Principles and Practice of
`
`
`
`~‘~}"Wl LEY
`
`0001
`
`Noven Pharmaceuticals, Inc.
`EX2008
`Mylan Tech., Inc. v. Noven Pharma, Inc.
`lPR2018-00174
`
`

`

`Principles and Practice
`of Skin Toxicology
`
`Editors
`
`Robert P. Chilcott
`
`Chemical Hazards and Poisons Division, Health Protection Agency, Chilton, UK
`
`and
`
`Shirley Price
`
`School of Biomedical and Molecular Sciences, University of Surrey, UK
`
`John Wiley & Sons, Ltd
`
`0002
`
`

`

`Copyright © 2003
`
`lohn Wiley 8: Sons Ltd, The Atrium, Southern Gate, Chichester,
`West Sussex P019 SSQ, England
`
`Telephone (+44) 1243 W9???
`
`Email [for orders and customer service enquiries}: cs-books@wi]ey.co.uk
`Visit our Home Page on www.wileyeurope.com or www.wiley.com
`
`All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any
`means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs
`and Patents Act [933 or under the terms ofa licence issued by the Copyright Licensing Agency Ltd. 90 Tottenham Court Road.
`London WIT 4LP. UK. without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the
`Permissions Department, [ohn Wiley & Sons Ltd, The Atrium. Southern Gate, Chichester. West Sussex P019 BSQ. England, or
`emailed to permreq®wiley.co.uk, or faxed to (+44) 1243i ”0620.
`
`Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names
`used in this book are trade names, service marks. trademarks or registered trademarks of their respective owners. The Publisher is not
`associated Iwith any product or vendor mentioned in this book.
`
`This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. it is sold on
`the understanding that the Publisher is not engaged in rendering professional services. lfprofessional advice or other expert
`assistance is required, the services ofa competent professional should be sought.
`
`Other Wiley Editorial Ofl'ices
`
`Iohn Wiley 8t Sons Inc., 111 River Street, Hoboken, N] 0?030. USA
`
`Iossey—Bass, 989 Market Street, San Francisco. CA 94103—IT41, USA
`
`Wiley—VCH Verlag Gth, Boschstr. [2, D—69469 Weinheim. Germany
`
`Iohn Wiley & Sons Australia Ltd, 42 McDougail Street. Milton, Queensland 4064, Australia
`
`Iohn Wiley 8( Sons (Asia) Pte Ltd. 2 Clementi Loop #02—01. [in King Distripark, Singapore 129809
`
`John Wiley 8r Sons Canada Ltd, 6045 Freemont Blvd, Mississauga, Ontario, LSR 4,13, Canada
`
`Wiley also publishes its books in a variety of electronic formats. Some content that appears
`in print may not be available in electronic books.
`
`Library ofCongress Cataloging-in-Pablicatr'on Data
`
`Principles and practice of skin toxicology l' editors. Robert P. Chiicott.
`Shirley Price.
`P. ; crn.
`
`Includes bibliographical references and index.
`ISBN 9r3-0-470-511r2-5
`
`l. Chilcott. Robert P. I]. Price. Shirley, Dr.
`l. Dennatotoxicology.
`[DNLM: 1. Skin Physiology. 2. Skin Absorption. 3. Skin Diseases. WR
`102 9957 2003]
`RL303.P}75 2008
`615’5’73 — dc22
`
`2008002901
`
`British Library Cataloguing in Publication Data
`
`A catalogue record for this book is available from the British Library
`
`ISBN 978—0—470—5] l?2—5
`
`Typeset in 10112 Minion by Laserwords Private Limited. Chennai, India
`Printed and bound in Singapore by Markono Ltd
`This book is printed on acid-free paper responsibly manufactured from sustainable forestry
`in which at least two trees are planted for each one used for paper production.
`
`0003
`
`

`

`Dedications
`
`(RC) For all my teachers, lecturers and professors. Especially the ones that
`were left in despair.
`
`For Emlyn Evans and Trefor Pedrick. True gentlemen of knowledge.
`
`For all of my family.
`
`For the young ladies in my life:
`
`Caroline, Florence Megan and Charlotte Rose.
`
`(SP) For my partner in crime, Rob Chilcott, the more verbose member of the
`partnership, and Carolyn, for her patience
`
`For Pete, my husband, and for Jessica and Jonathan for their patience
`during the editing of this document
`
`For my mentors who taught me the essence of Toxicology ~ I am still
`learningll
`
`0004
`
`

