Toxicology of the Skin (Target Organ Toxicology Series)

Target Organ Toxicology Series Series Editors A.Wallace Hayes, John A.Thomas, and Donald E.Gardner Toxicology of the Lung, Third Edition Donald E.Gardner, James D.Crapo, and Roger O.McClellan, editors, 668 pp., 1999 Neurotoxicology, Second Edition Hugh A.Tilson and G.Jean Harry, editors, 386 pp., 1999 Toxicant-Receptor Interactions: Modulation of Signal Transductions and Gene Expression Michael S.Denison and William G.Helferich, editors, 256 pp., 1998 Toxicology of the Liver, Second Edition Gabriel L.Plaa and William R.Hewitt, editors, 444 pp., 1997 Free Radical Toxicology Kendall B.Wallace, editor, 454 pp., 1997 Endocrine Toxicology, Second Edition John A.Thomas and Howard D.Colby, editors, 352 pp., 1997 Reproductive Toxicology, Second Edition Raphael J.Witorsch, editor, 336 pp., 1995 Carcinogenesis Michael P.Waalkes and Jerrold M.Ward, editors, 496 pp., 1994 Developmental Toxicology, Second Edition Carole A.Kimmel and Judy Buelke-Sam, editors, 496 pp., 1994 Immunotoxicology and Immunopharmacology, Second Edition Jack H.Dean, Michael I.Luster, Albert E.Munson, and Ian Kimber, editors, 784 pp., 1994 Nutritional Toxicology Frank N.Kotsonis, Maureen A.Mackey, and Jerry J.Hjelle, editors, 336 pp., 1994 Toxicology of the Kidney, Second Edition Jerry B.Hook and Robin J.Goldstein, editors, 576 pp., 1993 Cardiovascular Toxicology, Second Edition Daniel Acosta, Jr., editor, 560 pp., 1992 Ophthalmic Toxicology George C.Y.Chiou, editor, 352 pp., 1992 Toxicology of the Blood and Bone Marrow Richard D.Irons, editor, 192 pp., 1985 Toxicology of the Eye, Ear, and Other Special Senses A.Wallace Hayes, editor, 264 pp., 1985 Cutaneous Toxicity Victor A.Drill and Paul Lazar, editors, 288 pp., 1984

Toxicology of Skin Target Organ Toxicology Series

Target Organ Toxicology Series

Toxicology of Skin Edited by

Howard I.Maibach Department of Dermatology University of California San Francisco, California, USA

USA Publishing Office: TAYLOR & FRANCIS 325 Chestnut Street Philadelphia, PA 19106 Tel: (215) 625–8900 Fax: (215) 625–2940 Distribution Center: TAYLOR & FRANCIS 7625 Empire Drive Florence, KY 41042 Tel: 1–800–624–7064 Fax: 1–800–248–4724 UK TAYLOR & FRANCIS 27 Church Road Hove E.Sussex, BN3 2FA Tel: +44 (0) 1273 207411 Fax: +44 (0) 1273 205612 This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” TOXICOLOGY OF SKIN Copyright © 2001 Taylor & Francis. All rights reserved. Printed in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a database or retrieval system, without prior written permission of the publisher. Cover design by Curt Tow. A CIP catalog record for this book is available from the British Library. The paper in this publication meets the requirements of the ANSI Standard Z39.48–1984 (Permanence of Paper). Library of Congress Cataloging-in-Publication Data Toxicology of skin/edited by Howard I.Maibach. p.; cm. —(Target organ toxicology series) Includes bibliographical references and index. ISBN 1-56032-802-9 (alk. paper) 1. Dermatotoxicology. 2. Contact dermatitis. 3. Urticaria. I. Maibach, Howard I. II. Series. [DNLM: 1. Skin Diseases—metabolism. 2. Skin—metabolism. 3. Skin—physiopathology. 4.Skin Absorption. WR 140 T755 2000] RL803.T69 2000 616.5–dc21 00–037722 ISBN 0-203-36282-9 Master e-book ISBN

ISBN 0-203-37542-4 (Adobe eReader Format) ISBN 1-56032-802-9 (case)

Contents

I. Irritant Dermatitis 1.

Electron Paramagnetic Resonance Study for Defining the Mechanism of Irritant Dermatitis Yoshiaki Kawasaki, Jun-ichi Mizushima, Roger Cooke, and Howard I.Maibach

2

2.

Irritant Patch Test in Atopic Individuals Harald Loeffler and Isaak Effendy

17

3.

Quantification of Nonerythematous Irritant Dermatitis Veranne Charbonnier, Howard I.Maibach, Boyce M.Morrison, Jr., and Marc Paye

23

4.

Detergents Isaak Effendy andHoward I.Maibach

28

5.

Permeability of Skin for Metal Compounds Jurij J.Hostýnek

36

6.

Irritant Dermatitis: Subthreshold Irritation Ethel Tur

51

7.

Cytokines and Irritant Dermatitis Syndrome Isaak Effendy and Howard I.Maibach

59

8.

Assay to Quantify Subjective Irritation Caused by the Pyrethroid Insecticide Alpha-Cypermethrin Rudolf A.Herbst, Robert B.Strimling, and Howard I.Maibach

73

9.

Prediction of Skin Corrosivity Using Quantitative Structure-Activity Relationships Martin D.Barratt

79

II. Percutaneous Absorption 10.

Contamination and Percutaneous Absorption of Chemicals from Water and Soil Ronald C.Wester and Howard I.Maibach

87

11.

Determining an Occupational Dermal Exposure Biomarker for Atrazine through Measurement of Metabolites in Human Urine by HPLC-Accelerator Mass Spectrometry Bruce A.Buchholz, John S.Vogel, S.Douglas Gilman, Shirley J.Gee, Bruce D.Hammock, Xiaoying Hui, Ronald C.Wester, and Howard I.Maibach

98

12.

