This makes oral dosing at best impractical, and at worst impossible in the case of many compounds, even if the material is encapsulated or given in diet. Considerations of Strain Thus far we have focused on the differences between the different species of common laboratory animals and on how this should influence our choice of a model. However, for each of the two species that are used the most in toxicology (the rat and mouse), there is the additional level of complexity caused by differences between strains. There are three different genetic categories of strains of rodents used in toxicological research: random bred, inbred and F1 hybrids (or outbred). Random bred animals are produced in large colonies where mating occurs randomly among males and females from unrelated litters. Commercially performed random breeding should not be unplanned, but rather occur in such a manner as to minimize inbreeding. Inbred animals are the result of sister–brother or parent–offspring matings. Twenty or more generations of sister–brother or parent–offspring mating are necessary to establish an inbred strain. Outbred animals are the results of matings between two inbred strains and are usually more vigorous than either of the parental strains. Animals within an inbred or outbred strain are essentially identical genetically, serving to remove a significant source of biological variability. Strains also exist of the other laboratory animal species, but are generally neither as rigorously defined or of as great a concern, and these have been less studied as a source of variation within toxicity studies. That strain differences within species are a source of significant and broad differences in results has now been well established, in many cases with varying degrees of knowledge of the underlying mechanistic basis. The resistance of some strains of rabbits to atropine is believed to be due to the hydrolysis of the drug by atropine sterase, controlled by the gene A8, belonging to the group containing the gene that governs black pigmentation. As a result, resistance to atropine and black pigmentation are often associated (Sawin and Glick 1943). Likewise, some strains of rabbits possess a pseudococaine esterase that makes their insensitivity to this drug extreme. There are also varieties within strains. These result from various factors that in total are labeled genetic drift, and can lead to significant differences in response to toxicants. An example is the

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resistance that some wild rats have developed to the anticoagulant rodenticides (Gratz 1973; Zimmermann and Matschiner 1974), requiring that new forms of rodenticides be developed. Strain variations in response to biologically active agents arise from the same general mechanistic differences as do species differences. Although these (Hilado and Furst 1978) pharmacogenetics have been the subject of numerous reviews (Hathway 1970; Kalow 1962, 1965; Lang and Vesell 1976; Meier 1963a, 1963b; Moore 1972; Vesell 1969), it is still the case that few investigators have studied the mechanisms of variation in higher animals (Becker 1962). The differences in handling characteristics have generally not appeared in the literature before. Biological Variation There are also individual animal-to-animal variations in temperature, health, and sensitivity to toxicities that are recognized and expected by experienced animal researchers, but are only broadly understood. The resulting differences in response are generally accredited to individual biological variation. This same phenomenon has been widely studied and observed among humans, and is expected by an experienced clinician. Examples of such individual variations in human include isoniazid, succinylcholine, and glucose-6-phosphate levels or activities. In the first of these, slow inactivators who are deficient in acetyltransferase, and therefore acetylate agents such as isoniazid only slowly, and are thus more liable to suffer from the peripheral neuropathy caused by an accumulation of isoniazid. At the same time, people with more effective acetyltransferase require larger doses of isoniazid to benefit from its therapeutic effects, but in so doing are more likely to suffer liver damage. Likewise, individuals with low levels of serum cholinesterase might exhibit prolonged muscle relaxation and apnea following an injection of a standard dose of the muscle relaxant succinylcholine, and glucose-6-phosphate dehydrogenase deficiency is responsible for the increased probability of some individuals given primaquine or antipyrine to suffer from a hemolytic anemia.

ENVIRONMENTAL FACTORS Temperature Changes in temperature can alter the toxicity of a compound. As examples, at ambient temperatures colchicine and digitalis are more lethal to the rat than to the frog. However, the sensitivity of the frog can be increased by raising the environmental temperature of the two species. The duration of response also decreases as the temperature is raised, suggesting that a temperaturedependent biotransformation of these compounds is involved. Temperature can include both the background environmental temperature and the internal, physiologically regulated temperature of the animal itself. Many chemicals can profoundly alter body temperature to the acceleration of reaction rates, but rather were due to alterations in the rates of physical factors, such as the absorption rate. Keplinger et al. (1959) investigated the toxicity of 58 chemicals under different ambient conditions, including temperature. He found that many of the patterns of acute toxicity response were biphasic relative to ambient temperature, with some temperature in the ambient range being associated with a peak sensitivity in many cases. Humidity Selisko et al. (1963) investigated the effects of a number of environmental factors on the acute IP toxicity of nicotine to mice and found only humidity to have a significant influence. Humidity


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does not have a marked influence on absorption through the skin except at the extreme limits of its range (Neely et al. 1967), and the relationship between humidity and transdermal water loss in sweating animal species is not linear (Grice et al. 1972). The relationships in nonsweating species (which include all of our common laboratory species) is even more complex (Neely et al. 1967). The physiological status of the test animal in terms of hydration can markedly influence its response to toxicants. Muller and Vernikos-Danellis (1968) showed that the LD50s of caffeine and dextroamphetamine in mice were markedly affected by both ambient temperature and the animals’ hydration, with caffeine showing a large potentiation of toxicity at 30°C, whereas dextroamphetamine showed much less change. At lower temperatures (22°C and 15°C) the acute toxicity of both compounds was much less influenced by hydration (Cremer and Bligh 1969). Environmental temperature and humidity are generally closely related and as such have frequently been considered together (Lang and Vesell 1976). Understanding the basis for the temperature dependence of many of the actions of biologically active compounds has been an area of significant progress over the last 20 years. Belehradek (1957) successfully combined his own and other investigators’ research to produce a unified theory of cellular rate processes based on an analysis of the actions of temperature. He concluded that the rate of biological processes is primarily dependent on the resistance of cellular matter to the free movement of molecules within the cells rather than the rate of actual chemical reactions themselves. He was enthusiastic about the relationship between the rate responses of cellular systems and Slotte’s temperature–viscosity relationship formula. Brody (1964) has, however, reviewed the applicability of this last to vertebrate animals and pointed out its shortcomings. The relationship between responses to toxicants and ambient temperature in animals is sometimes paradoxical. Usinger (1957) investigated this in mice and found a series of biphasic relationships with optimal, or peak, ranges. Mean oxygen consumption per unit of body weight diminishes as temperature increases further. Likewise, he measured the rectal temperatures occurring when the ambient temperature was 25°C, increasing on either side of this temperature. Ahdaya et al. (1976) investigated thermoregulation in mice exposed to parathion, carbaryl, and DDT at temperatures of 1°C, 27°C, and 38°C. All three of the pesticides were found to be least toxic at 27°C. Doull (1972) has reviewed these temperature-dependent responses for many chemicals and presented the hypothesis that temperature is directly correlated with the magnitude and inversely correlated with the duration of the biological response to biologically active xenobiotics in many organisms. Although this temperature dependence stands as a general rule, there are a number of special case exceptions. It is also clear that the effect of temperature on one response variable might not necessarily be predictive of effects on other biological response variables. Baetjer and Smith (1956) found that the onset of death, rate of dying, and rate of recovery due to parathion in mice were more rapid, whereas the mortality was higher at 35.6°C than at 22.8°C. At 15.5°C, the onset of death was delayed and the total mortality was greater than at 22.8°C. They also investigated the influence of both pre- and postexposure temperatures on the response of the mice and determined that mortality varied directly with the pre- and inversely with the postexposure ambient temperatures. Their conclusion was that the results could not be attributed. Barometric Pressure Interest in the effect of atmospheric pressure on the toxicity of chemicals is fairly recent, arising from human activities in space and deep sea diving vessels. At high altitudes, the toxicity of digitalis and strychnine are decreased, whereas that of amphetamine is increased. The influence of atmospheric pressure seems to be mainly (but not entirely) attributable to altered physiological oxygen tension rather than a direct pressure effect (Brown 1980). Recently this interest has taken a new turn as concern as to the possible hazard of fires and atmospheric contaminants on submarines has surfaced.

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Light Whole body irradiation with electromagnetic radiation, including light, increases the toxicity of central nervous system stimulants and decreases that of central nervous system depressants. The toxicity of analgesics such as morphine does not seem to be altered. Many toxicants do exhibit a diurnal pattern of response in animals that is generally related to the light pattern, In rats and mice, P-450 enzyme activity is at its greatest at the beginning of the dark phase of the cycle. Social Factors A variety of social factors (interactions between individual animals and between animals and research workers) can modify the toxicities of chemicals in animals and undoubtedly also in humans (see table 14.27) Animal handling, housing (singly or in groups), types of cages, and laboratory routine are all important components of such considerations. Table 14.27

Psychological Functions Showing Time-Based Cyclic Variations

Activity/sleep Body temperature Pain threshold Adrenocortical function Skin histamine sensitivity Liver function Renal function Eosinophil count Mitotic rates Food consumption Body weight

Edwards (1982) should be consulted for a good overview of the factors to be considered in the design and operation of a laboratory in terms of both good science and economic and regulatory considerations. Temporal Factors Most biological organisms are influenced, directly or indirectly, by a stream of daily and annual variations in their environment. These variations include light, temperature, social interactions, and food, and have resulted in a notable cyclical variations in function. Many of these cycles can be considerably amplified or modified. Liver function, for example, includes liver glycogen (which is also seasonally variable), glycogen phosphorylase, tyrosine transaminase, tryptophan pyrrolase, and esterase; some of these rhythmic changes are reflected in hepatic cell ultrastructure. Renal function includes urine volume and pH, and excretion of sodium, potassium, chloride, phosphate, uric acid, adrenal cortex, and probably other tissues as well. Blood leukocytes other than eosinophils show daily variation, although the eosinophilic changes have probably been the most studied. Reviews of these topics can be found in Aschoff (1965), Bunning (1967), Conroy and Mills (1970), Mills (1973), and Gall (1977). Many unicellular organisms also show cyclically varying functions; for example, cell generation time, photosynthetic capacity, phototaxis, and luminescence. In higher plants, growth, leaf and petal movement, and CO2 fixation are rhythmic functions, and in fungi, spore discharge (Wilkins 1973). An interesting feature of these rhythms is that they continue when rhythmic external stimuli are removed. If the daily light–dark cycle is turned into continuous light and if temperature variation


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is suppressed, and if, in humans, clues like watches and clocks and fixed meal times are removed, the rhythmic variation in function continues, but with a period that might differ slightly from the original 24 hr. This is the so-called free-running period. A 24-hr rhythm might become, for example, 25.0 or 22.9 hr (usually, however, between 20 and 28 hr), and settles to this new period indefinitely until external clues are restored. This phenomenon is the origin of the expression circadian, meaning about a day’s length. Considerable work has gone into trying to identify the biological clock that maintains these rhythms, so far without clear-cut success. Does the clock reside in an organ (e.g., the central nervous system or the adrenal cortex), or is it a cellular function? The answer is probably both: The cell has mechanisms that allow it to entrain to environmental rhythms, and that in higher organisms these become systematized in regulatory organs to coordinate the functioning of the body as a whole in response to environmental changes (Mills 1966, 1973). Both unicellular organisms and avian and mammalian cells in culture show circadian rhythms in the absence of exogenous influences (Bruce 1965). Present arguments center around whether the cellular clock is based on sequential DNA transcription or biochemical networks with natural oscillatory periods. A more recent hypothesis suggests that the cell membrane, with its stable lipids and mobile protein ionic gates, might serve as an oscillator with the underlying slow periodically (Njus et al. 1974). The majority of the laboratory animals used in toxicology are nocturnal, and they do not change while in the laboratory. Our procedures rarely take the fact and implication of this underlying biological activity rhythm into account. Diurnal rhythms (more correctly nychthemeral; diurnal refers to daytime, nocturnal to the night) are probably the most important, but there are almost certainly other cyclical changes in physiological function, connected to season or to sexual function, which for the most part we disregard, and might occasionally be important. The significance of circadian variation for drug action and toxicology will depend very much on the nature of the drug, its absorption characteristics, and the way in which it is administered. Toxicity from continuous atmospheric exposure is not likely to show much circadian dependence, nor from a drug only slowly absorbed or slowly metabolized to an active component. These are perhaps the exceptions, however, and with the increasing range of drugs showing some clinical effectiveness, many of which also carry undesirable side effects, it would seem sensible to administer these in a regimen that allows them to exert their maximum desired effect with a minimum of side effects. Although drugs acting on the central nervous system, the cardiovascular system, and the kidney and the steroids are obvious candidates for such consideration, there is evidence that susceptibility to infection is also circadian dependent, and chemotherapeutic agents and the antibiotics might not be outside such an inquiry. In addition, a sizable proportion of the population has its biological clock thrown into potential disarray. Besides the pilots and crew members of long-distance flights and the jet-set traveler, the night-shift workforce is numerous enough, and includes not only the factory workers and truck drivers, but hospital staff and watchkeepers at sea, all carrying considerable responsibility. If there is a relationship between drug action and the circadian cycle. it deserves proper evaluation.