`

`Contents
`
`Foreword
`
`Preface
`
`Acknowledgements
`
`List of contributors
`
`PARTI Introduction
`
`1 Cutaneous anatomy and function
`Robert P. Chifcott
`
`1.1
`1.2
`
`1.3
`
`1.4
`
`Introduction and scope
`Surface features
`
`Functional histology of the epidermis and associated structures
`
`Species differences
`
`Summary
`References
`
`2 Biochemistry of the skin
`Simon C. Wilkinson
`
`2.1
`
`2.2
`
`2.3
`
`2.4
`2.5
`
`2.6
`
`Introduction and scope
`
`Protein synthesis and organisation during epidermal differentiation
`
`Lipid synthesis and organisation during epidermal differentiation
`
`Lipid classes in the stratum corneum
`Stratum corneum turnover
`
`Biotransformations in skin
`
`Summary
`References
`
`0005
`
`xv
`
`xvii
`
`xix
`
`xxi
`
`1
`
`3
`
`3
`3
`
`8
`
`13
`
`15
`15
`
`17
`
`17
`
`18
`
`19
`
`20
`23
`
`24
`
`42
`42
`
`

`

`vfii
`
`3
`
`3.1
`
`3.2
`
`3.3
`
`CONTENTS
`
`Skin photobiology
`Mark A. Birch-Mochin and Simon C. Wilkinson
`
`Introduction and scope
`
`Photoprotection and melanogenesis
`
`Increased environmental ultraviolet radiation exposure and its link with
`
`photoageing and skin cancer
`
`3.4 Mitochondrial DNA as a biomarker of sun exposure in human skin
`
`3.5
`
`3.6
`
`Apoptosis
`
`Sun protection
`
`Summary
`References
`
`PART 11 Skin Absorption
`
`4
`
`Skin as a route of entry
`Simon C. Wilkinson
`
`4.1
`
`Salient anatomical features of the stratum corneum — the ‘brick and mortar
`
`4.2
`
`4.3
`
`4.4
`
`4.5
`
`4.6
`4.7
`
`4.8
`
`model’
`
`Species and regional variation in skin structure
`
`Species and regional variation in skin permeability
`
`Intra- and inter-individual variation in percutaneous absorption
`
`Effect of age on skin barrier function
`
`Role of skin appendages
`The in vitro skin sandwich model
`
`Penetration of particles through appendages
`
`Summary
`References
`
`5
`
`Physicochemical Factors Affecting Skin Absorption
`Keith R. Brain and Robert P. Chilcott
`
`5.1
`
`5.2
`
`5.3
`
`Introduction
`
`Physicochemical properties
`
`Exposure consideratiOns
`
`Summary
`References
`
`6 Principles of Diffusion and Thermodynamics
`
`W. John Pugh and Robert P. Chilcott
`
`0006
`
`51
`
`51
`
`51
`
`55
`
`60
`
`61
`
`63
`
`65
`65
`
`69
`
`71
`
`71
`
`72
`
`74
`
`75
`
`76
`
`??
`?8
`
`79
`
`80
`80
`
`83
`
`83
`
`84
`
`39
`
`91
`91
`
`93
`
`

`

`CONTENTS
`
`6.1
`
`6.2
`
`6.3
`6.4
`
`6.5
`
`6.6
`
`6.7
`
`6.8
`
`6.9
`
`Introduction and scope
`
`Some definitions pertaining to skin absorption kinetics
`
`Basic concepts of diffusion
`Fick’s Laws of diffusion
`
`Thermodynamic activity
`
`Skin absorption of a substance from two different vehicles
`
`Partitioning
`
`Diffusivity
`
`Skin absorption data and risk assessments
`
`Summary
`References
`
`7
`
`In vivo measurements of skin absorption
`
`James C. Wakefield and Robert P. Chilcott
`
`7.1
`
`Introduction and scope
`
`12 Why conduct in vivo studies?
`
`7.3
`
`14
`15
`
`Ethics and legislation
`
`Standard methodology: DEED Guideline 427
`Alternative in vivo methods
`
`Summary
`References
`
`8
`
`In vitro percutaneous absorption measurements
`
`Ruth U. Pendlington
`
`Introduction and scope
`
`Regulatory guidelines
`
`Why assess percutaneous absorption in wire?
`
`Basic principle of in vitro percutaneous absorption measurements
`Choice of diffusion cell
`
`Skin membrane considerations
`
`Integrity measurements
`
`Choice of receptor fluid and sampling considerations
`Test material considerations
`
`8.1
`
`8.2
`
`8.3
`
`8.4
`8.5
`
`8.6
`
`8.?
`
`8.8
`8.9
`
`8.10 Application of test preparation to the skin
`
`Examples of results from in vitro skin absorption studies
`8.11
`8.12 What is considered to be absorbed?
`
`8.13 Micro-autoradiography
`
`Summary
`References
`
`0007
`
`ix
`
`93
`
`94
`
`97
`9?
`
`98
`
`99
`
`101
`
`102
`
`105
`
`106
`106
`
`109
`
`109
`
`110
`
`110
`
`115
`119
`
`126
`126
`
`129
`
`129
`
`129
`
`130
`
`131
`131
`
`136
`
`13?
`
`138
`139
`
`140
`
`142
`146
`
`147
`
`14?
`147
`
`