Partitioning of Chemicals from Water into Powdered Human Stratum Corneum (Callus) Xiaoying Hui, Ronald C.Wester, Philip S.Magee, and Howard I.Maibach

107

13.

Dermal Exposure of Hands to Pesticides Graham A.Matthews

121

14.

Iontophoresis Angela N.Anigbogu and Howard I.Maibach

124

15.

Penetration and Barrier Creams Hongbo Zhai and Howard I.Maibach

133

III. Allergic Contact Dermatitis 16.

Fiber Kathryn L.Hatch and Howard I.Maibach

139

vi

17.

Novel Topical Agents for Prevention and Treatment of Allergic and Irritant Contact Dermatitis John J.Wille and Agis F.Kydonieus

157

18.

Allergic Contact Dermatitis: Clinical Considerations Yung-Hian Leow and Howard I.Maibach

174

19.

Pharmacology, Toxicology, and Immunology of Experimental Contact Sensitizers Whitney Hannon, Tim Chartier, and J.Richard Taylor

179

20.

Modulation of the Dermal Immune Response by Acute Restraint Sally S.Tinkle and Melanie S.Flint

193

21.

Textile-Dye Allergic Contact Dermatitis Prevalence Kathryn L.Hatch and Howard I.Maibach

202

22.

Dose-Response Studies in Guinea Pig Allergy Tests Søren Frankild

212

23.

Operational Definition of Allergic Contact Dermatitis S.Iris Ale and Howard I.Maibach

228

24.

Predictive Immunotoxicological Risk Assessment B.Homey, P.Lehmann, and H.W.Vohr

235

25.

Allergic Contact Dermatitis from Transdermal Systems Cheryl Levin and Howard I.Maibach

244

IV. Contact Urticaria 26.

Animal Models for Immunologic Contact Urticaria and Nonimmunologic Contact Urticaria Antti I.Lauerma and Howard I.Maibach

252

27.

Diagnostic Tests in Dermatology Smita Amin, Antti I.Lauerma, Howard I.Maibach

256

V. Miscellaneous 28.

Wound Healing Products David W.Hobson

265

29.

Issues of Toxicity with Dermatologic Drugs During Pregnancy Barbara R.Reed

291

30.

Roles of Calcium Ions in Skin Barrier Hanafi Tanojo and Howard I.Maibach

322

31.

Nuclear Receptors for Psoralen in Cultured Human Skin Fibroblasts K.Milioni, E.Bloom, and H.I.Maibach

330

32.

In Vitro Viability Assays Mikyung Kwah

337

33.

Dermal Penetration of Calcium Salts and Calcinosis Cutis Stephen D.Soileau

349

Index

358

Contributors

S.Iris Ale Department of Dermatology, University Hospital, Republic University, Montevideo, Uruguay Smita Amin Department of Dermatology, University of California, San Francisco, CA, USA Angela N.Anigbogu Department of Dermatology, University of California, San Francisco, California, USA Martin D.Barratt Marlin Consultancy, Carlton, Bedford, United Kingdom E.Bloom Department of Dermatology, University of California, San Francisco, California, USA and Department of Dermatology, University of Montreal, Montreal, Quebec, Canada Bruce A.Buchholz Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California, USA Veranne Charbonnier Department of Dermatology, University of California, San Francisco, California, USA Tim Chartier School of Medicine, University of Miami, Miami, Florida, USA Roger Cooke Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA Isaak Effendy Department of Dermatology, University of Marburg, Germany Melanie S.Flint Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA Søren Frankild Department of Dermatology, Odense University Hospital, Denmark Shirley J.Gee Department of Entomology, University of California, Davis, California, USA S.Douglas Gilman Department of Entomology, University of California, Davis, California, USA (currently at Department of Chemistry, University of Tennessee, Knoxville, Tennessee, USA) Bruce D.Hammock Department of Entomology, University of California, Davis, California, USA Whitney Hannon School of Medicine, University of Miami, Miami, Florida, USA Kathryn L.Hatch College of Agriculture, The University of Arizona, Tucson, Arizona, USA Rudolf A.Herbst Department of Dermatology, University of California, San Francisco, California, USA and Department of Dermatology and Allergology, Hannover Medical University, Hannover, Germany David W.Hobson DFB Pharmaceuticals, San Antonio Texas, USA B.Homey Department of Dermatology, Heinrich-Heine University, Düsseldorf, Germany Jurij J.Hostýnek Euromerican Technology Resources, Inc., Lafeyette, California, USA and Department of Dermatology, University of California, San Francisco, California, USA Xiaoying Hui Department of Dermatology, University of California, San Francisco, California, USA Yoshiaki Kawasaki Department of Dermatology, University of California, San Francisco, California, USA Mikyung Kwah Department of Dermatology, University of California, San Francisco, California, USA Agis F.Kydonieus Samos Pharmaceuticals LLC, Kendall Park, New Jersey, USA Antti I.Lauerma Department of Dermatology, University of Helsinki, Helsinki, Finland P.Lehmann Department of Dermatology, Heinrich-Heine University, Düsseldorf, Germany Yung-Hian Leow National Skin Centre, Singapore Cheryl Levin Department of Dermatology, University of California, San Francisco, California, USA Harald Loeffler Department of Dermatology, University of Marburg, Germany