REFERENCES Ahdaya, S. M., Shah, P. V., and Guthrie, F. E. (1976). Thermoregulation in mice treated with parathin, carbaryl or DDT. Toxicol. Pharmacol. 35, 575–580. Aschoff, J. (ed.). (1965). Circadian clocks. Amsterdam: North-Holland. Baetjer, A. M., and Smith, R. (1956). Effect of environmental temperature on reaction of mice Jo parathion, an anticholinesterase agent. Am. J. Physiol. 186, 39–46.

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Baker, H. J., Cassell, G. H., and Lindsey, J. R. (1971). Research complicants due to Haemobartonella and Eperythrozoon infection in experimental animals. Am. J. Pathol. 64, 625–656. Becker, W. A. (1962). Choice of animals and sensitivity of experiments. Nature. 193, 1264–1266. Belehradek, J. (1957). A unified theory of cellular rate process based upon an analysis of temperature action. Protoplasma. 48, 53–71. Bleby, J. (1976). Disease-free (SPFE) animals. In The UFAE handbook on the care and management of laboratory animals (5th ed.). Edinburgh, Scotland: Churchill Livingstone. Brater, D. C., Sokol, P. P., Hall, S. D., and McKinney, T. D. (1992). Disposition and dose requirements of drugs in renal insufficiency. In The kidney: Physiology and pathophysiology (2nd ed.), eds. D. W. Seldin and G. Giebisch, 3671–3695. New York: Raven Press. Brody, S. (1964). Bioenergetics and growth. New York: Hafner. Brouwer, K. L. R., Dukes, G. E., and Powell, J. R. (1992). Influence of liver function on drug disposition. In Applied pharmacolkinetics (3rd ed.), eds. W. E. Evans, J. J. Schentag, and W. J. Jusko, 6-1–6-59. Vancouver, WA: Applied Therapeutics. Brown, V. K. (1980). Acute toxicity in theory and practice. New York: Wiley. Bruce, V. G. (1965). Cell division and the circadian clock. In Circadian clocks. Ed. J. Aschoff, 125–138. Amsterdam: North-Holland. Bunning, E. (1967). The physiological clock. New York: Longmans. Calabrese, E. J. (1983). Principles of animal extrapolation. New York: Wiley. Cawthorne, M. A. (1979). The use of animal models in the detection and evaluation of compounds for the treatment of obesity. In Animal models of obesity, ed. M. F. W. Festing, 79–90. London: Macmillan. Conroy, R. T. W. L., and Mills, J. N. (1970). Human rhythms. London: Churchill. Cremer, J. E., and Bligh, J. (1969). Body temperature and responses to drugs. Br. Med. Bull. 23, 299–306. Doull, J. (1972). The effect of physical environmental factors on drug response. In Essays in toxicology (Vol. 3), ed. W. J. Hayes, 82–96. New York; Academic Press. Eaton, G. J. (1972). Intestinal helminths in inbred strains of mice. Lab. Anim. Sci. 22, 850–853. Edwards, A. G. (1982). Animal care and maintenance. In Principles and methods of toxicology, ed. A. W. Hayes, 321–345. New York: Raven Press. Elmes, P. C., and Bell, D. P. (1963). The effects of chlorine gas on the lungs of rats with spontaneous pulmonary disease. J. Pathol. Bacteriol. 86, 317–327. Festing, M. F. W. (1978). Genetic variation and adaptation in laboratory animals. In Das Tier in Experiment, ed. W. H. Weihe, 16–32. Bern, Switzerland: Hans Huber. Festing, M. F. W. (ed.) (1979a). Animal models of obesity. London: Macmillan. Festing, M, F. W. (1979b). Inbred strains in biomedical research. London: Macmillan. Gall, D. (1977). Temporal variations in toxicity. In Current approaches in toxicology, ed. B. Ballantyne, 39–52. Bristol, England: John Wright. Gralla, E. S. (ed.) (1986). Scientific considerations in monitoring and evaluating toxicological research. Washington, DC: Hemisphere Publishing. Grant, L., Hopkins, P., Jennings, G., and Jenner, F. A. (1971). Period of adjustment of rats used for experimental studies. Nature. 232, 135. Gratz, N. G. (1973). A critical review of currently used single-dose rodenticides. Bull. World Health Org. 48(4), 469–477. Grice, K., Sattar, H., and Baker, H. (1972). The effect of ambient humidity on transepidermal water loss. J. Invest. Dermatol. 58, 343–346. Gruneberg, H. (1952). The genetics of the mouse (2nd ed.). The Hague, Netherlands: Nijhoff. Hathway, D. E. (1970). Species, strain and sex differences in metabolism. In Foreign compound metabolism and mammals, ed. D. E. Hathway. London: Chemical Society. Hilado, C. J., and Furst, A. (1978). Reproducibility of toxicity data as a function of mouse strain, animal lot and operator. J. Combust. Tox. 5, 75–80. Johnson, H. D., and Voss, E. (1952). Toxicological studies of zinc phosphide. J. Am. Pharm. Assoc. (Sri Ed.). 41, 468–472. Kalow, W. (1962). Pharmacogenetics: Heredity and the response to drugs. Philadelphia: Saunders. Kalow, W. (1965). Dose–response relationship and genetic variation. Ann NY Acad. Sci. 123, 212–218. Keplinger, M. L., Lanier, G. E., and Deichmann, W. B. (1959). Effects of environmental temperature on the acute toxicity of a number of compounds in the rat. Toxicol. Appl. Pharmacol. 1, 156–161.


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Lang, C. M., and Vesell, E. S. (1976). Environmental and genetic factors affecting laboratory animals: Impact on biomedical research. Fed. Proc. 35, 1123–1124. Lindsey, J. R., Baker, H. J., Overcash, R. G., Cassell, G. H., and Hunt, C. E. (1971). Murine chronic respiratory disease. Am. J. Pathol. 64, 675–716. Matzke, G. R., and Millikin, S. P. (1992). Influence of renal function and dialysis on drug disposition. In Applied pharmacokinetics (3rd ed.), eds. W. E. Evans, J. J. Schentaqg, and W. J. Jusko, 8-1–8-49. Vancouver, WA: Applied Therapeutics. Mayersohn, M. (1992). Special considerations in the elderly. In Applied pharmacokinetics (3rd ed.), eds. W. E. Evans, J. J. Schentag, and W. J. Jusko, 9-1–9-43). Vancouver, WA: Applied Therapeutics. Meier, H. (1963a). Experimental pharmacogenetics: Physiopathology of heredity and pharmacologic responses. New York: Academic Press. Meier, H. (1963b). Potentialities for and present status of pharmacological research in genetically controlled mice. In Advances in pharmacology (Vol. 2), eds. S. Garattini and P. A. Shore. New York: Academic Press. Mills, J. N. (1966). Human circadian rhythms. Physiol. Rev. 46, 128–171. Mills, J. N. (ed.). (1973). Biological aspects of circadian rhythms. London: Plenum Press. Moore, D. H. (1972). Species, sex and strain differences in metabolism, In Foreign compound metabolism in nammals, ed. D. E. Hathway. London: Chemical Society. Muller, P. J., and Vernikos-Danellis, J. (1968). Alteration in drug toxicity by environmental variables. Proc. West. Pharmacol. Soc. 11, 52–53. Neely, W. A., Turner, M. D., and Taylor, A. E. (1967). Bidirectional movement of water through the skin of a non-sweating animal. J. Surg. Res. 7, 323–328. Njus, D., Sulzman, E. M., and Hastings, J. W. (1974). Membrane model for the circadian clock. Nature. 248, 116–120. Oser, B. L. (1981). The rat as a model for human toxicology evaluation, J. Toxicol. Environ. Health. 8, 521–542. Pritchard, J. A. (1965). Changes in the blood volume during pregnancy and delivery. Anesthesiology 26, 393–399. Rowland, M., and Tozer, T. N. (1995). Clinical pharmacokinetics (3rd ed.). Baltimore: Williams and Wilkins. Sawin, P. B., and Glick, D. (1943). Atropinesterase, a genetically determined enzyme in the rabbit. Proc. Natl. Acad. Sci. 29, 55–59. Selisko, O., Hentschel, G., and Ackermann, H. (1963). Uber die abhangigkeit her mittleren todlichen dosis (LD50) von exogenen Faktoren. Arch. Int. Pharmacodyn. Ther. 45, 51–69. Simpson, F. O., Phelan, E. L., Clark, D. W. J., Jones, D. R., Gresson, C. R., Lee, D. R., and Bird, D. L. (1973). Studies on the New Zealand strain of genetically hypertensive rats. Clin. Sci. Mol. Med. 45, 15S–21S. Strong, L. C. (1942). The origin of some inbred mice. Cancer Res. 2, 531–539. Tedeschi, D. H., and Tedeschi, R. E. (1968). Importance of fundamental principles in drug evaluation. New York: Raven Press. Usinger, W. (1957). Respiratorischer stoffweschel and korpetemperature der weissen mans in thermoindefferenter umgebung. Pfugers Arch. 264, 520–535. Vesell, E. S. (1969). Recent progress in pharmacogenetics. In Advances in pharmacology and chemotherapy (Vol. 7), eds. S. Garattini, A. Goldin, F. Hawking, and I. J. Koplin, 162–187. New York: Academic Press. Wilkins, M. B. (1973). Circadian rhythms in plants. In Biological aspects of circadian rhythms, ed. J. N. Mills, 235–279. London: Plenum Press. Williams, R. L. (1984). Drugs and the liver: Clinical applications. In Pharmacokinetic basis for drug treatment, eds. L. Z. Benet, N. Massoud, and J. G. Gambertoglio, 63–76. New York; Raven Press. Zimmermann, A., and Matschiner, J. T. (1974). Biochemical basis of hereditary resistance to warfarin in the rat. Biochem Pharmacol. 23(6), 1033–1040.

CHAPTER

15

Laws and Regulations Governing Animal Care and Use in Research Shayne C. Gad Gad Consulting Services

CONTENTS Laboratory Animal Care and Welfare............................................................................................901 The Process ....................................................................................................................................902 The Law .........................................................................................................................................903 Animal Welfare Act..............................................................................................................905 Institutional Animal Care and Use Committee........................................................905 Exercise for Dogs .....................................................................................................909 Psychological Well-Being of Nonhuman Primates .................................................909 Other AWA Requirements ........................................................................................911 International Regulations......................................................................................................911 Occupational Health and Safety...........................................................................................912 Special Areas of Responsibility ...........................................................................................912 Pain and Distress ......................................................................................................913 Publications ....................................................................................................................................915 Code of Federal Regulations, Title 9, Chapter 1, Subchapter A: Animal Welfare.............915 Animal Care Policies............................................................................................................915 Professional Organizations.............................................................................................................915 American Association for Accreditation of Laboratory Animal Care ................................915 American Association for Laboratory Animal Science .......................................................916 American Veterinary Medical Association...........................................................................916 American College of Laboratory Animal Medicine............................................................916 References ......................................................................................................................................916 Additional Resources .....................................................................................................................918

LABORATORY ANIMAL CARE AND WELFARE This chapter seeks to summarize and consider the broad and general category of animal care and welfare. The concept of adequate animal care and the mandates either implied or specified by law are discussed. 901

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Quality science and research depends on quality animal care. In fact, state-of-the-art research demands first-class animal care. The commonly accepted ideas of animal welfare were in vogue long before the passage of the first version of the Laboratory Animal Welfare Act (AWA 1966). Federal and international regulations concerning the safety of the entire scope of products in commerce have had several safety objectives (see Gad 2001; Gad and Chengelis 1998), which have had a great impact on animal care. These safeguards are essential to assure the scientific staff that animal care meets certain standards. In no area of science is the need or the quest for knowledge more acute than in the area of animal welfare and animal rights. Immersed in this quest are the biological scientist, research administrators, regulatory officials, veterinarians, and the lay public in general. There is no question that animal welfare should be and perhaps already is the foremost consideration in the justification for using animals as experimental subjects (Acred, et al. 1994). There is long-standing and growing opposition to the use of animals, primarily because of the gray area of distinction between animal welfare and animal rights (British Cruelty to Animals Act 1876; Fano 1997; French 1975; Freudinger 1985; Hampson 1979). The question of animal rights suggests that you can elevate the so-called animal rights (if in fact there are such rights) to a position of equal moral value with human rights (Russell and Burch 1959). However, it is not possible to equate animal rights with human rights without diminishing human rights, especially the human right to improved health and safety in everyday life. Most biologically oriented scientists and veterinarians understand the need to provide the best available environment, nutrition, housing, and general care to all experimental subjects used in basic research, safety testing, and educational practices. There is no question, nor can there be any wavering concern, that animals used in these endeavors must be provided the best care available. If these practices are followed, the question of animal welfare is addressed. Without the use of animal models or animal subjects, major accomplishments in transplantation; surgical techniques; chemotherapy; or in pathogenesis of infectious diseases and cancer, diagnostic methods, or reproductive failures could never have occurred.