`

`x
`
`CONTENTS
`
`PART III Toxicological Assessment
`
`9
`
`Skin immunology and sensitisation
`David A. Basketter
`
`9.1
`
`9.2
`
`9.3
`
`9.4
`
`9.5
`
`9.6
`
`9.7
`
`Introduction
`
`Definitions
`
`Skin sensitisation
`
`Identification of skin sensitisers
`
`Risk assessment
`
`Other types of allergic skin reaction
`
`Future prospects
`
`Summary
`References
`
`10 In vitro phototoxicity assays
`
`Penny Jones
`
`10.1
`
`Introduction and scope
`
`10.2
`
`In vitro strategies for phototoxicity testing
`
`10.3 The UV/visible absorption spectrum as a pre-screen for phototoxicity
`
`10.4
`
`In vitro assays for phototoxicity using monolaye;r cultures
`
`10.5
`
`In vitro assays for photoallergenicity
`
`10.6
`
`In vitro assays for phototoxicity using human 3-D skin models
`
`Summary
`References
`
`149
`
`151
`
`151
`
`151
`
`152
`
`155
`
`160
`
`163
`
`164
`
`164
`165
`
`169
`
`169
`
`169
`
`III
`
`172
`
`114
`
`177
`
`181
`181
`
`11 In vitro alternatives for irritation and corrosion assessment
`
`185
`
`Penny Jones
`
`11.1
`
`Introduction and scope
`
`11.2 Acute dermal irritation/corrosion
`
`11.3 Validation/regulatory status of in who assays for skin corrosion
`11.4 In vitro tests for skin corrosion
`
`11.5 Validation/regulatory status of in vitro assays for skin irritation
`11.6
`In vitro tests for skin irritation
`
`Summary
`References
`
`12 Instruments for measuring skin toxicity
`
`Helen Taylor
`
`12.1
`
`Introduction and scope
`
`0008
`
`185
`
`185
`
`186
`188
`
`194
`195
`
`197
`198
`
`201
`
`201
`
`

`

`CONTENTS
`
`12.2
`
`Skin surface pH
`
`12.3 Biomechanical properties
`12.4 Sebum
`
`12.5
`
`Skin surface contours
`
`12.6 Thickness
`
`12.7 Desquamation
`
`12.8 Applications and measurement of transepidermal water loss
`12.9 Guidance for TEWL measurements
`
`12.10 Hydration measurement
`
`12.11 Guidance for hydration measurements
`
`12.12 Relationship between hydration and dermal toxicity
`12.13 Colour measurement
`
`12.14 Measurement of vascular perfusion
`12.15 A final word of caution
`
`Summary
`References
`
`PART IV Clinical Aspects
`
`13 Introduction to dermatology
`
`Manjunatha Kalavala and Alex Anstey
`
`13.1
`
`Introduction and scope
`
`13.2 Clinical assessment of patient with skin disease
`
`13.3 Cutaneous manifestations of disease following exposure to chemicals and
`pharmaceutical formulations
`13.4 Overview of standard treatments
`
`Summary
`
`14 Clinical aspects of phototoxicity
`
`Anthony D. Pearse and Alex Anstey
`
`Introduction and scope
`14.1
`14.2 UV-induced skin reactions
`
`Phototoxicity (photoirritancy) reactions
`14.3
`14.4 Photosensitive reactions
`
`Summary
`References
`
`15 Occupational skin diseases
`
`Jon Spiro
`
`0009
`
`xi
`
`202
`
`204
`205
`
`205
`
`205
`
`205
`
`206
`208
`
`209
`
`212
`
`213
`213
`
`215
`216
`
`21?
`21?
`
`221
`
`223
`
`223
`
`224
`
`234
`241
`
`243
`
`245
`
`245
`24?
`
`24?
`251
`
`256
`256
`
`259
`
`