viii

Philip S.Magee Department of Dermatology, University of California, San Francisco, California, USA Howard I.Maibach Department of Dermatology, University of California, San Francisco, California, USA Graham A.Matthews Imperial College at Silwood Park, Ascot, Berkshire, United Kingdom K.Milioni Department of Dermatology, University of California, San Francisco, California, USA Jun-ichi Mizushima Department of Dermatology, University of California, San Francisco, California, USA Boyce M.Morrison, Jr. Colgate Palmolive Company, Piscataway, New Jersey, USA Marc Paye Colgate Palmolive R & D, Milmort, Belgium Barbara R.Reed University of Colorado Health Sciences Center, Denver, Colorado, USA Stephen D.Soileau Gillette Medical Evaluation Laboratories, Needham, Massachusetts, USA Robert B.Strimling Department of Dermatology, University of California, San Francisco, California, USA Hanafi Tanojo Department of Dermatology, University of California, San Francisco, California, USA J.Richard Taylor School of Medicine, University of Miami, Miami, Florida, USA Sally S.Tinkle Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Morgantown, West Virginia, USA Ethel Tur Department of Dermatology, Tel Aviv Sourasky Medical Center and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel John S.Vogel Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California, USA H.W.Vohr Research Toxicology, Bayer AG, Wuppertal, Germany Ronald C.Wester Department of Dermatology, University of California, San Francisco, California, USA John J.Wille Bioderm Technologies Incorporated, Trenton, New Jersey, USA Hongbo Zhai Department of Dermatology, University of California, School of Medicine, San Francisco, California, USA

SECTION I Irritant Dermatitis

Toxicology of Skin Edited by Howard I.Maibach Copyright © 2001 Taylor & Franc

1 Electron Paramagnetic Resonance Study for Defining the Mechanism of Irritant Dermatitis Yoshiaki Kawasaki and Jun-ichi Mizushima Department of Dermatology, University of California, San Francisco, California, USA Roger Cooke Department of Biochemistry and Biophysics, University of California, San Francisco, California, USA Howard I.Maibach Department of Dermatology, University of California, San Francisco, California, USA

Introduction Materials and Methods Materials Stratum Corneum Preparation, Spin Labeling, and Surfactant Treatment for Cadaver Skin Stratum Corneum Preparation, Spin Labeling, and Surfactant Treatment for Stripped Stratum Corneum from Volunteers EPR Spectral Measurement Clinical Testing Results and Discussion Effect of Surfactant Mixtures (SLS/SLG) on Intercellular Lipid Cadaver Skin Effect of Surfactants on Intercellular Fluidity of Stratum Corneum Obtained from Fluidity of Stratum Corneum Obtained from Cadaver Skin Effect of Incubation Time and Concentration of SLS on the Intercellular Lipid Fluidity of Cadaver Stratum Corneum EPR Spectrum Measurement of Human-Stripped Stratum Corneum Conclusion INTRODUCTION The intercellular lipid lamellae in the stratum corneum constitute the main epidermal barrier to the diffusion of water and other solutes (Elias and Friend, 1975; Elias, 1981, 1983; Landman, 1986; Wertz and Downing, 1982). These lipids, arranged in multilayers between the corneocytes (Swartzendruber et al., 1989; Wertz et al., 1989), consist of ceramides (40%–50%), free fatty acids (15%–25%), cholesterol (15%–25%), and cholesterol sulfate (5%–10%) (Gray et al., 1982; Long et al., 1985; Swartzendruber et al., 1987). Information on the molecular structure of these lipids is important in elaborating a rational design of effective penetration enhancers for transdermal drug delivery (Woodford and Barry, 1986) and to understand the mechanism of irritant dermatitis and other stratum corneum disease. This information has been obtained by thermal analysis (Bouwstra et al., 1992; Golden et al., 1987; Van Duzee, 1975), X-ray diffraction study (Bouwstra et al., 1994; Bouwstra et al., 1991; Bouwstra et al., 1991; Garson et al., 1991; Vilkes et al., 1973; White et al., 1988), FT-IR spectroscopy (Bommannann et al., 1990; Krill et al., 1992), and electron paramagnetic resonance spectroscopy (EPR) (Rehfeld et al., 1988, 1990). Several investigators (Barker et al., 1991; Faucher and Goddard, 1978; Froebe et al., 1990; Fulmer and Kramer, 1986; Giridhar and Acosta, 1993; Imokawa, 1980; Mokawa et al., 1975; Rhein et al., 1986, 1990; Wilmer et al., 1994) demonstrate that stratum corneum swelling, protein denaturation, lipid removal, inhibition of cellular proliferation, and chemical mediator release contribute to irritation reaction; however, the mechanism of irritant dermatitis has not been understood and defined completely yet.

ELECTRON PARAMAGNETIC RESONANCE STUDY

3

On the other hand, permeability is increased by an increase in fluidity, both in biological and artificial membranes, suggesting the correlation between flux and fluidity (Golden et al., 1987; Knutson et al., 1985). The dynamic properties of intercellular lipid of stratum corneum are incompletely characterized; the effect of surfactants has not been studied in detail. EPR-employing nitroxide spin probes, the spin labeling method, has been utilized as a valuable spectroscopic method to provide information about the dynamic structure of membranes (Curtain and Gorden, 1984; Sauerheber et al., 1977). Spin probes are specifically incorporated with the lipid or lipid part of biological membranes. Thus, each label reflects the properties of different membrane regions. EPR spectra of membrane-incorporated spin probes are sensitive to the rotational mobility, orientation of the probes, and polarity of the environment surrounding the probes. This chapter investigates the influence of surfactants on the intercellular lipid structure of cadaver stratum corneum and the possibility of EPR spectra measurement on the stripped stratum corneum, which might reflect the actual skin lipid conditions. MATERIALS AND METHODS Materials 5-Doxyl stearic acid (5-DSA), purchased from Sigma Chemical Co. (St. Louis, MO), was used as the spin-labeling reagents without further purification. TABLE 1.1. Table of surfactants Category