THE PROCESS The regulatory process typically begins with a Public Law or Act passed by the U.S. Congress. The legislation generally requires that an agency (e.g., U.S. Department of Agriculture) develop regulations and standards to ensure that the wishes of Congress stated in the law are carried out. The regulations and standards basically define to whom the regulations apply and the requirements for compliance with the regulations. The agency generally publishes the proposed regulations in the Federal Register for a period of written public comment. The process can also involve public hearings at which the agency gathers additional oral and written input from interested parties. Both of these mechanisms offer ways in which institutions can influence the regulations that affect them. Following the public comment period, the agency reviews all comments and issues the final rule, which specifies the effective date of the regulation. The extent and applicability of the regulations for animal facilities varies somewhat based on the species housed and type of research, teaching, and testing procedures performed. There are, however, fairly general regulations that apply to most facilities, as well as professionally accepted standards of practice that apply. It is these more general, or commonly encountered, regulations and practices that are the focus of this chapter. State and local requirements, which can also have an impact on animal facility management (e.g., health and safety ordinances), must also be considered but are beyond the scope of this publication. International regulations are also considered.

LAWS AND REGULATIONS GOVERNING ANIMAL CARE AND USE IN RESEARCH

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THE LAW Historically, regulations covering the care and use of animals for experimental research have been developed from two main sources: the scientists themselves and humane societies that were formed to protect companion, farm, and laboratory animals. The National Institutes of Health (NIH), since its inception in 1887 (as the Hygienic Laboratory at the U.S. Marine Hospital in Staten Island), has led the way in developing guidelines for the proper care and use of laboratory animals. In the 1920s the director of NIH was personally responsible for decisions concerning the use of animals in any given experiment. During World War II, the Committee for Medical Research and the National Research Council of the National Academy of Science joined in the effort to reduce war-related injury and disease. This research required the use of animals, which also led to standards for animal care. By war’s end, the major oversight of animal care issues was transferred to NIH from the Wartime Office of Scientific Research. By 1958, NIH’s Division of Research Grants began a peer-review system of selecting the most meritorious grant applications for funding. Included in the examination process was the issue of care and use of laboratory animals. In 1963, the first edition of the Guide for the Care and Use of Laboratory Animals was issued by the Animal Care Panel, later renamed the American Association for Laboratory Animal Science (AALAS 1997). The most recent edition of the Guide was published in 1996 (National Research Council 1996) and remains the primary reference for research animal care and use in the United States. In 1966 Congress passed the Pet Protection Act in the wake of public outcry over the alleged misuse of animals. This act was the first version of what is now the Animal Welfare Act (1966). The AWA was revised in 1970 and in 1976 and underwent major revisions in 1985 and 1991. The Act gave the U.S. Department of Agriculture (USDA) responsibility for implementing the new law, which applied to dogs, cats, rabbits, monkeys, guinea pigs, and hamsters. Recent changes in the interpretation of the law have brought all animals used for research purposes under the umbrella of the law, but the courts have blocked implementation of this as regards rats, mice, and birds. The USDA has issued Parts 1, 2, and 3 of the final regulations implementing the 1985 amendments of the AWA (NIH 1985), which cover definitions (Part 1), regulations (Part 2), and standards (Part 3). It is Part 3 that gives specific guidelines for the humane handling, care, treatment, and transportation of animals used in research and teaching programs. The Animal and Plant Health Inspection Service (APHIS), part of the USDA, was given responsibility for facility registration, licensing of animal suppliers, and inspection of licensed facilities to assure compliance with the AWA. Inspectors for APHIS inspect licensed facilities on at least an annual basis (unannounced) to ascertain the status of the facilities’ physical plants, training of animal care personnel, and the overall care and welfare of research animals covered under the AWA. Table 15.1 presents current housing standards for laboratory animals. Prior to 1985, the Public Health Service (PHS) policy on humane care and use of laboratory animals use was governed by Awardee Institution (National Research Council 1996). The amended policy, which is in agreement with 9 Code of Federal Regulations (CFR), lists nine areas of concern that must be addressed and adhered to by the responsible institutional official (NIH 1985; APHIS 1989). Briefly, these areas are as follows: 1. Care and use of animals must be in accordance with the AWA. 2. Experiments involving animals must be designed with consideration of their relevance to human or animal health, advancement of knowledge, or the good of society. 3. Animals selected for a procedure must be of the appropriate species, quality, and minimum number required to provide valid results.

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Table 15.1 Recommended Housing Conditions for Selected Laboratory Animal Speciesa

Species

Housing Typeb

Mouse

Group

Rat

Group

Hamster

Group

Guinea pig

Group

Rabbit

Single

Rabbit (nursing)c Cat

Single (plus litter) Single

Dog

Single

Monkey

Single

Ape

Single

Poultrye

Single

Sheep/goat

Single

Swine

Single

Cattle

Single

Horse

Single

a

b

c d e

Size

Minimum Cage/Pen Floor Area

Minimum Cage/Pen Height (cm)

Room Temperature (°C)

10–15 g 15–25 g 25 g 100 g 100–200 g 200–400 g 400–500 g 500 g 60–80 g 80–100 g 100 g 350 g 350 g 2 kg 2–4 kg 4–5.4 kg 5.4 kg 2–4 kg 4–5.4 kg 4 kg 4 kg 15 kg 15–30 kg 30 kg 1 kg 1–10 kg 10–25 kg 25–30 kg 30 kg 20 kg 20–35 kg 35 kg < 0.25 kg 0.25–1.5 kg 1.5–3.0 kg > 3.0 kg < 25 kg 25–50 kg < 15 kg 15–50 kg 50–200 kg > 200 kg < 75 kg 75–350 kg 350–650 kg > 650 kg Adult

39–52 cm2 52–77 cm2 97 cm2 110 cm2 110–148 cm2 148–258 cm2 258–387 cm2 452 cm2 65–84 cm2 84–103 cm2 123 cm2 387 cm2 651 cm2 0.14 m2 0.14–0.27 m2 0.27–0.36 m2 0.45 m2 0.46 m2 0.56 m2 0.27 m2 0.36 m2 0.72 m2 1.1 m2 2.2 m2 0.14 m2 0.14–0.39 m2 0.39–0.72 m2 0.72–0.90 m2 1.4 m2 0.90 m2 0.90–1.4 m2 2.3 m2 0.02 m2 0.02–0.09 m2 0.09–0.18 m2 0.27 m2 0.9 m2 0.9–1.8 m2 0.7 m2 0.7–1.4 m2 1.4–4.3 m2 > 5.4 m2 2.2 m2 2.2–6.5 m2 6.5–11.2 m2 > 13.0 m2 13.0 m2

13 13 13 18 18 18 18 18 15 15 15 18 18 36 36 36 36 36 36 61 61 Species-specificd Species-specificd Species-specificd 51 51–76 76–91 91–117 117 140 140–152 213 — — —

18–26 18–26 18–26 18–26 18–26 18–26 18–26 18–26 18–26 18–26 18–26 18–26 18–26 16–22 16–22 16–22 16–22 16–22 16–22 18–29 18–29 18–29 18–29 18–29 18–29 18–29 18–29 18–29 18–29 18–29 18–29 18–29 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27 16–27

— — — — — — — — — —

Recommendations for primary enclosure from the Guide (National Research Council 1996); larger animals might also require secondary enclosures for exercise, mating, or other activities. Typical preference for housing; listed group-housed animals are frequently single housed according to study protocol. Space recommendation from the Animal Welfare Act (USDA 1995). Recommended cage height for dogs is 6 in. above the head during normal standing position. Based primarily on the chicken.


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4. 5. 6. 7. 8.

Use must avoid or minimize discomfort, stress, and pain to the animals. Appropriate sedation, analgesia, or anesthesia must be provided to the animals. Animals that suffer severe or chronic pain must be euthanized as soon as possible. Housing conditions must be appropriate for the species. Investigators must be appropriately qualified and experienced for conducting procedures on living animals. 9. Exceptions to the principles of animal care and use should not rest with investigator(s), but with the Institutional Animal Care and Use Committee (IACUC).

Since 1989, great strides have been made in setting and applying standards for animal care and welfare across all research facilities, regardless of funding sources. These standards are more “performance-based” than the previous “engineering-based” application of the guidelines; that is, exact cage size, air flow, and painted surfaces within animal rooms. Also, suitable means of euthanasia have been characterized (see table 15.2). Animal Welfare Act The AWA, passed by the U.S. Congress in 1966, addressed a variety of activities involving animals in interstate and foreign commerce. The specific intent was to ensure that animals used in research, for exhibition, or in production for the pet trade were provided with humane care and treatment and appropriate transportation, and also to protect owners of animals from theft. Subsequent amendments to the AWA, the most recent in 1991, have focused increased attention on the use of animals in research, testing, and teaching. The regulations and standards published by the USDA pursuant to the AWA include very specific requirements for research facilities. The AWA itself is quite specific regarding some of the requirements that were to be addressed by the USDA when writing the regulations. In addition to the development of minimum standards for housing, feeding, watering, sanitation, and other standards for most species, the AWA specifies that minimum standards be developed for the exercise of dogs, and for a physical environment adequate to promote the psychological well-being of nonhuman primates. It also specifies that research facilities must do the following: • • • • • • •

Consider procedures to minimize pain and distress. Consider the use of alternatives to animals. Require veterinary consultation for use of anesthetics and for pre- and postsurgical care. Prohibit the use of paralytics without anesthesia. Restrict the use of animals in more than one major operative procedure. Provide training. Establish an IACUC with specific responsibilities to oversee its animal-related activities.

A detail included by Congress in the 1985 Amendments to the AWA was considered unusual at the time and provided the Secretary of Agriculture and the USDA little flexibility in developing the regulations and standards in some areas. However, in many areas, the standards have taken the approach of performance expectations, rather than more specific engineering-based standards. The following discussion focuses on a few of the requirements of the Animal Welfare Act Regulations (AWAR) that have an impact on management of animal facility programs today. Institutional Animal Care and Use Committee The AWA (USDA 1995) requires the animal research facility to form an IACUC for oversight of animal-related issues, following the standards set forth by the Guide (NIH 1985). An active,

Most Most Several small laboratory animals Most small Mice, rats Fish, amphibians Birds, small rodents, rabbits Most small Large farm or wildlife species Foxes, sheep, swine, mink Small amphibians Most small Several Several small Several Several Several Several Several Several

Barbiturate injection Anesthetic inhalation

Carbon dioxide inhalation

Carbon monoxide inhalation

Microwave exposure

Tricaine/bensocaine injection Cervical dislocation

Gunshot

Electrocution

Nitrogen/argon inhalation

Exsanguination

Rapid freezing

Air embolism injection

Drowning Strychnine dosing

Chloroform injection Cyanide dosing Stunning (blow to head)

Possibly convenient Possibly convenient Rapid, no drug residue

Safe, cheap Possibly convenient

Safe, cheap

Safe, cheap

Safe, cheap

Rapid, safe, readily available

Rapid, no drug residue

Rapid, cheap, no drug residue

Rapid, ease for certain species

Rapid, safe Rapid, safe, cheap, no drug residue Rapid, no drug residue

Rapid, safe, brain enzymes fixed

Rapid, safe, cheap, multiple animals Rapid loss of consciousness

Rapid, safe, cheap Rapid, multiple animals exposed

Major Advantages

Causes convulsions, other signs of distress Very stressful, slow Causes convulsions, painful contractions Very hazardous to staff Unpleasant, hazardous Unpleasant, sufficient force required

Very stressful

Very stressful

Stressful in some species, must limit O2

Requires training, some hazard, unpleasant Requires training, dangerous, unpleasant Requires special equipment, hazardous, unpleasant, severe contractions Requires training, unpleasant

Costly Requires training, unpleasant for staff

Specialized training, equipment, costly

Hazardous to staff, difficult to detect

Some species stressed or very tolerant

Requires training, restraint, drug control Initial irritation, hazardous to staff

Major Limitations

Source: Summarized from the Report of the American Veterinary Medical Association (AVMA) Panel on Euthanasia (1993).