`

`xii
`
`15.1
`
`15.2
`
`15.3
`
`15.4
`
`15.5
`
`15.6
`
`15.?
`
`15.8
`
`15.9
`
`CONTENTS
`
`Introduction and scope
`Dermatitis
`
`Development of occupational dermatitis
`
`Patterns of occupational dermatitis
`
`Incidence of occupational dermatitis
`Effects of dermatitis on work
`
`The outlook in occupational dermatitis
`
`Identification of occupational dermatitis
`
`Other occupational skin disorders
`
`15.10
`
`Investigation of a case of dermatitis at work
`
`Summary
`References
`
`16 Prevention of occupational skin disease
`Chris Pockham
`
`16.1
`
`16.2
`
`16.3
`
`16.4
`
`16.5
`
`16.6
`
`16.7
`
`16.8
`
`16.9
`
`Prevention of occupational skin disease
`
`Defining the problem
`
`Material safety data sheets
`
`Chain of responsibility
`
`Managing dermal exposure
`
`Selection and use of per50nal protective equipment
`
`Protective or ’barrier' creams: do they have a role?
`
`The role of education and training
`Conclusions
`
`Summary
`References
`
`PART V Regulatory
`
`17 Occupational skin exposures: legal aspects
`Chris Packham
`
`17.1
`
`17.2
`
`17.3
`
`17.4
`
`12.5
`
`17.6
`
`Introduction and scope
`
`Brief overview of current United Kingdom legislation
`
`The employer's perspective
`Hazard identification
`
`Risk assessment
`
`Gloves: a note of caution
`
`Summary
`References
`
`0010
`
`259
`
`260
`
`263
`
`264
`
`265
`
`265
`
`266
`
`266
`
`26?
`
`2?0
`
`226
`
`276
`
`279
`
`229
`
`280
`
`282
`
`283
`
`284
`
`289
`
`294
`
`294
`
`294
`
`294
`
`294
`
`297
`
`299
`
`299
`
`300
`
`303
`
`304
`
`306
`
`309
`
`310
`
`310
`
`

`

`CONTENTS
`
`18 Safety assessment of cosmetics: an EU perspective
`Jo Larner
`
`18.1
`
`Introduction and scope
`
`18.2 Overview and scope of Cosmetics Directive 76/768/EC
`
`18.3 Overview of the requirements of the EU Cosmetics Directive
`18.4 Scientific advice
`
`Influence of other legislation
`18.5
`18.6 Adverse effects from cosmetics
`
`18.7 Toxicity of cosmetic ingredients
`
`18.8 The safety assessment
`18.9
`A final consideration
`
`Summary
`References
`
`Appendix 18.1 Additional obligations for cosmetic suppliers
`
`xiii
`
`311
`
`311
`
`312
`
`315
`316
`
`31?
`318
`
`320
`
`326
`328
`
`329
`329
`
`330
`
`19 Regulatory dermatotoxicology and international guidelines
`
`333
`
`Adam Woolley
`
`19.1
`
`Introduction
`
`19.2 Regulatory context
`
`19.3
`
`Product groups and the human context
`
`19.4 Dermal toxicology with the different product groups
`
`19.5
`
`Factors in dermal toxicity
`
`19.6 Repeat dose dermal toxicology
`
`19.? Classic short-term dermal toxicity studies
`
`19.8
`
`Pragmatic considerations
`
`Summary
`References
`
`20 Glossary of main terms and abbreviations
`
`James C. Wakefield
`
`Index
`
`333
`
`334
`
`335
`
`336
`
`338
`
`339
`
`341
`
`344
`
`345
`345
`
`347
`
`358
`
`0011
`
`

`

`5 Physicochemical Factors
`Affecting Skin Absorption
`
`Keith R. Brain1 and Robert P. (Ihilcott2
`
`'Wetsh Schoot of Pharmacy, Cardifi University, CF10 3XF and An—eX, Capital.r Business Park,
`
`Cardijj‘, CF3 2PX, UK
`
`lChemicat Hazards and Poisons Division, Centre for Radiation, Chemical and Environmental
`
`Hazards, Chittan, Oxfordshire 0X11 0R0, UK
`
`Primary Learning Objectives
`
`0 Refating the concept of‘dose’ to skin absorption.
`
`0 Identifying the major physicocbemicai determinants ofskin absorption and a consideration of
`how other modulatingfactors (principaiiy exposure conditions) can afiect dermal absorption.
`
`Introduction
`5.1
`
`
`The primary factor that dictates the percutaneous toxicity of a chemical is its ability to penetrate the
`skin. This dictum, whilst relatively straightforward, is complicated by the fact that there is a myriad
`of factors which can affect skin absorption.
`
`The central dogma of toxicology was formulated by Paracelsus (a.k.a. Theophrastus Phillippus
`Aureolus Bombastus von Hohenheim; Box 5.1), who understood that it is the dose of a
`
`chemical which ultimately dictates its toxicity; ‘sola dosis facit venenum’ (User 1987). Thus,
`factors which influence skin absorption (and thus ‘dose’) are necessarily factors which affect
`percutaneous toxicity: a t0xic chemical which is unable to traverse normal skin will not be
`toxic via the percutaneous route. For example, botulinum toxin is one of the most poisonous
`substances known. However, it does not penetrate healthy skin and so is essentially non-toxic
`following skin contact.
`Skin absorption of chemicals is a passive process. Unfortunately, this does not mean that
`the process of dermal absorptiOn is simple and highly predictable, as there are a diverse range
`of factors that can affect the rate and extent to which a chemical is absorbed. These include
`
`(amongst others) vehicle effects (Hilton et at. 1994), ageing (Roskos and Maibach 1992), race
`(Kompaore et at. 1993), gender (Bronaugh et at. 1983}, disease (Moon and Maibach 1991),
`
`Principies and Practice offikin Toxicningy Edited by Robert P. Chilcutl and Shirley Price
`if) 2008 [01111 Wiley & Sons. Ltd
`
`0012
`
`