Abbreviation Chemical name

Purity

Supplier

Anionic

SLS SL SLES SLEC SLG MSAC

>99% >98% Commercial grade Commercial grade >98% >98%

Sigma Chemical Co. (USA) Junsei Chemical Co. (Japan) Kao Co. (Japan) Sanyo Kasei Ltd. (Japan) Ajinomoto Co., Inc. (Japan) Tokyo chemical Industry Co., Ltd. (Japan) Ajinomoto Co., Inc. (Japan)

Cationic

Amphoteric HEA

Sodium lauryl sulfate Sodium laurate Sodium lauryl POE(3) ether sulfate Sodium lauryl POE(3) ether carboxylate Monosodium lauroyl glutamate Monostearyl trimethyl ammonium chloride N-[3-alkyl (Elias and Feingold, 1992; Bouwstra et al., 1992) oxy-2hydroxypropyl]-L-arginine hydrochloride

>98%

The surfactants shown in Table 1.1 were used without further purification. Purity of all materials were as stated by the suppliers. Test solutions were prepared with deionized water (MILLI-Q reagent water system (Millipore Co., Bedford, MA)). Deionized water was the vehicle control. Chemical structures of surfactants and spin label used in this study are described in Figure 1.1. Stratum Corneum Preparation, Spin Labeling, and Surfactant Treatment for Cadaver Skin Human abdominal skin was obtained from fresh cadaver skin with a dermatome. Epidermis was separated from dermis by immersing the skin in 60°C water bath set for 2 minutes followed by mechanical removal. Then, the epidermis was placed stratum corneum side up on the filter paper and floated on 0.5% wt trypsin (type II; Sigma) in a Tris-HCl buffer solution (pH 7.4) for 2 hours at 37°C. After incubation, any softened epidermis was removed by mild agitation of the stratum corneum sheet. Stratum corneum was dried and stored in a desiccator at −70°C for 3–4 days. Details are described by Quan (Quan and Maibach, 1994; Quan et al., 1995). 5-DSA was used as a stearic acid spin-labeling agent. One slice of dry stratum corneum sheet (approximately 0.49 cm2; ) was incubated in 1.0 mg/dl 5-DSA aqueous solution ( ) for 2 hours at 37°C and washed gently with deionized water to remove the excess of spin label. Surfactant treatment was as follows: a spin-labeled stratum corneum was immersed in surfactant aqueous solutions and incubated at 37°C for hours. The stratum corneum was taken out of the surfactant solution at indicated times. After rinsing with deionized water and removing the excess water, stratum corneum was mounted on the flatsurface EPR cell and EPR spectra were recorded. The control EPR spectrum was recorded for the spin-labeled stratum corneum kept in the deionized water at 37°C instead of the surfactant solution.

4

TOXICOLOGY OF SKIN

FIG. 1.1. Chemical structure of spin probe and surfactants (a) 5-DSA (5-Doxyl Steraric Acid) (b) Monoodium Lauroyl Glutamate (SLG) (c) Sodium Lauroyl POE (3) Ether Carboxylate (SLEC) (d) Sodium Lauryl Sulfate (SLS) (e) Sodium Lauryl POE (3) Ether Sulfate (SLES) (f) Sodium Laurate (SL) (SL) (g) Monostearylammonium chloride (MSAC) (h) N-[3-alkyl (12.14) oxy-2-hydroxypropyl]-L-arginine hydrochloride (HEA).

Stratum Corneum Preparation, Spin Labeling, and Surfactant Treatment for Stripped Stratum Corneum from Volunteers Stripped stratum corneum was obtained from the patch test sites of three healthy volunteers (3 male; mean age 34, ranging 30– 38) after giving their informed consent. Stratum corneum was removed from volar side of forearm skin by a single stripping

ELECTRON PARAMAGNETIC RESONANCE STUDY

5

with one drop of cyanoacrylate resin onto a quartz glass (0.5 cm×1.5 cm; Nihon Denshi CC, Tokyo, Japan) in accordance with the method of (Imokawa et al., 1991). Stripped stratum corneum, which is attached on a quartz glass, was spin labeled with a drop (approximately 30 µl) of 1.0 mg/dl 5-DSA solution for 30 minutes at 37°C and then washed with deionized water to remove excess spin probe on the stripped skin surface. EPR Spectral Measurement One slice of stratum corneum previously labeled with 5-DSA was mounted on the flat surface EPR cell, whose active cell area is approximately 1 cm2. EPR measurements were performed at approximately 23–25°C. EPR spectra were obtained with a Bruker ER200D EPR spectrometer (Bruker Inc., Billierica, MA) with microwave power output of 20 mW; spectrum data were collected by an IBM PC using software written in PC/FORTH (ver. 3.2, Laboratory Microsystems, Marina del Rey, CA). The hyperfine splittings of labeled skin samples were determined with 100 gauss scan width, 2.0×105 receiver gain, and 100 msec TABLE 1.2. EPR spectral data and clinical data of SLS/SLG mixtures Sample name

Averaged S (mean±S.D.) n=3

Human patch (mean±SD)

Visual

TEWL (g H2O/m2/h)

Control 0.856±0.028 0.53±0.08 13.0±1.0 0.25wt % SLS 0.700±0.021 0.73±0.08 22.3±1.7 0.50wt % SLS 0.662±0.038 0.70±0.10 22.3±1.7 0.75wt % SLS 0.644±0.027 0.87±0.14 22.7±1.5 1.00wt % SLS 0.560±0.034 1.03±0.15 25.4±2.6 0.25wt % SLS+0.75wt % SLG 0.809±0.070 0.73±0.08 20.0±1.7 0.50wt % SLS+0.50wt % SLG 0.714±0.004 0.67±0.08 20.7±1.9 0.75wt % SLS+0.25wt % SLG 0.656±0.040 0.87±0.11 21.2±2.6 0.25wt % SLS+1.00wt % SLG 0.808±0.047 NA NA 0.50wt % SLS+1.00wt % SLG 0.790±0.054 NA NA 0.75wt % SLS+1.00wt % SLG 0.744±0.040 NA NA 1.00wt % SLS+1.00wt % SLG 0.663±0.051 NA NA 1.00wt % SLG 0.819±0.023 0.67±0.08 15.8±1.1 Error bars=Mean±SD, n=3 for order parameters, Mean±SD, n=15 for clinical data. NA=Not available in reference (Lee et al., 1994).