Pithing

Decapitation

Species

Method

Table 15.2 Common Methods of Euthanasia in the Toxicology Laboratory

Unacceptable method Unacceptable method Unacceptable method without other lethal procedure

Acceptable (preferred) method Acceptable method for small species Acceptable at high concentrations Acceptable method with appropriate generation Acceptable method with appropriate equipment Acceptable method Conditionally acceptable method when justified Conditionally acceptable method when justified Conditionally acceptable method when necessary Conditionally acceptable method in specialized instances Conditionally acceptable method in specialized instances Conditionally acceptable method is specialized instances Unacceptable method without anesthesia Unacceptable method without anesthesia Unacceptable method without anesthesia Unacceptable method Unacceptable method

AVMA Recommendation

906 ANIMAL MODELS IN TOXICOLOGY


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well-informed IACUC is one of the best guarantees that animal welfare policies are supported and adhered to. Although research facilities are sometimes criticized for self-policing (IACUC members are chosen by the management of the facility), there are numerous checks and outside (USDA, American Association for Accreditation of Laboratory Animal Care [AAALAC]) inspections that, along with the practical considerations already noted, make most IACUCs effective. The required membership of an IACUC has been noted previously, and the total number of members depends on the size of the facility and the extent of its research. Beyond the mandated composition of the groups, it is desirable to include members from diverse backgrounds among scientific and nonscientific disciplines. Outside experts can also serve as consultants. There might be need for subcommittees to carry out the many functions of the IACUC. As noted in the Guide, the IACUC oversees and assesses the animal care program and the use of animals in research. At least every 6 months, it inspects the facility (with emphasis on all animal housing, testing, and supply areas), reviews protocols, and carefully records all findings. A written report (containing meeting minutes, documents reviewed, areas inspected, and items discussed) signed by most or all members is issued to principal investigators and facility management detailing any deficiencies. Specifically, protocols and subsequent animal use and euthanasia are evaluated on the following bases (the Guide): • • • • • • • • • • •

Rationale of proposed animal use Justification of the species used Justification of the number of animals used Availability of alternative methods (Bennett 1994) Adequacy of staff training and experience Atypical housing or husbandry requirements Consideration of sedation, analgesia, and anesthesia Unnecessary duplication of testing Use of repeated or major surgical techniques Criteria for excessive pain or distress Method of euthanasia

Standardized procedures in addition to specialized research projects must be considered by the IACUC. Standardized protocols should not require a full review each time they are employed, but any revisions affecting animal use must be considered by the IACUC. Specialized protocols should each be reviewed by the IACUC. There should be a review and approval form for each project, which should be maintained as part of study records. These records should also be maintained in facility files, available for independent review, such as a USDA inspection. The IACUC report must reflect adherence to the AWA, with minor and major deviations being noted. A major deficiency is one that threatens the heath, well-being, or safety of the animals. Identification of animal welfare deficiencies need not only originate from formal inspections, however. Any staff member should be encouraged to present concerns to the IACUC. Technical or nontechnical personnel, for example, should feel free (without any negative consequences) to inform a direct supervisor or an IACUC member of any excessive pain or distress witnessed during the course of a toxicology project. Such information can be kept confidential, but careful documentation must be made. It then becomes the responsibility of site management and the IACUC to investigate and take appropriate corrective action as warranted. This process is discussed by Silverman (1994). There must be a reasonable and specific plan, with an outlined schedule, for the correction of significant deficiencies. Resolution of any major deviation should occur through a joint effort of the researcher, facility management officials of the area of concern (animal care supervisor, environmental or maintenance staff, purchasing agent, etc.), veterinary staff, and the IACUC. If major deficiencies are not resolved according to the plan and schedule, the remaining deficiencies “shall be reported in writing by the IACUC, through the Institutional official, to APHIS and any Federal agency funding that activity” (AWA). The IACUC will also withhold approval for the specific

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studies affected until concerns are resolved. Of course, conflicts might occur among staff, researchers, management, and study sponsors. These will have to be resolved in the light of the principles presented earlier—ethics, science, social pressures, and legal requirements. Secondary responsibilities of the IACUC are training staff members, providing information on animal use, interacting with other personnel (health and safety officers, managers, legal staff, etc.), developing a liaison with outside organizations promoting humane research, and engaging in public education (Dell 1994). Any complaints or charges of animal misuse made by the public or the media should be investigated by the IACUC. This should aid facility management in arriving at an appropriate response. Part 2 of 9 CFR, Chapter 1 (Regulations) states: Each institution which falls under the authority of the AWA and/or received PHS support for research or teaching involving laboratory animals must operate a program with clear lines of authority and responsibility for self monitoring the care and welfare of such laboratory animals.

The mechanism for such monitoring was given to the IACUC. To monitor the care and use of animals, the AWA requires that an appropriate administrative official at each facility appoint the members of the IACUC. The IACUC serves as the watchdog for the welfare of animals in the same manner as does the institutional review board in the hospital or medical center. Membership of the IACUC must include a scientist from the institution experienced in research involving animals; a doctor of veterinary medicine who either is certified by the American College of Laboratory Animal Medicine or has had experience in laboratory animal medicine; a person who is not affiliated with the facility or institution; and other members as required by federal, state, or local regulations. Humane care and treatment of animals used in research, testing, and education requires a certain amount of scientific and professional judgment. The husbandry needs of each species and the special requirements for the species, including humane handling, must be considered in all programs in which these animals are to be used. Each facility or institution must establish a written policy for the humane care and use of a given species. This program must be in compliance with applicable federal, state, and local laws and regulations. Depending on the fund or funding source used to provide for these animals or to provide the basic research, other regulations might be enforced. The established program must follow Guide for the Care and Use of Laboratory Animals, published by the U.S. Department of Health and Human Services and the regulations provided by the U.S. Department of Agriculture found in 9 CFR part 3 entitled “Animal Welfare Standards.” The responsibility for directing such a program is usually given to a veterinarian or another qualified professional. At least one veterinarian must be associated with the program. The IACUC is responsible for evaluating the animal care and use program. The duties of the IACUC must include: 1. Meeting at regular intervals to ensure compliance with the Guide (the regular intervals can be no less than annually). 2. Ensuring that a mechanism exists to review the humane care and use of animals in research, testing, and education. 3. Providing a written report at least annually to the responsible administrative official on the status of the animal care and use program. 4. Reviewing and/or investigating concerns involving the care and use of animals at the research facility resulting from public complaints received and from reports of noncompliance received from laboratory or research facility personnel. 5. Reviewing, approving, and requiring modifications or withholding approval of those components of proposed or ongoing activities related to the care and use of animals. 6. Other duties as assigned.


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Although the duties of the IACUC just outlined fall into a very broad and general category, other duties and responsibilities have historically been assigned to IACUC. In many institutions the IACUC assures that adequate veterinary care is provided. This care can either be provided by an on-site veterinarian or by a contract veterinarian who makes periodic visits to the facility. There must be a written program for animal use and care. Although it is the responsibility of the institution to ensure that people caring for and using laboratory animals are qualified to do so, it often falls on the IACUC to make sure that these individuals are qualified and possess the proper qualifications and expertise to conduct the various phases of research that are being considered. Because animal care and use programs require professional, technical, and husbandry support, the IACUC must assure that the institution or facility employs people trained in laboratory animal science and provides the opportunity for continuing education and properly supervised on-the-job training to ensure effective implementation of the program. Exercise for Dogs The requirement for exercise of dogs has been the subject of considerable discussion since the AWA was passed. In the case of dog exercise, the AWA provided little guidance other than that the exercise program be determined by the attending veterinarian and be in accordance with the standards to be developed. The subsequent AWAR (Part 3: Subpart A) establishes several requirements worthy of comment. The facility is required to develop, document, and follow a plan (approved by the attending veterinarian) to provide dogs with the opportunity for exercise. In practice, and in certain parts of the standard, this has been interpreted to require involvement of the IACUC in the process. The plan must be a written program with specific procedures for implementation. The expected approach is to define program-specific ways of achieving the goal of exercise. The “opportunity for exercise” need only be addressed if certain minimum requirements for housing space are not met. For dogs housed individually, additional opportunity for exercise need not be provided if the floor space provided in the enclosure is at least twice the minimum space required by the AWAR for individually housed dogs. When housed in groups, no additional exercise is required if the total floor space is at least 100% of the AWAR required space for each dog if maintained separately. This standard has generated significant controversy. The standards provide little guidance for the provision of exercise if these space requirements are not met, other than that dogs be provided access to a run or open area. The frequency and duration of exercise is not specified, but is to be determined by the attending veterinarian. Most institutions have been able to comply with the regulations by first developing a written plan that simply outlines or specifies how dogs will be housed in their facilities (i.e., space requirements) and how they will be exercised, if required. The plan is typically approved by the IACUC and reviewed on a schedule consistent with the review of protocols (annually) or in conjunction with the IACUC’s semiannual program review. Because of the lack of specific requirements in the AWAR, many facilities have selected a frequency and duration of exercise opportunity consistent with the available exercise space, the average daily canine population, and personnel resources. With that as a starting point, periodic observation (with documentation) of the behavior of the animals serves as a means of evaluating the success of the program. A conscientious effort to plan, implement, evaluate, and improve the exercise program for dogs, if appropriately documented, is generally sufficient to demonstrate compliance to the standard. Additional guidance can be found in the National Research Council publication, Laboratory Animal Management: Dogs. Psychological Well-Being of Nonhuman Primates The original AWA states that facilities housing nonhuman primates must provide a “physical environment” adequate to promote the psychological well-being of primates, the AWAR (Part 3:

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Subpart B) specifies that “environmental enhancement” be provided. An environmental enhancement plan must be developed that is consistent with appropriate professional standards as cited in appropriate professional journals or reference guides. Initial plans developed by institutions utilized numerous and varied strategies. Although it was clear that a certain degree of flexibility was essential to developing and implementing effective programs, the need for guidelines was recognized. The National Research Council, Institute for Laboratory Animal Research (ILAR) subsequently published guidelines specifically addressing this requirement. That report, The Psychological WellBeing of Nonhuman Primates, provides considerable information on environmental enhancement strategies for different species and for different situations. The report supports the performancebased standard approach as distinct from engineering-based standards, but emphasizes the need for scientific approaches to meeting the standards so that results can be measured and evaluated. When using this document as a guideline, it is important that professional judgment is used in interpreting and applying the recommendations. As is the case with the care of any animal species, the specific knowledge, experience, and skills of the facility personnel involved in the implementation of the program are critical. The AWAR for environmental enhancement do specify areas that must be addressed, including social grouping, environmental enrichment, special considerations, and restraint devices. The facility plan must address the social needs of any nonhuman primate species known to exist in social groups in the world. Exceptions to the requirement for social interaction can include animals that exhibit overly aggressive behavior or those that are debilitated as a result of age or other conditions. It is recognized that animals suspected of having contagious disease would need to be isolated from other animals, as would animals undergoing diagnosis and treatment of a disease condition. Compatibility of animals housed in groups is stressed. If housed individually, the animals must be able to see and hear others of their species unless this is contraindicated for medical or scientific reasons and so documented. The AWAR suggest several approaches to environmental enrichment designed to provide a means for the animals to express species-typical activities. Examples include the following: • • • • •

Perches, swings, mirrors, and various cage complexities Objects to manipulate Varied food items and foraging opportunities Interaction with caretakers where appropriate Housing with compatible conspecifics

It is recognized that special circumstances could exist that will influence the approach to housing and environmental enrichment. These include provisions for infants and juveniles, research-based protocol restrictions, and the housing of large great apes. The AWAR prohibit the use of restraint devices unless required for health reasons (determined by the attending veterinarian) or as part of a research protocol approved by the IACUC. The restraint should be for the shortest time possible. If restraint exceeds 12 hr, the animal should periodically be provided with the opportunity for unrestrained exercise if the research protocol or situation permits. The institution’s approach to meeting the requirement for environmental enhancement can be very similar to that for exercise for dogs. A written plan is developed, taking into consideration the species, existing facility and equipment, and personnel resources. The plan is typically approved by the IACUC and reviewed on a regular basis. The plan should include methods of data collection that allow for evaluation of the success of the program. The ILAR report, The Psychological WellBeing of Nonhuman Primates, provides guidance not only on strategies for implementation, but also on measures that can be used to evaluate success. By using the ILAR report and other available information, compliance with the standard can be achieved with diligent planning, implementation, evaluation, and improvement that is well documented.