`

`84
`
`CHOS: PHYSICOCHEMICAL FACTORS AFFECTING SKIN ABSORPTION
`
`Box 5.1 Paracelsus
`
`
`did not particularly endear him to his peers!
`
`
`
`Paracelsus, also known as Theophrastus Phillippus Aureolus
`Bombastus von Hohenheim (circa 1493 — 1541), was a largely
`salt-taught polymath who recognised the dose-response
`relationship which is an underpinning principal of
`modern toxicology.
`
`A somewhat interesting character, he roamed Europe. north
`Africa and parts of Asia in his pursuit of alternative medical
`knowledge. His published works. personal activities and
`outspoken criticism of contemporaneous medical practices
`
`A full—colour version of this figure appears in the colour plate section of this book
`
`chemical damage (Wahlberg 1972), lipid content (Elias 1981), hydration (Behl et ai. 1980),
`pH (Allenby er ai. 1969), stress (Denda er al. 2000) and physicochemical properties of the
`pEHetrant (Lien et at. 1973); one could even imagine that the services of an astrologer may
`be a useful adjunct to predicting skin absorption! However, the problem can be simplified
`by considering just a relatively small number of factors which exert the greatest influence
`over skin absorption. The most pertinent to dermal toxicology include the physicochemical
`properties of the penetrant and the exposure conditions.
`Other important factors influencing percutaneous absorption (e.g. effects of metabolism,
`species and regional variation, thermodynamic factors and methodological considerations)
`are considered in Chapters 2, 4, 6 and 8, respectively. A number of authoritative texts can
`also be consulted (Bronaugh and Maibach 1999; Schaefer and Redelmeier 1996; Wester and
`Maibach 1983).
`
`Physicochemical properties
`5.2
`
`
`A major determinant of skin absorption relates to the physicochemical properties of the applied
`chemical, in particular, size, solubility, charge and hydrogen bonding capacity. An understanding
`of these factors can allow an approximation of the extent to which a given chemical will cross the
`stratum corneum.
`
`The primary factors affecting skin absorption are concerned with the physicochemical
`properties of the penetrant. The most important physicochemical parameters are arguably
`molecular weight, solubility, charge and hydrogen bonding capacity. A basic understand—
`ing of these relatively simple factors will enable even the least experienced toxicologist
`to be able to make a reasonable judgement as to the dermal bioavailability of a given
`chemical.
`
`0013
`
`