time constant. Each sample was scanned several times; EPR signals were averaged to give a single estimate for the sample. Triplet signals, which are sharp, can be observed when the spin probe (doxyl group) moves freely, as shown in Figure 1.2(a); however, the spectrum becomes broader (Figure 1.2[b]) when spin probe mobility is restricted by interaction with other components. When the spin probe is incorporated in the highly oriented intercellular lipid structure in normal skin, the probe cannot move freely due to the rigidity of lipid structure, and its EPR spectrum represents the broad profile, such as in Figure 1.2(b). Once the normal structure is completely destroyed by chemical and/or physical stress, there is nothing to inhibit probe mobility and the EPR spectrum profiles become sharp as in Figure 1.2(a). EPR spectral profile represents the rigidity of the environment of the spin probe. To express the rigidity quantitatively, an order parameter is calculated from the EPR spectrum. Order parameters (S) were calculated according to (Griffith and Jost, 1976), (Hubbel and McConnell, 1971), and (Marsh, 1981): where 2A || is identified with the outer maximum hyperfine splitting; Amax (Figure 1.2) and A minimum hyperfine splitting Amin (Figure 1.2). a0 is the isotropic hyperfine splitting constant for nitroxide molecule in the crystal state:

is obtained from the inner

The values used to describe the rapid anisotropic motion of membrane-incorporated probes of fatty acid type are Similarly, the isotropic hyperfine coupling constant for the spin label in the membrane (

) is given by

6

TOXICOLOGY OF SKIN

FIG. 1.2. Typical EPR spectrum of 5-DSA labeled stratum corneum—Indication for reading huperfine.

values are sensitive to the polarity of the environment of the spin labels, as increases in values reflect an increase in the polarity of the medium. The order parameter (S) provides a measure of the flexibility of the spin labels in the membrane. It follows that S=1 for highly oriented (rigid) and S=0 for completely isotropic motion (liquid). Increases of order parameter reflect decreases in the segmental flexibility of the spin label, and, conversely, decreases in the order parameter reflect increases in the flexibility (Curtain and Gorden, 1984). Clinical Testing Healthy volunteers who were free of skin disease and had no history of atopic dermatitis were recruited for patch testing. After 30 minutes, test sites were marked on the subject’s back or forearms and baseline of TEWL was measured. Two

ELECTRON PARAMAGNETIC RESONANCE STUDY

7

hundred microliters of each test solution was applied using polypropylene chambers (Hilltop; Cincinnati, Ohio) secured with paper tape (Scanpor, Norgesplaster, Oslo, Norway). Application sites were randomized to minimize anatomical bias (Cua et al., 1990; Van der Valk and Maibach, 1989). Patches were removed after 24 hours and the test sites were exposed to air at least 30 minutes in order to allow deconvolution of excess water. Each site was visually graded in accordance with the following system: 0=normal skin or no reaction; 0.5=faint, barely perceptible erythema or slight dryness; 1= definite erythema or dryness; 2=erythema and induration; 3=vesiculation. TEWL was measured quantitatively with an evaporimeter EP-1 (Servo Med, Stockholm, Sweden) or with a Tewameter TM-20 (Courage+Rhazaka, Cologne, Germany) at 30 minutes after patch removal. Readings were performed at a stable level 30 seconds or more after application of the TEWL probe on the skin. TEWL values were expressed as g/m2/h (Lee, 1994). Statistical Analysis Statistical analysis for EPR spectral data was conducted with using unpaired t-test with single tail. Clinical data were statistically analyzed by two-way analysis of variance (ANOVA, Fisher’s test). The significance level was taken as pamphoterics>anionics>nonionics (Rieger, 1997). These two compounds have a plus charge. Their interaction with stratum corneum might be different from that of anionics, such as SLS. A plus charge might have more attractive interaction to proteins electrically because proteins are generally believed to be negatively charged. The change of order parameter corresponds to the structural changes of lipid layers. Two phases can be speculated to increase fluidity of lipid structure (decreasing the order parameter). The first phase is an effect of surfactant incorporated into the lamellar structures. If surfactant interferes or decreases lateral interactions between lipids, mobility increases similar to the phase conversion from liquid crystal to gel in the lamellar layers. The second phase is the destruction of lammelar structure by micellization or solubilization of lipid by surfactant. In this case, lipids no longer have dimensional restrictions and gain much higher mobility.