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Other AWA Requirements The AWAR (Part 3: Subparts A–F) contains the majority of the requirements specific to the care of various species including requirements for facilities, operations, animal health and husbandry, and transportation. Detailed standards are provided for areas such as indoor and outdoor housing, primary enclosures (cages and pens), feeding, watering, cleaning and sanitation, and pest control. In some cases the standards are quite specific (engineering-based standards). However, the concept of performance-based standards and use of professional judgment is evident throughout the section. In 1998 the USDA amended the regulations under the AWA to address issues of comfort and foot injury associated with housing of dogs and cats on suspended mesh or slatted flooring. Following a phase-in period for new purchases, the requirements of this amendment became effective for all primary enclosures in January 2000. The standards for primary enclosures for dogs and cats (Part 3, Subpart A, Section 3.6) state that the enclosures must have “floors that are constructed in a manner that protects the dogs’ and cats’ feet and legs from injury, and that, if of mesh or slatted construction, do not allow the dogs’ or cats’ feet to pass through any openings in the floor.” The standards require that suspended flooring for dogs and cats either be constructed of metal strands greater than 1/8 in. (9 gauge) in diameter or coated with a material such as plastic or fiberglass. Primary enclosures with such flooring must also be constructed so that the floor does not bend or sag between the structural supports. The intent of this amendment was to address concerns that certain types and designs of suspended flooring contributed to foot injuries, and that appropriate modification would decrease the incidence of such problems. In addition, sagging, unstable floors were thought to cause psychological trauma for dogs trying to balance on the floors. The modifications to the standards were intended to improve comfort for dogs and cats housed in primary enclosures with suspended floors. With the exception of the most recent changes, requirements included in the AWAR were incorporated into the National Research Council’s (1996) Guide for the Care and Use of Laboratory Animals. International Regulations Internationally, animal welfare concerns are greater (Europe) or lesser than in the United States, as reflected in their current laws (AAALAC 1997b, 1997c; Bayne and Martin 1998; Cooper 1990a, 1990b; Council Directive 1986; Howard-Jones 1985; Miller 1998; Netherlands Animal Welfare Society 1991; Nevalainen 1999). In the United Kingdom, the equivalent regulations are called the Animal (Scientific Procedures) Act and are administered by the Home Office. Corresponding laws are in force in all major countries, as listed here: • Australia • Australian Council 1990 • Canada • Guide to the Care and Use of Experimental Animals (Vol. 1, 2nd ed. 1993; Vol. 2 1984) Rowsell 1991 • China • Animal Protection Law • Germany • German Animal Welfare Act (May 1998) • German Animal Welfare Act (May 1998) [English translation] • Japan • Law Concerning the Protection and Control of Animals (October 1973) • New Zealand • Animal Welfare Act of 1999 (October 1999)

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• Code of Recommendations and Minimum Standards for the Care and Use of Animals for Scientific Purposes (August 1995) • Code of Recommendations and Minimum Standards for the Emergency Slaughter of Farm Livestock (December 1996) • Code of Recommendations and Minimum Standards for the Sale of Companion Animals (April 1994) • Code of Recommendations and Minimum Standards for the Welfare of Animals in Boarding Establishments (August 1993) • Code of Recommendations and Minimum Standards for the Welfare of Animals at Stockyards (May 1995) • Code of Recommendations and Minimum Standards for the Welfare of Animals at the Time of Slaughter and Licensed and Approved Premises (July 1996) • Code of Recommendations and Minimum Standards for the Welfare of Bobby Calves (August 1993) • Code of Recommendations and Minimum Standards for the Welfare of Deer During the Removal of Antlers (January 1992) • Code of Recommendations and Minimum Standards for the Welfare of Dogs (May 1998) • Code of Recommendations and Minimum Standards for the Welfare of Layer Hens (September 1996) • Code of Recommendations and Minimum Standards for the Welfare of Livestock from Which Blood Is Harvested for Commercial and Research Purposes (April 1996) • Code of Recommendations and Minimum Standards for the Welfare of Ostriches and Emus (August 1993) • Code of Recommendations and Minimum Standards for the Welfare of Pigs (January 1994) • Code of Recommendations and Minimum Standards for the Welfare of Sheep (July 1996) • Norway • Animal Welfare Act (December 1974) • Sweden • Swedish Animal Welfare Act (February 1998) • United Kingdom • Animals (Scientific Procedures) Act of 1986

Occupational Health and Safety An occupational health program is mandatory for personnel working in laboratory animal facilities. This program must include a physical examination and a medical and work history prior to work assignment. Periodic physical examinations are required for people in some job categories, especially those who handle hazardous materials. In addition, an educational program must be provided to teach personnel about zoonoses, personal hygiene, and other considerations that could be of a prime safety concern to those individuals involved in the program, such as special precautions to be taken by pregnant women (Acha and Szyfrei 1980). Other occupational hazards including animal bites and allergies to animal dander or hair must be considered. Of special concern is the area of B virus exposure to those individuals handling primates. In some areas there might be requirements for special clothing or breathing apparatus, which can be determined by the risk of exposure to the individual employee. Special Areas of Responsibility Recently, two areas of consideration have caused the IACUC a great deal of discomfort and disagreement in their deliberations. This issue of pain and distress caused by physical restraint and multiple or invasive surgical procedures has caused many IACUC members to question the use of these procedures with specific concern for the overall welfare of the animals involved. Brief physical restraint of animals for administration of drugs, collection of samples, or other experimental


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procedures is certainly considered justifiable. The physical restraint can be accomplished manually or with simple restraint devices such as slings, racks, or squeeze cages. Prolonged restraint (longer than 4 hr) of any animal, including the use of chairs for nonhuman primates, should be avoided unless absolutely essential to the research objectives. Prolonged restraint must be specifically spelled out within the research protocol, along with the justification for prolonged restraint in these specific restraint devices. One of the major concerns of the IACUC is that these restraint devices should not be used simply as a convenience to the investigator in handling or managing animals. In many cases the use of such devices must be specifically approved by the IACUC. Consideration must also be given the possibility of lesions or illness associated with restraint, such as decubitus ulcers, dependent edema, and weight loss. If these problems develop, adequate veterinary care must be provided to the animals and in some cases might mandate a temporary or permanent removal of animals from such restraint devices. Multiple or invasive surgical procedures on a single animal are generally discouraged. In any case, the multiple major surgical procedures must be approved by the IACUC. It might be necessary for the researcher to develop alternate procedures that do not require the use of multiple surgical procedures. In no case can cost savings be a consideration or adequate reason for performing multiple surgical procedures. Pain and Distress Whereas such areas as lighting, food, water, cage size, ventilation, environmental temperature, humidity, and exercise are mandated by regulation, the minimization of stress, discomfort, and pain is poorly defined or understood. The understanding of stress, discomfort, and pain in animals is based primarily on professional and aesthetic judgment made by the investigtors and members of the IACUC. Without pre- and postsurgical medication, stress and pain will result. What criteria can be used to assure that pain and distress are minimized? In most research units, pain is recognized by the action of the species under study. By understanding the normal posture, movement, and attitude of the species, animal care technicians can recognize abnormal posture, movement, or lack thereof; attitude (aggressive vs. submissive); grooming, eating, or drinking habits; or vocalization. Any of these signs can suggest to the investigator or veterinarian that the animal is experiencing pain or discomfort. These observations are straightforward, and judgments concerning their meaning or significance are fairly easy to make, assuming pain in the experimental animal subject is perceived in the same manner as is pain in the human subject. Behavioral changes as just described can be short term, in which case the changes might be adaptive. However, long-term behavioral changes can lead to maladaptive mannerisms that are important signs of distress (not necessarily pain) in laboratory animals. These maladaptive behavioral traits indicate that medical intervention is required. Lack of grooming or excessive hair removal in rodents might suggest organic disease before other clinical symptoms are observed. Nonhuman primates are wasteful eaters and, as such, can become emaciated even though the animal appears to be consuming the appropriate diet. The areas of stress and discomfort are much harder to quantify, and there is therefore a great need to understand and determine how best to alleviate the causes. It is often impossible to determine whether an animal is undergoing a normal process of adapting to a stressing agent (stressor). However, an aggressive attempt to alleviate the stress for this individual animal might be ill advised and counterproductive. The research objective might be compromised to the extent that valid results are not obtained and could require the study to be repeated with more experimental animals involved. There is a great deal of disagreement on the meaning of terms such as comfort, well-being, discomfort, stress, fear, anxiety, pain, and distress. The mechanisms that contribute to the functional and recognizable state of well-being versus that of distress involve biochemical, physiological, and psychological changes that are poorly understood in animals. However, certain factors have been

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recognized to cause pain and stress and are outlined here. These factors generally apply to all mammalian species, as summarized in table 15.3. Table 15.3 Factors Recognized to Cause Pain and Stress in Laboratory Animals Causes of Pain

Causes of Stress

Injury Surgical manipulation Disease

Restraint Noise Odors and pheromones Presence of other species, including humansa Starvation and/or dehydration Fear, anxiety, boredom, and group housing of the same sexb

a b

Dogs enjoy the presence of humans. Especially in primates.

Many of the signs of stress (distress) listed in table 15.4 can become maladaptive responses and can become permanent parts of animals’ activities and seriously threaten their overall well-being. Any behavior that relieves the intensity or duration of stress is likely to become habitual. Such behaviors include coprophagy, hair pulling, self-trauma, and repetitive movements (cage circling). Table 15.4 Observable Signs of Severe Pain or Distress Local effectsa Skin corrosion Ocular injury Extremity injury Systemic effectsa Nervous signs Locomotor/muscular signs Respiratory signs Cardiovascular signs Gastrointestinal signs Behavioral effectsb Excessive vocalization Atypical actions Direct response to pain

Absence of actions a b

Severe erosion or ulceration penetrating most or all of dermis, large areas of severe necrosis Severe corneal opacity, corneal ulceration, purulent or bloody discharge, severe periocular necrosis Severe swelling, ulcerative lesions, apparent fractures, severe cutaneous erosion/sloughing, gangrenous appearance Severe or persistent tremors, convulsions, narcosis, catalepsy Ataxia, paralysis, prostration Gasping or labored breathing, very slow or rapid breathing, audible breathing (rales, wheezing) Very slow or rapid heart rate, severe pallor, redness or cyanosis of extremities Persistent vomiting, severe diarrhea, anorexia Squealing, grunting, growling, whimpering, howling (especially during movement or handling) Self-mutilation, stereotypic activity, restlessness, head shaking, apparent apathy to stimulants Licking, biting, or scratching of affected area; unusual posture to relieve pressure on affected area (hunched or stretched appearance); excessive struggling or biting during handling; grimacing or baring teeth Failure to groom, decreased socialization, poor reflexes, marked decrease in feed or water intake

Myers and DePass (1993). Mroczek, (1992); USDA (1995); see also Baumans (1994).

There is a requirement, mandated in 9 CFR, for the opportunity to exercise for dogs kept in individual cages. If these cages provide less than twice the space required for the size of the dog (size of dog is determined using formula found in 9 CFR, Part 3 of Subchapter A), these animals must be provided the opportunity to exercise. The exact exercise procedure is determined by the attending veterinarian and approved by the IACUC. In those institutions or facilities housing or using primates, there is a mandated requirement to provide “environmental enrichment to promote psychological well-being” of the species involved. These institutions and facilities must have a written plan that addresses the following:


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1. Social grouping. Based on the social needs of species as it exists in nature. 2. Environmental enrichment. Primary enclosures must be enriched to provide species-typical activities, such as, perches, swings, and foraging or task-oriented feeding methods.

Interaction with the primary care individual is an important element of this enrichment program. The attending veterinarian can exempt an individual primate from participation in the enrichment program based on medical considerations. The IACUC can also exempt an individual from participation in the program based on scientific reasons set forth in the protocol. Regardless of the reason for exemption, the exemption must be reviewed at least every 30 days by the veterinarian and at least annually by the IACUC.

PUBLICATIONS Code of Federal Regulations, Title 9, Chapter 1, Subchapter A: Animal Welfare. This code is available from USDA, APHIS/Animal Care, 4700 River Road, Unit 45, Riverdale, MD 20737–1234. This current version of the regulations developed by the USDA specifies how to comply with the AWA and its amendments. The subchapter is divided into four sections: Definitions, Regulations, Standards, and Rules of Practice Governing Proceedings Under the Animal Welfare Act. The Definitions section describes exactly what is meant by terms used in the legislation. “Animal,” for example, specifically excludes rats of the genus Rattus and mice of the genus Mus, as well as birds used in research. The Regulations section includes subparts for licensing, registration, research facilities, attending veterinarians and adequate veterinary care, stolen animals, records, compliance with standards and holding periods, and miscellaneous topics such as confiscation and destruction of animals and access and inspection of records and property. The bulk of the subchapter is the third section, which provides standards for specific species or groups of species. Included are sections for cats and dogs, guinea pigs and hamsters, rabbits, nonhuman primates, marine mammals, and the general category of “other warm-blooded animals.” Standards include those for facilities and operations, health and husbandry systems, and transportation. The final section sets forth the Rules of Practice applicable to adjudicating administrative proceedings under Section 19 of the Animal Welfare Act. Animal Care Policies The policy manual gives policies issued by APHIS/Animal Care that clarify the AWA regulations. Among the topics covered are “Written Narrative for Alternatives to Painful Procedures,” “Space and Exercise Requirements for Traveling Exhibitors,” and “Annual Report for Research Facilities.” Originally issued in April 1997, new policies can be added at any time and are included in the manual.