`

`5.2: PHYSICOCHEMICAL PROPERTIES
`
`85
`
`5.2.1 Molecular weight
`
`As a general rule of thumb, chemicals with a molecular weight greater than ~500 Da do not
`penetrate the skin. This is known as the ‘rule of500’ (805 and Meinardi 2000). This upper limit
`on molecular size mainly results from the physical arrangement of lipids between adjacent
`corneocytes of the stratum corneum (see Chapters 1 and 2). However, there is some evidence
`to suggest that large, linear, ‘wiggly’ molecules (such as heparin and DNA) may be able to
`traverse the stratum corneum, albeit in relatively small quantities.
`
`5.2.2 Solubility
`
`The solubility of a chemical is commonly quantified in terms of how it partitions between
`two immiscible liquids, such as water and ether. The more common measure of solubility
`is the octanol—water partition coefficient (Log P, also known as Kow). The Log P value can
`be experimentally derived (Figure 5.1) or estimated using commercially available computer
`softwarel. Clearly, an experimentally derived value represents the gold standard. The value of
`Log P is calculated using Equation (5.1).
`
`KW = Log |: [octanol]]
`
`[water]
`
`(5-1)
`
`Where [octanol] and [water] represent the concentration ofa chemical in octanol and water,
`respectively.
`Being hydrophobic, octanol represents a lipophilic environment. In contrast, water is
`(perhaps rather obviously!) a hydrophilic environment. Thus, the tendency of a chemical
`to partition into octanol rather than water is reflected in a positive Log P value, whereas
`preferential partitioning into the water phase results in a negative value (Table 5.1). An equally
`amphiphilic compound would have a Log P of 0.
`The relationship between solubility and the rate of skin absorption stems primarily from
`the ability of a chemical to partition into the stratum corneum. If a chemical is excessively
`hydrophilic, it will not partition into the predominantly lipid environment of the stratum
`corneum (Chaptersl and 4). In contrast, if a chemical is too strongly lipophilic,
`it will
`readily partition into the stratum corneum but will not partition out into the predominantly
`hydrophilic environment of the underlying epidermal tissue. Put simply, it will remain stuck
`within stratum corneum. Thus, in order to penetrate the skin, the solubility of a chemical
`requires a balance between these two extremes. In general, 3 Log P of between 1 and 3 is
`considered to be optimal for skin absorpti0n (Figure 5.2).
`The importance of lipophilicity and molecular size on skin permeation has been well
`established and incorporated into a series of models, the best known of which is the ‘Potts
`and Guy’ equation (Potts and Guy 1992); EquatiOn (5.2). In recent years, considerable efforts
`have been put into refinement of these models, driven both by innate scientific curiosity and
`an increased requirement for cost-effective methods of generating dermal safety data on a
`large range of existing chemicals. However, it is important to appreciate that these models
`
`1It should be noted that Log P can only be used to describe the solubility of uncharged species. Log D (distribution
`coefficient) should be used for charged molecules. However, as most charged molecules do not readily penetrate the
`skin Log P is the most commonly used parameter.
`
`0014
`
`

`

`86
`
`CHOB: PHYSICOCHEMICAL FACTORS AFFECTING SKIN ABSORPTION
`
`Sample of
`chemical
`
`' .
`
`octanol —b-
`
`Constant stirring—,
`
`water fl -
`
`
`
`Aliquot samples taken
`from octanol and water
`
`phases and concentration 4——
`of chemical determined in
`each
`
`
`
`Centritugationfsettling
`
`
`
`Kow
`
`is introduced into a vial containing octanol and water (which are immiscible and so
`The chemical
`separate on standing into to distinct layers). The mixture is then stirred for a period before centrifugation
`(to separate out the octanol and water layers). Samples of the upper (octanol) and lower (water) phases
`are then carefully obtained and the concentration of chemical
`in each phase determined using an
`appropriate analytical method. The K0W is then calculated by dividing the concentration of the chemical
`in the octanol phase by the concentration in the water (Equation (5.1)).
`
`Figure 5.1
`
`Summary of method to measure Log P (KW) of a test compound
`
`are based on a limited data set on permeation from saturated aqueous solutions and neither
`formulation effects nor physiological factors are considered. Obvious anomalies, such as
`differential permeation of stereo-isomers with identical MW and log P values, demonstrate
`that predictions generated by such models should be used with caution.
`
`Log Kp = 0.71 Log K0,, — 0.006.mw — 2.74
`
`(5.2)
`
`Where Kp = permeability coefficient (see Chapter 6}, K0W is the octanol—water partition
`coefficient (Log P) and mw is molecular weight.
`
`5.2.3 Charge
`
`The presence of proteins (such as keratin) endows the stratum corneum with both positively
`and negatively charged groups. This characteristic, in combination with the lipophilic nature
`of the stratum corneum provides an effective barrier against charged molecules (ions). Thus,
`in general, ions are (at best) poorly absorbed across the stratum corneum. Indeed, there is
`
`0015
`
`

`

`5.2: PHYSICOCHEMICAL PROPERTIES
`
`8?
`
`A small selection of chemicals and their associated Log P values obtained from the EDETUX
`Table 5.1
`database2 and Flynn list (Reproduced from Flynn, G.L. (1990). Physicochemical determinants of skin
`absorption, in Principles of route to route extrapolation for risk assessment (eds Gerrity, T.R. and Henry,
`Cl), Copyright © 1990, Elsevier Science)
`
`Chemical
`
`glucose
`sucrose
`water
`
`butanediol
`ethanol
`
`scopolamine
`Nicotine
`nicotine
`
`paroxon
`diethyltoluamide
`oestradiol
`
`meperidine
`testosterone
`
`fentanyl
`Chlorpyrifos
`
`Log P
`
`—3.24
`—2.25
`—1 .38
`
`—{].92
`—0.31
`
`0.98
`1.17
`1.17
`
`1.98
`2.18
`2.69
`
`2.72
`3.32
`
`4.05
`=4 .96
`
`(Arbitrary)
`
`SkinAbsorptionHate
`
`Solubility (Log F')
`
`Figure 5.2 Representation of the theoretical effect of solubility (expressed as Log P) on the rate
`of skin absorption through skin (Note that this is an empirical generalisation! In reality, the actual
`relationship will varyr according to the particular group of chemicals being studied (e.g. alkanols,
`phenols, esters etc))
`
`evidence to suggest that appendageal routes (Chapter 4} may be the predominant pathway for
`diffusion for charged molecules, especially hydrophilic ions.
`The presence of negatively charged groups outnumbers those that are positive and so the
`stratum corneum carries a net negative charge. For this reason, the penetration of positively
`
`2httpzi'iede(ox.ncl.ac.ul<firrdex.html
`
`0016
`
`