8

TOXICOLOGY OF SKIN

The results shown in Table 1.1 indicate that mobility increase by SLG can be attributed to the phase one structural changes in the lipid layers, and SLS might cause further disruption of the structures of lipid layers. The role of water in the stratum corneum must also be considered for the effects of surfactant to lipid layers. Treatment with anionic surfactants might influence water penetration and/or skin swelling (Takino et al., 1996). (Rhein et al., 1986, 1990) examined the swelling of stratum corneum caused by surfactants and reported that swelling effect of surfactants suggest mechanism of action as the basis for in vivo irritation potential. The correlation between order parameter S obtained from EPR spectrum and the clinical readings are summarized in Figure 1.3. The correlation coefficients (r2) of visual score and TEWL values were 0.76 and 0.83, respectively. The order

ELECTRON PARAMAGNETIC RESONANCE STUDY

9

FIG. 1.3. Correlation between clinical data of 24 hour patch and order parameter S of 5-DSA labeled cadaver stratum corneum incubated in surfactants solution for 1 hour at 37°C. (a) Correlation between order parameter and visual score and (b) Correlation between order parameter and TEWL (Error bars: Mean±SD, n=3 for order parameter; Mean±SD, n=14 for clinical data).

parameter S correlates better to TEWL values than to visual scores. This difference may be explainable in that TEWL is a direct measure of water barrier function, whereas visual scores represent total skin reactions, including physical or structural changes of skin tissue due to the physiological or biological reactions with surfactant. The order parameter might not predict the following skin reaction after the disorder of lipid structure, such as denaturation of proteins or mucosaccarides in dermis. Order parameter measurement of stratum corneum may predict the minimal difference of irritating potential among various kinds of chemicals.

10

TOXICOLOGY OF SKIN

FIG. 1.4. Order parameter of 5-DSA labeled cadaver stratum corneum treated with water, SLS, SLG, and SLS/SLG mixtures (total concentration 1.00 wt%, 1.00 wt% SLG addition to the SLS solutions).

Effect of Surfactant Mixtures (SLS/SLG) on Intercellular Lipid Fluidity of Stratum Corneum Obtained from Cadaver Skin Consumers are exposed daily to anionic surfactants in cleansing products, which may be irritating, especially when applied to sensitive skin sites. Although the properties of detergency and mildness seem to be contradictory, it is possible to reconcile these opposites by choice of surfactants that can reduce irritation potential. Some materials may reduce irritation potential of harsher surfactants, such as sodium lauryl sulfate (Kanari et al., 1992; Lee et al., 1994; Rhein et al., 1986, 1990). SLS was the most severely irritating and SLG is the mildest amongst the anionic surfactants tested. The influence of surfactant mixtures (SLS/SLG) on intercellular lipid fluidity of stratum corneum obtained from cadaver skin is discussed in this section. The profile of EPR spectra of 5-DSA labeled depends on the SLS concentration. The order parameters obtained from each EPR spectrum of stratum corneum treated with water, 0.25% wt, 0.50% wt, 0.75% wt, 1.00% wt SLS solutions, SLS/SLG mixtures (total concentration is constant at 1.00% wt) and 1.00% wt SLG solution were summarized in Table 1.3. The order parameter of water-treated stratum corneum (vehicle control) was 0.86± 0.03. Anionic surfactants as an amphiphilic molecule might be incorporated into structured lipids (lamellar structure). Order parameter (S) calculated from 1. 00% wt SLS-treated stratum corneum was 0.56±0.03, indicating lipid structure disordering. On the contrary, the high S value (0.82±0.02) for 1.00% wt SLG means less lipid structure disordered; 1.00% wt SLG almost equals to water. Treatment with 0. 25% wt, 0.50% wt, and 0.75% wt SLS solutions revealed intermediate levels between 1.00% wt SLG and SLS. Each order parameters of 5-DSA-labeled stratum corneum treated with SLS/SLG mixtures (total concentration is constant at 1.00% wt) showed higher values than that of 0.25% wt, 0.50% wt, 0.75% wt SLS, respectively. There were no statistically significant difference between 0.50% wt SLS and 0.50% wt SLS/0.50% wt SLG, and between 0.75% wt SLS and 0.75% wt SLS/0.25% wt SLG (p>0.05). These results suggest that SLG-inhibited SLS induced lipid fluidization. To confirm the antifluidization of SLG, the SLS/SLG mixture solutions were prepared with making SLG concentration constant at 1.00% wt and measured EPR spectra of 5-DSA labeled stratum corneum treated with them. The calculated order parameters S are plotted in Figure 1.4. Order parameters at each SLS concentration (0.25, 0.50, 0.75, and 1.00% wt SLS) with 1.00% wt SLG showed higher values than those of SLS only solutions. There were statistically significant differences between with and without 1.00% wt SLG (pISE Soap>SUC SLS>SLES>CPAB>LESS>RM SS>PEG (each 1%) 0.5% SLS>0.5% dodecyl trimethyl ammonium bromide > potassium soap N-alkyl-sulfate C12 >C8–10, C14–

One-time occlusive test Repeated short-time occlusive Repeated short-time open 2-day soap chamber test

Tupker et al., 1999

24-hour patch test

Visual scoring Visual scoring and TEWL Visual scoring and TEWL TEWL, skin reflective color (SRC/chromameter) TEWL, capacitance

24-hour patch test

TEWL, SRC

Wilhelm et al., 1994

5-day repeated occlusive application test (2 times daily)

Spectroscopic and visual scoring, TEWL, SRC, capacitance, skin replica Skin surface water loss (SSWL)

Zhou et al., 1991

Korting et al., 1994 Wilhelm et al., 1994

16

2% SLS>2.9% LAS>7.9% PEG-20 glyceryl monotallowate

7% SLS>7% CAPB>1% 24-hour plastic occlusion stress Berardesca et al., 1990 BAC>10% sorbitan monolaurate test 5% SLS>0.5% BAC>100% PG 48-hour patch test Visual scoring Willis et al., 1989 5% SLS=0.5% BAC>100% PG Histology SLS>cocobetaine>CAPB (each 48-hour patch test TEWL van derValk et al., 1985 2%) AEOS-3E0=alkyl (C12–14 average) ethoxy sulphate s, BAC=benzalkonium chloride; CAPB=cocoamidopropyl betaine; ISE=sodium cocoyl isethionate; LAS=linear alkyl (C12 average) benzene sulfonate; LESS=disodium laureth sulphate; PEG=polyethylene glycol; PG=propylene glycol; RMSS=disodium ricinoleamido monoethanolamido sulfosuccinate; SLES=sodium lauryl ether sulphate; SLS=sodium lauryl sulphate; SUC= disodium lauryl 3-ethoxysulfosuccinate. Modified from Effendy and Maibach, 1995. TABLE 4.3. In vitro toxicity ranking of frequently used surfactants Toxicity ranking