PROFESSIONAL ORGANIZATIONS There are several professional groups that provide special information to the institution and the IACUC in areas of education, training, or certification. A brief description of some of these organizations follows. American Association for Accreditation of Laboratory Animal Care The AAALAC promotes high-quality animal care and use through a voluntary accreditation program. Institutions using, maintaining, or breeding laboratory animals for biological research can

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apply for accreditation. The NIH accepts full accreditation by AAALAC as assurance that the animal facilities are in compliance with Public Health Service policy. AAALAC is accordingly a nongovernmental organization. American Association for Laboratory Animal Science The AALAS is made up of individuals and institutions professionally concerned with the production, care, and use of laboratory animals. The organization collects and exchanges information on all phases of laboratory animal care and management through the publication of a journal (Laboratory Animal Science), bulletins, and other documents. The AALAS animal technician certification is an important method to develop uniform requirements and training programs that lead to certification of technicians based on their experience and knowledge. American Veterinary Medical Association The AVMA is a major international organization of veterinarians. Its objective is to advance the science and art of veterinary medicine, including its relationship to public health and agriculture. It sponsors specialization in veterinary medicine through the formal recognition of specialty certifying organizations, including the American College of Laboratory Animal Medicine (ACLAM) and the American College of Veterinary Pathologists (ACVP). American College of Laboratory Animal Medicine The ACLAM is a specialty board founded in 1957 to encourage education, training, and research in laboratory animal medicine (ACLAM 1996). Several professional societies such as the Society of Toxicology (SOT), the ACVP, and the Federation of American Societies for Experimental Biology have developed and published “position” papers on the use of animals in experimentation (i.e., Guiding Principles in the Use of Animals in Toxicology, adopted by the SOT in July 1991).

REFERENCES Acha, P. M. and Szyfrei, B. (1980). Zoonoses and communicable diseases common to man and animals (Scientific Publication No. 354). Washington, DC: World Health Organization. Acred, P., et al. (1994). Guidelines for the welfare of animals in rodent protections tests: A report from the Rodent Protection Test Working Party. Lab Anim. 28, 13–18. American Association for Laboratory Animal Science. (1997). AALAS technician certification program: Candidate bulletin. Cordova, TN: AALAS. American College of Laboratory Animal Medicine. (1996). Report of the American College of Laboratory Animal Medicine on adequate veterinary care in research, testing, and teaching. Chester, NH: Author. American Veterinary Medical Association (AVMA) Panel on Euthanasia. (1993). 1993 report of the AVMA Panel on Euthanasia. J. Am. Vet. Med. Assoc. 202, 229–249. Animal and Plant Health Inspection Service, U.S. Department of Agriculture. (1989, August 31). Fed. Reg. 54, 36112–36163. Association for Assessment and Accreditation of Laboratory Animal Care International. (1997b). Snapshots of animal research regulations across Europe. AAALAC Int. Conn. August 11. Association for Assessment and Accreditation of Laboratory Animal Care International. (1997c). U.S. and European guidelines more alike than different: A side-by-side comparison. AAALAC Int. Conn. August 11. Baumans, V. (1994). Pain and distress in laboratory rodents and lagomorphs: Report of the Federation of European Laboratory Animal Science Association (FELASA) Working Group on Pain and Distress. Lab. Anim. 28, 97–112.


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Bayne, K., and Martin, D. (1998). AAALAC International: Using performance standards to evaluate an animal care and use program. Lab Anim. 27, 32–35. Bennett, B. T. (1994). Alternative methodologies. In Essentials for animal research: A primer for research personnel (2nd ended.), eds. B. T. Bennett, M. J. Brown, and J. C. Schofield, 9–17. Beltsville, MD: National Agricultural Library. British Cruelty to Animals Act of 1876. (1876). Cooper, J. E. (1990a). Management, health and welfare of laboratory animals in developing countries. SCAW News. 13(1), 14–15. Cooper, J. E. (1990b). Management, health and welfare of laboratory animals in developing countries: Part Two. SCAW News. 13(2), 12–14. Council directive on the approximation of laws, regulations, and administrative provisions of the member states regarding the protection of animals used for experimental and other scientific purposes. (1986). Council of European Union, Brussells, Belgium. Dell, R. B. (1994). Interacting with the IACUC. Lab. Anim. 23, 34–35. Fano, A. (1997). Lethal laws. New York: Zed Books. French, R. D. (1975). Antivivisection and medical science in Victorian society. Princeton, NJ: Princeton University Press. Freudinger, U. (1985). Public initiative for the abolition of vivisection, called the “Weber Initiative” [English translation]. Schweiz Arch. Tierheilkd. 127, 635–649. Gad, S. C. (2001). Regulatory toxicology (2nd ed.). Philadelphia: Taylor & Francis. Gad, S. C., and Chengelis, C. P. (1998). Acute toxicology testing perspectives and horizons (2nd ed.). San Diego, CA: Academic Press. Hampson, J. (1979). Animal welfare: A century of conflict. New Scientist. 84, 280–282. Howard-Jones, N. (1985). A CIOMS ethical code for animal experimentation. WHO Chron. 39, 51–56. Laboratory Animal Welfare Act. (1966). United States P.L. 89-544, amended by the Animal Welfare Act of 1970 (P.L. 91-597), 1976 (P.L. 94-279), 1985 (P.L. 99-198) and 1990 (P.L. 101-624). Miller, J. (1998). International harmonization of animal care and use: The proof is in the practice. Lab Anim. 27(5), 28–31. Mroczek, N. S. (1992). Recognizing animals suffering and pain. Lab. Anim. 21, 27–31. Myers, R. C., and DePass, L. R. (1993). Acute toxicity testing by the dermal route. In Health risk assessment: Dermal and inhalation exposure and absorption of toxicants, eds. R. G. M. Wang, J. B. Knaak, and H. I. Maibach, 167–199. Boca Raton, FL: CRC Press. National Institutes of Health. (1985). Guide for the care and use of laboratory animals (NIH Publication No. 86-23). National Research Council. (1998). The psychological well-being of nonhuman primates. Washington, DC: Institute for Laboratory Animal Research. National Academy Press. National Research Council. (1996). Guide for the care and use of laboratory animals. Washington, DC: National Academy Press. National Research Council. (1994). Laboratory animal management: Dogs. Washington, DC: National Academy Press. Netherlands Animal Welfare Society. (1991). Dutch animal welfare office makes progress. ATLA News Views. 19, 389. Nevalainen, T. (1999). FELASA guidelines for education of specialists in laboratory animal science (Category D). Lab Anim. 33, 1–15. Russell, W. S., and Burch, R. L. (1959). The principles of humane experimental technique. London: Methuen & Co. Society of Toxicology. (1991). Guiding principles in the use of animals in toxicology. Reston, VA: Author. Silverman, J. (1994). IACUC handling of mistreatment or non-compliance. Lab. Anim. 23, 30–32. U.S. Department of Agriculture. (1995). Subchapter A: Animal Welfare. 9 CFR Ch. 1, 1.1-4.11. Washington, DC: U.S. Department of Agriculture.

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ADDITIONAL RESOURCES American Association for Accreditation of Laboratory Animal Care 11300 Rockville Pike, Suite 121, Rockville MD 20852-3035, USA American Association of Laboratory Animal Science 70 Timber Creek Drive, Suite 5, Cordova, TN 38018, USA American College of Toxicology 9650 Rockville Pike, Bethesda, MD 20814-3998, USA American Society of Primatologists Regional Primate Center University of Washington, Seattle, WA 98195, USA American Veterinary Medical Association 1931 North Meachum Road, Suite 100 Schaumburg, IL 60173-4360, USA Animal and Plant Health Inspection Service U.S. Department of Agriculture 4700 River Road, Unit 84 Riverdale, MD 20737-1234, USA Animal Welfare Information Center National Agricultural Library U.S. Department of Agriculture 10301 Baltimore Boulevard, Room 205 Beltsville, MD 20705, USA Australian and New Zealand Council for the Care of Animals in Research and Teaching, Limited P.O. Box 19, Glen Osmond SA 5064, Australia Canadian Association for Laboratory Animal Science CALAS National Office Biosciences Animal Service University of Alberta Edmonton, Alberta T6G 2E9, Canada Canadian Council on Animal Care Constitution Square 350 Albert Street, Suite 315 Ottawa, Ontario K1R 1B1, Canada European Center for Validation of Alternative Methods TP 580, JRC Environmental Institute 21020 Ispra (VA), Italy


Foundation for Biomedical Research 818 Connecticut Avenue NW, Suite 303 Washington, DC 20006, USA Institute for Laboratory Animal Resources National Academy of Sciences 2101 Constitution Avenue NW Washington, DC 20418, USA Institute of Laboratory Animal Science University of Zürich Winterhurerstrasse 190 8057 Zürich, Switzerland International Council for Laboratory Animal Science University of Kuopio SF-70211 Kuopio 10, Finland Public Responsibility in Medicine and Research 132 Boylston Street, Fourth Floor Boston, MA 02116, USA Scientists Center for Animal Welfare Golden Triangle Building One 7833 Walker Drive, Suite 340 Greenbelt, MD 20770, USA Society of Toxicology 1767 Business Center Drive Suite 302 Reston, VA 20190-5332, USA The Johns Hopkins Center for Alternatives to Animal Testing 111 Market Place, Suite 840 Baltimore, MD 21202-6709, USA Universities Federation for Animal Welfare 8 Hamilton Close South Mimms, Potters Bar Hertfordshire EN6 3QD, UK

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APPENDIX

Commercial Sources of Laboratory Animals Shayne C. Gad Gad Consulting Services

Ace Animals Alder Ridge Animal Technologies Ltd. B&K Universal Ltd. Barton’s West End Buckshire Corp Camm Research Lab Animals CEDS Charles River Covance Research Products Cytogen Davidson’s Mill Ellegard Minipigs Elm Hill Gemini Research Harlan Haycock Kennels Hazleton Hilltop Isoquimen Kiser Lake Kennels Kralek Farms Liberty Research Lone Star Laboratory Swine Marshall BioResources Millbrook Breeding Lab Myrtle’s Rabbitry Osage Research Primates Primate Products Rana Ranch Bullfrogs

Minipig

Primate

Guinea Pig

X X

Rabbit

X

Dog

X

Ferret

X

Hamster

Mouse

Source

Rat

Species

X X X

X X

X

X

X

X

X

X X

X

X X

X X

X X X X X

X

X

X

X

X X X

X X

X

X

X X

X X

X X

X X

X X

X X X X X

X

X

X

921

922


Ridglan Farms Robinson Services S&S Farms Sasco Scientific Resources Intl. Simonsen Laboratories Sinclair Research Center Taconic The Jackson Laboratory Triple F Farms Western Oregon Rabbit Co White Eagle Worldwide Primates Xenogen Zivic Laboratories a

Minipig

Primate

Guinea Pig

Rabbit

Dog

Ferret

Hamster

Source

Mouse

Rat

Species

X X X X

X

X

X

X

X

X X

X X

X X X

X X X X X X

X X

This list is representative and is not meant to be exhaustive. The reader is referred to http://guide.labanimal.com/guide/index.html for a more complete, searchable listing.

CONTACT INFORMATION Ace Animals Inc. PO Box 122 Boyertown, PA 19512 USA Phone: 610-367-6047 Fax: 610-367-6048 E-Mail: [email protected] Web: http://www.aceanimals.com

B&K Universal Ltd. Grimston, Aldbrough Hull, East Yorkshire HU11 4QE UK Phone: +44 1964 527555 Fax: +44 1964 527006 E-Mail: [email protected] Web: http://www.bku.com

Alder Ridge Farms Inc. PO Box 290 Lakewood, PA 18439 USA Phone: 717-727-3458 Fax: 717-727-3459

Barton’s West End 161 Janes Chapel Rd. Oxford, NJ 07863 USA Phone: 908-637-4427 Fax: 908-637-4268 E-Mail: [email protected]

Animal Technologies Ltd. 4435 First St., #350 Livermore, CA 94550 USA Phone: 510-490-3036 Fax: 510-656-1921 Toll Free: 800-USA-MICE E-Mail: [email protected] Web: http://www.atbk.net

Buckshire Corp. PO Box 155 Perkasie, PA 18944 USA Phone: 215-257-0116 Fax: 215-257-9329 E-Mail: [email protected]

COMMERCIAL SOURCES OF LABORATORY ANIMALS

Camm Research Lab Animals 414 Black Oak Ridge Rd. Wayne, NJ 07470 USA Phone: 201-694-0703 Fax: 201-694-8905 CEDS Les Souches Mezilles, 89130 France Phone: +33 386 454058 Fax: +33 386 454058 E-Mail: [email protected] Charles River Laboratories 251 Ballardvale St. Wilmington, MA 01887 USA Phone: 978-658-6000 Fax: 978-658-7132 Toll Free: 800-522-7287 E-Mail: [email protected] Web: http://www.criver.com Covance Research Products PO Box 7200 Denver, PA 17517 USA Phone: 717-336-4921 Fax: 717-336-5344 Toll Free: 800-345-4114 E-Mail: [email protected] Web: http://www.crpinc.com Cytogen Research and Development, Inc. 89 Bellevue Hill Rd. West Roxbury, MA 02132 USA Phone: 617-325-7774 Fax: 617-327-2405 Davidson’s Mill Breeding Laboratories 231 Fresh Pond Rd. Jamesburg, NJ 08831 USA Phone: 732-821-9094 Fax: 732-821-5922 Toll Free: 888-252-6790 Ellegaard Minipigs USA, Inc. 2025 Ridge Rd. Perkasie, PA 18944 US Phone: 215-258-5090/215-258-5091 Fax: 215-257-9329 E-Mail: [email protected] or [email protected]