`

`88
`
`CHOS: PHYSICOCHEMICAL FACTORS AFFECTING SKIN ABSORPTION
`
`{arbitrary}
`
`
`Relativepenetrationrate
`
`Figure 5.3 Theoretical effect of vehicle pH on the skin absorption of a weak acid (A) and a weak
`base (B) (This illustration assumes a pKa or pr of 5 for the acid and base, respectively. The fraction
`of non-ionised acid increases at low pH, resulting in an increase in skin absorption. Conversely, the
`fraction of unionised base decreases at lower pH, leading to reduced penetration)
`
`charged molecules (cations) is generally faster than negatively charged molecules (anions). In
`other words, the stratum corneum is ‘cation selective’, which has implications for transdermal
`delivery of drugs (Walters 2002).
`The case is slightly more complicated for chemicals whose ionisation state is pH—dependent,
`such as weak acids, bases and zwitterions (molecules which have both acid and base groups). In
`general, unionised moieties penetrate better than ionised (Figure 5.3) and so a vehicle pH that
`favours the formation of non-ionised molecules will result in more extensive skin absorption.
`Furthermore, uniouised molecules tend to be more lipophilic than ionised forms and so a pH
`that favours the formation ofnon-ionised moieties may also promote skin absorption through
`a change in solubility.
`Given that the pH of the stratum corneum ranges from around 4 to 6, then molecules which
`are predorninarltiy non-ionised will tend to be absorbed more extensively than chemicals
`which are predominantly ionised within this pH range.
`
`5.2.4 Hydrogen bonding
`
`The stratum corneum contains a wealth of hydrogen bonding groups arising from its lipid
`and protein c0mpositi0n. These can form reversible bonds with chemicals as they diffuse
`through the stratum Corneum, provided that the penetrant has complementary hydrogen
`bonding groups. Diffusion of a chemical through the stratum corneum can be retarded if
`it undergoes hydrogen bonding within the stratum corneum. Put simply, hydrogen bonds
`between a penetrant and the components of the stratum corneum can be thought of as brief
`molecular handshakes.
`
`There are essentially two factors that affect the extent to which hydrogen bonding will slow
`down diffusion of a molecule through the stratum corneum. The first is the potential strength
`
`0017
`
`

`

`5.3: EXPOSURE CONSIDERATIONS
`
`89
`
`100
`
`2:;
`K
`
`3:
`av
`EU
`.9
`
`1:5
`LL
`
`1000 so
`
`10
`
`1
`
`0.1
`
`A
`
`B
`
`C
`
`D
`
`E
`
`F
`
`G
`
`resorcinol; D: hydroquinone; E: pyrogallol;
`A: phenol: B: catechol: C:
`F: benzenetrioi; G: phloroglueinol.
`
`The effect of the number and position of hydrogen bonding groups for a range of phenol
`Figure 5.4
`derivatives on penetration through a surrogate biological membrane (Du Plessis et at. 2001, page ?,
`Copyright 2001, reprinted with permission from Eisevier)
`
`of the hydrogen bond; some groups interact more strongly than others — the handshaking is
`firmer and longer. For example, hydrogen bonding between a nitrogen atom and an alcohol
`(OH) group is roughly twice as strong as that between a nitrogen atom and an amine [NHzl
`group. Secondly, the number of hydrogen bonding groups (and their relative position on
`the penetrating molecule] is also important: more hands mean more handshakes! This is
`illustrated by the differential skin absorption of phenol derivatives (Figure 5.4). A quantitative
`consideration of the effects of hydrogen bending on skin absorption is given in Chapter 6.
`
`5.3 Exposure considerations
`
`The way in which a chemical is presented to the skin can have a substantial impact on the subsequent
`rate of absorption and this must be accounted for in the experimental design of skin absorption
`
`studies.
`
`Whilst the physicochemical properties of a molecule can strongly influence skin absorption,
`the way in which skin exposure occurs is also important. Of relevance to toxicological studies
`are such considerations as the solvent (vehicle) in which the chemical is dissolved, whether
`the exposure site is covered (ocduded) or left open to the environment {unoccluded) and the
`general condition of the skin.
`
`5.3.1 Vehicle effects
`
`The influence of a vehicle on the skin absorption of a chemical cannot be overstated. Even
`apparently small changes to a topical formulation can have a profound influence on the
`
`0018
`
`