In vitro test (cell culture)

Assessment

BAC>SLS>between 80

Human primary keratinocytes

CTAB>SLS (at concentration: 3g/ mg) BAC>SLS (at concentration: 1×10 −5 M) Cationic=amphoteric>anionic>no n-ionic surfactants N-alkyl-sulfate C12 > C14>C10>C16>C8 BAC>SLS>between 20

Normal human epidermal keratinocytes (NHEK) Normal human oral and foreskin keratinocytes NHEK, HaCaT cells, and 3T3 cells HaCaT cells

Arachidonic acid and lnterleukin-1 Müller-Decker et al., 1994 release, MTT (mitochondrial metabolic activity) assay MTT assay Bigliardi et al., 1994 MTT assay and lactat dehydrogenase (LDH) release Neutral red release and cell growth/protein Neutral red release

References

Eun et al., 1994 Korting et al., 1994 Wilhelm et al., 1994

Commercial human skin model* MTT assay, LDH and PGE2 Osborne et al., 1994 (Skin2) release 0.2% BAC>0.5% SLS>0.5% Commercial human skin model* MTT assay Harvell et al., 1994 CAPB> 30% PG (Skin Equivalent) *Commercial human skin model=human dermal fibroblasts in a collagen-gel or a nylon-mesh matrix co-cultured with NHEK that have performed a stratified epidermis. CTAB: cetyltrimethylammonium bromide. Modified from Effendy and Maibach, 1995.

32

TOXICOLOGY OF SKIN

short-time occlusive, and repeated short-time open test) can vary the outcome of irritancy testing in mans (Table 4.3). The concordance among the different exposure methods has been found to be high when evaluated by transepidermal water loss (TEWL) but not by visual scoring, implying somewhat the superiority of the bioengineering assessment; however, visual scoring seems to be a “gold standard” in everyday use. This is one of the reasons when conducting irritancy test among the various methods that an exposure method which stimulates most in-use situations should be chosen. To predict the irritant potential and the irritancy ranking order of detergents in men, certain aspects have to be considered (e.g., type of detergent, mode of exposure, in-use situation, choice of irritancy testing). It has been proposed that the repeated open test is the best way to imitate most real-life situations where the uncovered skin is exposed to detergents. The repeated occlusive test or the one-time patch test may be suitable to mimic situations in which the skin is occluded after irritation by detergents (Tupker et al., 1999). Finally, one should keep in mind that in vivo irritancy testing in humans remains crucial as long as in vitro tests do not provide a comparable predictor value. REDUCED IRRITANT POTENTIAL OF MIXED SURFACTANT SYSTEMS Blends of surfactants have been used in cosmetic and pharmaceutical formulas, particularly, in order to increase the acceptance of the product due to its reduced irritant potential, mildness, and comfort. For instance, there is antagonism or mutual inhibition in an acid-base neutralization and in anionic-cationic surfactant reaction. SLS as well as linear C9–13 alkylbenzene sulphonate (LAS), when applied each alone at 20% to human skin, induced a noteable erythema. Nevertheless, a mixture of 20% SLS and 10% sodium lauryl ether-2E0 sulfate (SLES), or 10% cocoamidopropyl betaine (CAPB), or 10% cocodiethanolamine, caused significantly less erythema (Table 4.4). Similarly, a blend of 20% LAS+10% SLES+10% C9–11 alcohol 8 EQ (nonionic), a total surfactant level of 40%, was substantially less irritant than 20% LAS alone. Probably, irritant responses are not simply linked with the total concentration of surfactants used, but rather to the contents of the mixture (Dillarstone et al., 1993). TABLE 4.4. Reduced irritancy of mixed surfactant systems Mixture of surfactants vs. single surfactant

References

SLG+SLS<SLS Kawasaki et al., 1999, Lee et al., 1994 SLS+DDAB<SLS Hall-Manning et al., 1998 20% SLS+10% SLES, or 10% CAPB, or 10% CDEA98%, 51 mCi/mmol), [8–14C] theophylline (>98%, 51 mCi/mmol), and [U-14C] polychlorinated biphenyls (PCB, aroclor 1254, 54% chlorine, 32 mCi/mmol) were obtained from Amersham Corporation (Arlington Heights, IL) [U-14C] glycine (98%, 63 mCi/mmol), and [14C] urea (98%, 56 mCi/mmol) were obtained from ICN Biomedical Inc (Irvine, CA). [4–14C] Hydrocortisone (97.6%, 52 mCi/mmol) and [4–14C] estradiol (99%, 54.1 mCi/mmol) were obtained from Du Pont Company (Wilmington, DE). [Ring 14-C(U)] alachlor (21 mCi/mmol) was a gift from Monsanto Company (St. Louis, MO). [7-14C] Acitretin (>98%, 58.2 mCi/mmol) was a gift from Roche Dermatologics (Nutley, NJ). The pH values of these chemical solutions in distilled deionized water were determined by a Corning pH/ion Analyzer 250 (Corning Science Products, Corning, NY). Preparation of Powdered Human Stratum Corneum (Callus) The procedure was based on the method of (Wester et al., 1987). Adult foot callus was ground in the Micro-Mill Grinder (BelArt Products, Pequannock, NJ) in the presence of liquid nitrogen to form a powder. Particles of the PHSC that passed through a 50-mesh sieve but not an 80-mesh were used. Particle sizes within this range were shown to favor the experimental conditions. The sample was stored in the freezer until using. Depleting Lipid Content of the PHSC PHSC was mixed with n-hexane for 30 minutes, followed by chloroform and methanol mixture (2:1, v/v) overnight for extraction of intercellular lipids (Raykar et al., 1988; Kurihara-Bergstrom et al., 1990). The delipidized PHSC was stored in the freezer. The lipid content of the PHSC was determined by the change in weight before and after solvent extraction (Raykar et al., 1988). Prewetting of the PHSC with Water or Ethanol To determine effect of hydrated or perturbed PHSC on compound partitioning, powdered stratum corneum was wetted by 10 µl water or ethanol for 30 minutes prior to incubation. Incubation Procedure The experiment was performed by modifying the method of (Wester et al., 1987). A given dosage of radiolabeled chemical in 4.0 ml vehicle (distilled deionized water) was mixed with 10.0 mg of the powdered callus in a glass vial and incubated in a water bath with moderate shaking at 35°C for a given time period. The mixture then was separated by centrifugation at 1, 500×g for 15 minutes and the supernatant carefully removed. The PHSC pellet was resuspended three times in the same volume of deionized distilled water to remove any excess material clinging to the surface. Experiments indicated that further washing (four or five times) did not significantly change the PC value. The radioactivity of the chemical bound to the PHSC and that remaining in the vehicles was determined by scintillation counting. Five samples were used for each test.