923

Elm Hill Breeding Labs, Inc. 7 Kidder Rd. Chelmsford, MA 01824 USA Phone: 978-256-2322 Fax: 978-256-2545 Toll Free: 800-941-4349 E-Mail: [email protected] Web: http://www.elmhilllabs.com Gemini Research of Alabama 125 Aspen Ln. Odenville, AL 35120 USA Phone: 205-629-3229 E-Mail: [email protected] Web: http://www.brownfamilyenterprises.com Harlan PO Box 29176 Indianapolis, IN 46229 USA Phone: 317-894-7521 Fax: 317-894-1840 E-Mail: [email protected] Web: http://www.harlan.com Haycock Kennels, Inc. 629 Apple Rd. Quakertown, PA 18951 USA Phone: 215-536-8228 Fax: 215-536-7337 Hilltop Lab Animals PO Box 183, RD #1, Hilltop Scottdale, PA 15683 USA Phone: 724-887-8480 Fax: 724-887-3582 Toll Free: 800-245-6921 E-Mail: [email protected] Web: http://hilltoplabs.com Isoquimen SL E-Mail: [email protected] Kiser Lake Kennels PO Box 541 St. Paris, OH 43072 USA Phone: 937-362-3193/937-362-4071 Fax: 937-362-3193 E-Mail: [email protected]

924


Kralek Farms, Inc. 630 W. Clausen Turlock, CA 95380 USA Phone: 209-634-3609 Fax: 209-634-3609 E-Mail: [email protected] Web: http://kralek.com

Primate Products, Inc. 7780 NW 53 St. Miami, FL 33166 USA Phone: 305-471-9557 Fax: 305-471-8983 E-Mail: [email protected] Web: http://www.primateproducts.com

Liberty Research, Inc. PO Box 107, Rte. 17C Waverly, NY 14892 USA Phone: 607-565-8131 Fax: 607-565-7420 E-Mail: [email protected]

Rana Ranch Commercial Bullfrogs PO Box 1043 Twin Falls, ID 83303 USA Phone: 208-734-0899 Fax: 208-734-0899 Toll Free: 877-737-FROG E-Mail: [email protected] Web: http://www.ranaranch.com

Lone Star Laboratory Swine 1252 West Kingsbury Seguin, TX 78155 USA Phone: 830-401-5958 Fax: 830-401-5920 E-Mail: [email protected] Web: http://www.miniswine.com Marshall BioResources 5800 Lake Bluff Rd. North Rose, NY 14516 USA Phone: 315-587-2295 Fax: 315-587-2109 E-Mail: [email protected] Millbrook Breeding Labs PO Box 513 Amherst, MA 01004 USA Phone: 413-253-5083 Fax: 413-253-0562 E-Mail: [email protected] Myrtle’s Rabbitry, Inc. 4678 Bethesda Rd. Thompson Station, TN 37179 USA Phone: 615-790-2349 Fax: 615-794-9263 Toll Free: 800-424-9511 E-Mail: [email protected] Web: http://www.myrtles.com Osage Research Primates, LLC 54 Hospital Dr. Osage Beach, MO 65065 USA Phone: 573-348-8002 Fax: 573-348-1622

Ridglan Farms, Inc. PO Box 318 Mt. Horeb, WI 53572 USA Phone: 608-437-8670 Fax: 608-437-4731 E-Mail: [email protected] Robinson Services, Inc. PO Box 1057 Mocksville, NC 27028 USA Phone: 336-940-2550 Fax: 336-940-5260 E-Mail: [email protected] S & S Farms 1650 Warnock Dr. Ramona, CA 92065 USA Phone: 760-788-7007 Fax: 760-788-7042 E-Mail: [email protected] Web: http://www.snsfarms.com Sasco, Inc. 251 Ballardvale St. Wilmington, MA 01887 USA Phone: 978-658-6000 Fax: 800-255-8964 Toll Free: 800-228-4919 E-Mail: [email protected] Web: http://www.criver.com Scientific Resources International, Ltd. 432 Ridge St. Reno, NV 89501 USA Phone: 775-786-3244 Fax: 775-786-2767

COMMERCIAL SOURCES OF LABORATORY ANIMALS

Simonsen Laboratories, Inc. 1180-C Day Rd. Gilroy, CA 95020 USA Phone: 408-847-2002 Fax: 408-847-4176 E-Mail: [email protected] Web: http://www.simlab.com Sinclair Research Center, Inc. PO Box 658 Columbia, MO 65205 USA Phone: 573-387-4400 Fax: 573-387-4404 E-Mail: [email protected] Web: http://www.sinclairresearch.com Taconic 273 Hover Ave. Germantown, NY 12526 USA Phone: 518-537-6208 Fax: 518-537-7287 Toll Free: 888-822-6642 E-Mail: [email protected] Web: http://www.taconic.com The Jackson Laboratory 600 Main St. Bar Harbor, ME 04609 USA Phone: 207-288-6000; 207-288-5845 Fax: 207-288-6150 Toll Free: 800-422-MICE (for orders only) Web: http://www.jax.org/jaxmice Triple F Farms, Inc. Rd. #2, Box 432 Sayre, PA 18840 USA Phone: 570-888-4874 Fax: 570-888-5017 Toll Free: 866-817-1756 E-Mail: [email protected]

925

Western Oregon Rabbit Co. PO Box 653 Philomath, OR 97370 USA Phone: 541-929-2245 Fax: 541-929-2654 White Eagle Laboratories, Inc. 2003 Lower State Rd. Doylestown, PA 18901 USA Phone: 215-348-3868 x123 Fax: 215-348-5081 E-Mail: [email protected] Web: www.whiteeagletox.com Worldwide Primates, Inc. PO Box 971279 Miami, FL 33197 USA Phone: 305-378-9585 Fax: 305-232-3838 E-Mail: [email protected] Xenogen Biosciences 5 Cedar Brook Dr. Cranbury, NJ 08512 USA Phone: 609-860-0806 Fax: 609-860-8515 E-Mail: [email protected] Web: http://www.xenogen.com Zivic Laboratories, Inc. PO Box 101084 Pittsburg, PA 15237 USA Phone: 724-452-5200 Fax: 800-626-7287 Toll Free: 800-422-5227 E-Mail: [email protected] Web: http://www.zivic.com

DK2079_C017.fm Page 927 Monday, September 25, 2006 7:06 AM

Index 2-acetylaminofluorene, 478 2,4-toluene diisocyanate, 400 6-aminonicotinamide, 472 48-hour contact test, 776 α2u globulin, 202

A Absorption, 8 Absorption, distribution, metabolism, and excretion (ADME), 8, 25, 27 Accutane, 4, 5 Acetaminophen, 4, 245, 737, 766 Acetylation, 237 Acrylamide, 4 Acute toxicity studies, 48–49 Adenocarcinoma, 83, 93, 151 Adrenal cortex, 243 Adrenal-associated endocrinopathy, 548 Age, 853–854 AHH, 129 AIDS, 666 Alcohol metabolism, 8 Aleutian mink virus, 7, 510, 543 Alopecia, 100, 345, 393, 450, 587, 592 Ambulatory intravenous infusion, 687 Amphetamine, 125 Amyloidosis, 100, 119 Anal musk gland removal, 508 Anemia, 793 Anesthesia, 437 Animal and plant health inspection service (APHIS), 903–905 Animal Welfare Act, 571, 666, 667, 678, 905 Animals and man, 4 Antibiotic toxicity, 380, 401 Antibodies, 224 Antipyrine, 870 Aplastic anemia, 511, 795 Armadillo, 774 Armstrong test, 338, 367 Aromatic amines, 374, 720 Aromatic hydrocarbon hydroxylases (AHHs), 126 Arthritis, 209, 745 Artifacts, 792 Asbestos, 4 Astrocytoma, 110

Atenolol, 766 Atherosclerosis, 666 Atrial thrombosis, 79 Audiology, 336 AZT, 4, 832

B B6C3F1, 72 Bacterial pneumonia, 708 Benzo(a)pyrene, 478 Bladder calculi, 117 Blood pressure, 186 Body surface area, 841 Body weights, 692 Bone modeling and remodeling, 595 Bronchial-associated lymphoid tissue (BALT), 751 BSL-1, 671 BSL-4, 671

C Cage cards, 37 Caging, 5 Campylobacteriosis, 708 Canine distemper virus (CDV), 510, 543, 575 Canine hepatitis, 575 Cannulation, 190 Capsule, 171, 578 Carcinogenesis, 541 Carcinogenicity studies, 51–53, 164, 166 Carcinogenicity testing, 226 Cardiac disease, 745 Cardiac puncture, 189 Cardiomyopathy, 78, 548 Cardiotoxic effects of catecholamines, 402 Cardiovascular safety assessment, 703 Cardiovascular toxicity, 738 Cataractogenesis, 338 Cataract(s), 113, 390 Catatonia, 8 Cats, 8 CD-1, 5, 27–28, 72–74 Cellular immune response, 705 CF-1, 27–28 927


928


Chair restraint technique, 686 Changes in kidney function in pregnancy, 876 Cholangiocarcinoma, 108 Cholangiofibrosis, 105 Cholangioma, 108 Choosing species and strains, 5 Chronic toxicity studies, 51, 52 Cirrhosis, 105 Classification systems used to characterize liver impairment, 869–873 Clearance of selected benzodiazepine in young males and females, 884 Clinical chemistry values, 196 Clotting factors, 523 Coagulation, 799 Collection site, 791 Common methods of euthanasia in the toxicology laboratory, 906 Common routes of administration, 8 Comparative human acute lethal doses and animals LD 50s, 891 Comparative xenobiotic metabolism, 12 Complement, 337 Complement-4 deficiency, 400 Conjugate reactions, 236 Conjugative metabolism, 649 Conversion of animal doses to HEDs, 843, 845 Coronavirus, 461, 543 Cortical hyperplasia, 84 Creatinine clearance, 865–868 Cross-species extrapolation, 839 Cyclosporine, 4 CYP-450 oxidative metabolism, 870 CYP1A, 760 CYP1A1, 401 CYP1A2, 401 CYP2A, 761 CYP2B, 761 CYP3A, 762 CYP3A4, 219 CYP3A14, 401 CYP3A15, 401 CYP3A15, 401 CYP4A13, 401 CYP11B2, 401 CYP17, 401 Cystadenocarcinoma, 89 Cystadenoma, 89 Cytochrome P-450, 11, 125, 217, 218, 228, 246, 476–477, 716

D Deficiencies in vitamin E, 453 Delayed contact dermal sensitization, 338 Delayed neuropathology, 853 Delayed-type hypersensitivity (DTH), 705 Dermal carcinogenesis, 28, 57 Dermal toxicity, 738 Dermatosis of the ear, 593

Developmental toxicity assessment, 353, 541, 701 Diabetes mellitus, 666 Diarrhea, 676 Diazepam, 125, 351 Dietary admixture, 60 Dietary method, 168 Disease, 854–855 Distribution, 10 Dog(s) 4-week toxicity study, 585 13-week toxicity study, 585 beagle, 567 CYP-specific metabolic activities in, 647 feed consumption, 568 growth rate curves in, 569 clinical observations in normal dogs, 580 domestication, 567 exercise, 909 nutrient requirements for, 574 DNA vaccines, 499 Dosage, 7 Dose, 7 Dosimetry, 7 Draize scale for scoring skin reaction, 439 Drinking water studies, 61–62

E Ear notching or punching, 37–38 Ear tags, 37 Earthworm(s), 774 48-hr contact test, 776 toxicity rating in, 777 ECG waveform, 184, 697 Effect of diet, 791 Electrocardiography, 183, 581 Elixir of sulfanilamide, 3 Emesis, 511, 587 Endocardiosis, 616 Endometrial adenocarcinoma, 91 Environmental factors, 894–897 Epoxide hydrolase, 235 Erythrocytes, 793 Erythron, 617 Excretion, 14–15 Extrahepatic metabolism, 722 Extrahepatic xenobiotic metabolism, 243 Extramedullary hematopoiesis, 94, 103

F Fentanyl, 351 Ferret-legging, 498 Ferret(s), 7 acute dermal testing in, 540 acute inhalation toxicity in, 540 acute oral testing in, 540 black-footed, 497


INDEX

929

domesticated, 497 growth curve in, 515 hematology values for adult, 521 hepatic xenobiotic metabolizing enzymes in, 551 normative physiological data, 517 P-450 levels in, 550 sample-size requirements, 526 subchronic toxicity testing in, 541 Fish, 776 examples of carcinogenicity, 782 Flavine-dependent mixed function oxidase (FMFO), 234, 721 Follicular cell adenoma, 87 Follicular center cell lymphoma, 96 Food and Drug Administration (FDA), 2, 438, 508, 733, 834, 842, 848, 867 Food restriction, 158 Footpad injection, 348 Four Rs, 2 Fragmentation anemia, 796 Functional observational battery (FOB), 181, 182