`

`90
`
`CHOS: PHYSICOCHEMICAL FACTORS AFFECTING SKIN ABSORPTION
`
`rate and extent of skin absorption; this is reflected by international toxicological or cosmetic
`testing guidelines, which generally require that a topical formulation being assessed should be
`as close as possible to that intended to be marketed (see Chapters 7, 8 and 19). It is pointless,
`for example, to develop a margin of safety factor for a compound that is formulated for use
`in an aqueous based gel, based on skin permeation data of the test compound applied in an
`ethanolic solution. Cumulative permeation of the compound will probably be totally different
`over set exposure periods and the margin of safety may be completely over or underestimated.
`Vehicle effects are also of relevance to the pharmaceutical industry {transdermal drug
`delivery); whilst outside the remit of this book, a number of good texts are available on this
`subject {Barry 2003; Delgado-Charm and Guy 2001; Walters 2002). One reason why vehicles
`can so profoundly affect skin absorption is that they can alter the thermodynamic activity or
`fugacity of a penetrant. A more detailed overview of vehicle effects on the thermodynamic
`activity of a penetrant is given in Chapter 6.
`
`5.3.2 Volatility
`
`Volatility can affect the duration over which a chemical remains in contact with the skin
`and this can have a considerable influence on the rate and extent of skin absorption (and
`thus percutaneous toxicity). This is particularly apparent for highly toxic substances, such as
`chemical warfare agents, where systemic tOxicity is directly proportional to the volatility of a
`given substance within a chemical series (Chilcott 2007).
`
`5.3.3 Occlusion
`
`Occlusion can have two effects. The first [rather obvious) consequence of occlusion relates
`to volatile chemicals: preventing evaporative loss from the skin surface can enhance skin
`absorption and thus increase percutaneous toxicity. For example, contamination of skin with
`benzene results in very little systemic absorption as the vast majority of the applied dose
`(}99.9%) is lost through evaporation under normal circumstances. However, occlusion (for
`example, with a plastic film) can significantly reduce vapour loss and consequently increases
`skin absorption (Figure 5.5), potentially resulting in greater local or systemic toxicity.
`A second effect of occlusiOn is to increase skin hydration by preventing the normal loss
`of water from the skin surface from sweating or transepidermal water loss [TEWL; see
`Chapter 12). Water is essential for the maintenance of skin barrier function and, in normal
`skin, accounts for around 10% of the weight of the stratum corneum. Occlusion can increase
`the amount of water to 50% (wiw) and this excess hydration is generally associated with
`disruption of the normal structure of the stratum corneum with a corresponding loss of
`skin barrier function. However, the general rule that increased hydration leads to increased
`skin absorption has some notable exceptions, especially hydrophilic chemicals, which do not
`appear to be affected by occlusion-induced hydration (Zhai and Maibach 2001).
`
`5.3.4 Skin treatments
`
`Many guidelines for dermal toxicity assessment prescribe clipping, shaving or depilation as
`steps for preparing skin exposure sites (see Chapters 7 and 19). Such measures are necessary
`
`0019
`
`

`

`REFERENCES
`
`91
`
`III occluded
`
`El unocoluded
`
`20
`
`18
`
`16 -
`
`
`
`
`
`PercentageDosePenetrated
`
`14-
`
`12-
`
`10-
`
`
`
`
`10
`
`50
`
`100
`
`Amount Applied (pl)
`
`the amount
`Figure 5.5 Effect of occlusion on the evaporative loss of benzene from pig skin;
`penetrating occluded skin is consistently greater than unoccluded skin, regardless of the amount
`originally applied to the skin surface (Hattersley 2002)
`
`to provide an even site for application of a test substance and application chamber (where
`applicable). It should be noted that depilatories and shaving (wet and dry) both cause
`cousiderable damage to the stratum corneum (Marti et al. 2003; Wahlberg 1972) and so their
`use should be avoided or accounted for with suitable controls where appropriate.
`
`
`
`
`Summary
`
`0 The ‘dose makes the poison’ and so the extent to which a chemical can penetrate the skin will
`largely dictate its percutaneous toxicity.
`
`0 Skin absorption is largely influenced by the physicochemical properties of the penetrant:
`
`0 Whilst biological factors can be a source of significant variation, physicochemical properties
`generally control the magnitude of skin absorption.
`
`- The way in which a chemical is applied to the skin surface can also have