PARTITIONING OF CHEMICALS FROM WATER INTO POWDERED

109

Scintillation Counting The scintillation cocktail was Universal-ES (ICN, Costa Mesa, CA). Background controls, and test samples were counted in a Packard model 4640 counter (Packard Instruments). The data was transferred to a computer program (Appleworks/Apple IIE computer; Apple Computer Co., Mountain View, CA) that subtracted background control samples and generated a spreadsheet for statistical analysis. The counting process and a computer program have been verified to be accurate by a quality assurance officer at the University of California at San Francisco. Powdered Human Stratum Corneum/Water Partition Coefficient The value is determined by the following equation: (1) where CPHSC is the amount (µg) of the chemical absorbed in 10 mg of the PHSC and Cwater is the amount (µg) remaining in 4000 mg of the water after removing the PHSC pellet. Because the micrograms of the chemical in the PHSC or water is proportional to the counts (dpms) determined by the scintillation counter and partitioning of chemical into the PHSC exactly equaled the decrease of the chemical concentration in the vehicle (water), an alternative calculation method was also used: (2) where dpmi is the initial chemical concentration in the vehicle (water). These two calculation methods were shown to give similar results, and the reported experiments are calculated by the second method. Statistical Analysis Statistical analysis (students’ t-test, linear and multiple regressions) were performed in version 6.1 of MINTTAB (Minitab Inc, State College, PA) on an IBM PC-compatible computer. When P value was smaller than 0.05, it was considered as statistical significance. RESULTS Table 12.1 shows the effect of varying initial chemical concentrations on the PC PHSC/w of 12 test compounds. Under the fixed experimental conditions (2 hours incubation time and 35°C incubation temperature), the concentration required to reach a peak value of the partition coefficient varied from chemical to chemical. After reaching the maximum, increasing the chemical concentration in the vehicle did not elevate TABLE 12.1. Effect of initial aqueous phase chemical concentration on powdered human callus/water partition coefficient Chemical

Concentration* (%, w/v)

Partition (mean)

Coefficient† (S.D.)

Dopamine 0.46 0.92 Glycine 0.10 Urea 0.06 0.12 Glyphosate 0.04 0.08 Theophylline 0.36 0.54 Aminopyrine 0.14 Hydrocortisone

0.23 6.04 5.74 0.05 0.40 0.03 0.15 0.17 0.02 0.68 0.70 0.18 0.43 0.42 0.07 0.46 0.09

5.42 0.28 0.28 0.36 0.02 0.26 0.02 0.02 0.79 0.04 0.01 0.37 0.03 0.02 0.44 0.03 0.37

0.22

0.01 0.02

0.04

0.02

0.09 0.01

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TOXICOLOGY OF SKIN

FIG. 12.1. Effect of multiple doses on hydrocortisone partitioning from water into the powdered human stratum corneum. Group A was preincubated with 2.0 ml of saturated nonlabeled hydrocortisone for 30 minutes. Group B was preincubated with the same vehicle as group A. Then both groups were incubated with 2.0 ml of various concentration of [14-C] labeled hydrocortisone for 2 hours. *The values for group B were significantly higher than for group A (p0.05) were observed for untreated TABLE 12.2. Lipid content and water uptake of powdered human stratum corneum Water uptake (µg/mg dry PHSC) Stratum corneum source

Lipid content (%w/w dry PHSC)

Untreated PHSC

Lipid*

1 2.38 2 2.21 3 2.39 4 2.69 5 2.08 6 2.01 Mean 2.29 SD 0.25 *Lipid part extracted from the PHSC. †Rest part of the PHSC after lipid extraction.

Delipidized PHSC Protein†

Total

495.85 452.49 585.62 554.27 490.04 381.61 493.31 72.66

26.44 39.26 23.09 40.05 49.86 14.82 32.26 12.97

452.40 364.96 498.40 492.31 363.30 324.18 415.92 74.50

478.84 404.22 521.49 532.36 413.16 339.00 448.18 75.47

TABLE 12.3. Effects of water and ethanol pretreatment on the water-binding capacities of untreated and delipidized PHSC Water absorbed by the PHSC (µg equivalent/mg PHSC) Sample pretreatment

Untreated PHSC

Dry sample 468.00±71.80 Water prewetted 314.40±65.89* Ethanol prewetted 222.14±17.87* *Statistical significance between prewetted and dry untreated PHSC (p