G Gender differences, 791 General reviews of xenobiotic metabolism, 8 Genetic definition, 886–890 Genital herpesvirus, 399 Genobiotic metabolism, 864–868 Giardia, 577 Glomerular nephropathy, 207 Glucuronide conjugates, 14, 128, 238 Glutathione, 240, 241 Glutathione S-transferase, 481 Glutathione transferase activity, 480 Glycine conjugation pathway, 482 Glycogenic vacuolation, 104 Good laboratory practice (GLP), 36–37, 45, 212 Gossypol, 737 Göttingen, 740 Guide for the care and use of laboratory animals, 903 Guinea pig maximization test, 358 weight curves, 342 Gunn rat, 229, 240 Gut-associated lymphoid tissue (GALT), 454, 751

H Hair ball, 449 Hanford, 740 Harber test, 338, 365 Harderian gland, 114, 473 Hardian gland adenocarcinoma, 114 Hardian gland adenoma, 114 Harvey, William, 24 Heart rate, 185 Heartworm, 577 Heinz body anemia, 794

Helicobacter mustelae, 544 Hemangioma, 78 Hemangiosarcoma, 78 Hemodynamic parameters throughout pregnancy, 876 Hemorrhagic syndrome, 740 Hemosiderosis, 104 Hepatic clearance, 877 Hepatic impairment, 868 Hepatitis, 711 Hepatoblastoma, 108 Hepatocellular adenoma, 107 Hepatocellular carcinoma, 107 Heritable translocation assay, 55–56 Herpes simiae B virus, 710 Herpesvirus tamarinus, 711 High-dose toxicity testing, 838 Histamine, 378 Holtzman, 230 Hormel, 740 Host resistance test, 370 Host-mediated assay, 56 How species are actually selected, 835 Human equivalent dose (HED), 834, 842, 848 Human influenza virus, 499 Humoral immune response, 705 Hyaline glomerulonephropathy, 115 Hybrid vigor, 24 Hydra, 774 Hydrocephalus, 109 Hydrometra, 90 Hydronephrosis, 115 Hyperbilirubinemia, 807 Hypercalcemia, 808 Hyperglycemia, 800–801 Hyperphosphatemia, 808 Hyperproteinemia, 802 Hypervitaminosis D, 397 Hypocalcemia, 808 Hypoglycemia, 800–801 Hypophosphatemia, 808 Hypoproteinemia, 802–803

I Immediate hypersensitization, 338 Immune-mediated hemolytic anemia, 794 Immunology, 336 Immunomorphological classification of murine lymphomas, related leukemias, 96 Immunotoxicology, 704 Impaired GI absorption, 873 Implantable electronic microchips, 39 In vivo skin permeability, 425 Influence of gender on xenobiotic absorption, disposition, and toxicity, 878–885 Influence of liver disease on pharmacokinetics, 871 Influenza, 509 Infusion, 174 Inhalation, 68–69


930


Inhalation route, 179 Innate immune response, 706 Institutional Animal Care and Use Committee (IACUC), 667, 905–909 Institutional policy and regulatory issues, 667 Internal regulations, 911–913 Interstitial cell tumors, 99 Interstitial pneumonia, 758 Intradermal injection, 67, 346, 434 Intramuscular injection, 65–66, 348 Intramuscular route, 177, 434, 579, 689 Intranasal route, 178, 690 Intraocular dosing, 691 Intraperitoneal injection, 64–65, 348, 434 Intraperitoneal route, 176, 579, 690 Intratracheal administration, 180, 433 Intravasive cardiovascular procedures, 700 Intravenous infusion volumes and rates, 512 Intravenous injection, 62–64 Intravenous route, 172 Iron deficiency anemia, 795 Islet cell adenoma, 88 Islet cell carcinoma, 88 Ivermectin, 676

J Jacket and tether infusion technique, 686 Japanese medaka, 779, 783

K Karyomegaly, 104 Keratoacanthoma, 103 Ketamine, 351, 506 Ketosis, 397

L Laboratory Animal Welfare Act, 902 Laboratory measurements of age-related changes, 790 Large fibroma, 215 Lash lure, 3 Lassa fever, 399 Latex allergy, 400 Lavoisier, 337 Lead, 737 Leading causes of death in women, 880 Leewenhoeck, 424 Leiomyoma, 83, 91, 467 Leiomyosarcoma, 83, 91, 467 Leptospirosis, 575 Leukemia, 798 Leukocytes, 796 Leukon, 617 Licensing, 678

Limitations of models, 837 Lipofuscin, 84, 89, 104 Liver disease, 604 Liver lymphocyte index, 537 Locomotor activity, 183 Long-Evans, 230 Lymphosarcoma, 470, 547

M Magnusson and Kligman, 358 Mast cell tumor, 102 Mastitis, 593 Maximum tolerated dose (MTD), 162, 842 Measles, 678 Medaka carcinogenicity study, 783 Median survival rate, 27 Medullary hyperplasia, 84 Megaloblastic anemia, 796 Meningioma, 110 Mercury, 4 Metabolism, 10 Metastatic tumors, 113 MFO, 764 Microbiological definition, 885–886 Microgranulomas, 104 Microsomal multifunction oxidase (MMFO), 10, 11, 219, 221, 225, 228, 230–235, 243, 245, 246, 550, 648 induction, 224 inhibition, 223 Minipig clinical chemistry parameters, 735 CYP-450 enzyme activities in, 760 hematological parameters, 735 in toxicity testing, 733 reproductive toxicity in, 736 teratogenicity in, 736 Modified Beuhler procedure, 354 Monkey New World monkey, 665 Old World monkeys (cynomolgus), 665 xenobiotic metabolism in, 717 Morphine, 8, 240 Mouse (mice) C57B6/F1 (mice), 45 CYP activity in, 124 disease models in inbred strains of, 888 genetically engineered, 120 hepatic enzymes in, 123 induction of mammary tumors in, 93 p53+/– knockout, 120 RasH2 transgenic mouse, 122 Tg.AC mouse, 121 Mouse ear swelling test (MEST), 57 Mouse micronucleus assay, 54–55 Mucoid enteritis, 460 Mucoid enteropathy, 447 Murine mammary tumor virus infection (MuMTV), 92


INDEX

931

Mycoplasmosis, 199 Myocardial mineralization, 76

N N-alkylnitrosureas, 736 Nasal cavity, 608 Nasogastric dose administration, 682 Neonatal administration, 171 Nephroblastoma(s), 116, 465 Neurobehavioral examination, 181 Neurofibroma, 101 Neurofibromasarcoma, 101 Neurological examination grading system, 696 NMDA, 701 Noise, 32, 155, 427 Nonhuman primates blood chemistry, 694 dosing volumes, 680 hematology values, 693 psychological well-being of, 909–910 Normal body weights, 28 Normal physiological values, 26 Normal range, 789 NSAID, 736 Nutrient requirements, 40 Nutritional condition, 890

O Ocular administration, 433 Ocular toxicology, 704 Oligodendroglioma, 110 Ophthalmologic examinations, 696 Oral administration, 681 Oral route, 167 Organ weights and transformations, 532 Otoxicity, 338, 372 Ovarian cysts, 88

P Pain and distress, 913–915 Pair housing, 672 Pancreas, 81 Patellar reflexes, 696 Periocular dosing, 691 Peroxisomal proliferation, 233 Peroxisomal-inducing agents, 720 Pharmacokinetic evaluations, 700 Phenacetin, 4 Phenol, 10 Pheochromocytoma, 85 Photoirritation/toxicity, 442 Photosensitization, 338, 365 Physiological state, 855

Pinworms, 82, 198 Pituitary adenoma, 216 Pododermatitis, 209 Polyarteritis, 78 Polybrominated biphenyls, 737 Polycythemia, 796 Polysorbate-induced histamine reaction, 587 Postrenal azotemia, 803 Preanesthetics, 507 Pregnancy, 875–878 Prerenal azotemia, 803 Priestley, 2 Primary tumors, 75 Primate cytochrome P-450 isoenzymes, 717 Proliferative colitis, 510 Pyrogen test, 443

Q Quarantine, 676

R Rabbit(s) acute dermal toxicity studies, 442 acute primary dermal irritation studies, 441 acute primary eye irritation studies, 440 CYP-450 isoenzyme activities, 400, 476, 717 dermal toxicity studies with rabbits, 444 teratology studies, 445 urine, 428 xenobiotic drug metabolizing enzymes in, 475 Rabies, 544, 575 Radiculoneuropathy, 208 Randomization, 586 Rat(s) 14- or 28-day repeat dose toxicity study, 163 embryo-fetal development, 165 Fischer 344 rats, 6, 150, 245 background tumor incidences in, 214 functional observational battery (FOB) in, 182 growth rates, 153 hematology values of common strains of, 197 liver microsomes (36), 220 maximum tolerated dose study in, 162 metabolizing enzymes in, 220 Norway rat, 150 P-450 CYP-specific metabolic activities in, 218 Sprague-Dawley rat, 6, 150, 230, 245 Recommended housing conditions for selected laboratory animal species, 904 Rectal route, 178, 580 Reference ranges, 789 Regenerative anemia, 793 Renal azotemia, 803 Renal hyaline droplets, 202 Renal impairment, 864–868


932


Renal tubular mineralization, 203 Reproductive toxicology, 701 Retinal atrophy, 113 Rhabdomyolysis, 666 Rhesus baboon, 665 Rotavirus, 461

S S7A (ICH), 703 Safety factor, 849 Salmonellosis, 708 Scent glands, 497 Scurvy, 345, 387 Secondary source of hematopoiesis, 619; see also Oral route; Species differences; Strain differences Segment I, 164 Segment II, 164 Segment III, 164 Selecting an animal model, 5 Sendai virus, 112, 198 Serous atrophy of bone marrow fat cells, 753 Serum chemistry values for adult ferrets, 524 Sex, 851–852 Sex-related differences, 227 Shalita test, 338, 365 Shedding, 587 Sheep, 774 Shigellosis, 707 Shoebox caging, 155 Short-term toxicity studies, 50 Sialodacryoadenitis, 197 Signs of severe pain or distress, 914 Simian immunodeficiency virus (SIV), 713 Simian retroviruses (SRVs), 678, 712 Sinclair pig, 740 SKF 525A, 129 Skin painting studies, 28, 57 Snuffles, 463 Social housing, 672 Socialization, 572 Solid-bottom cages, 34–35 Special populations, 850–851 Species cases in species selection, 836 Species differences, 151, 812 Species peculiarities, 891 Species variation, 891 Species-specific toxic effects, 857–858, 891 Specific pathogen free (SPF), 161, 509 Split adjuvant test, 361 Spontaneously neoplastic lesions, 29 Squamous cell carcinoma, 81 Squamous cell hyperplasia of the forestomach, 82 Squirrel marmosets, 665 Squirrel monkey, 665 Steatosis, 209 Strain, 24 Strain differences, 151, 228, 477, 791 Straub tail, 8

Stress, 791, 852–853 Subcutaneous injection, 66–67, 434, 578, 689 Subcutaneous route, 177 Suicide substrates, 223 Sulfanilamide, 3 Sulfate conjugates, 237 Sunburn response, 738 Susceptibility factors, 890 Suspended caging, 157 Synthesis inhibitors, 223

T Tail vein infusions, 173 Tape stripping, 349–350 Tattoos, 38 Teratogenicity, 441, 736 Teratology studies, 53–54 Thalidomide, 2, 3, 4, 438, 666 Thrombocytopenia, 799 Thrombocytosis, 799 Thrombon, 618 Thymic atrophy, 93 Thymoma, 98 Toe clipping, 39 Toxicokinetic evaluations, 700 Trichobezoar, 449 Trimethyltin, 151 Triorthotolyl phosphate, 853 Troll minipig, 750 Trophoblastic giant cells, 385 Tuberculosis (TB), 676, 709 Tumors in B6C3F1 mice, 76–77 Tweens, 587 Tyzzer’s disease, 447, 459

U UDP-glucuronate, 239 UDP-glucuronosyltransferase, 128, 479, 649 Uncommonly used animal species, 774 Urine sediment evaluation, 811 UVA light, 357

V Vaginal administration, 580 Valproic acid, 4 Vascular access port, 687 Vascular ectasia, 74 Vasodilating antihypertensive, 745 Vermin resistance, 33 Vertebrae and spinal cord segments, 637 Viral pneumonitis, 197 Vitamin C, 344, 396 Vitamin E deficiency, 345


INDEX

933

W Water, 45–47 Water bottles, 46 Wire-bottom cages, 34 Wistar (rat), 230, 240 Wistar Institute, 150

Yolk sac carcinomas, 90 Yucatan, 740

Z X

Xenobiotic metabolism by the lung, 246 in monkeys, 717

Y

Zbinden, 832 Zinker’s, 397 Zucker, 229


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