WHO Dermal Absorption

This report contains the collective views of an international group of experts and
does not necessarily represent the decisions or the stated policy of the United
Nations Environment Programme, the International Labour Organization or the
World Health Organization.
Environmental Health Criteria 235
DERMAL ABSORPTION
First draft prepared by Drs Janet Kielhorn, Stephanie Melching-
Kollmu, and Inge Mangelsdorf, Fraunhofer Institute of Toxicology and
Experimental Medicine, Hanover, Germany
Published under the joint sponsorship of the United Nations
Environment Programme, the International Labour Organization
and the World Health Organization, and produced within the
framework of the Inter-Organization Programme for the Sound
Management of Chemicals.
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tives of the IPCS are to establish the scientific basis for assessment of the risk to human
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assistance in strengthening national capacities for the sound management of chemicals.
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of the United Nations, WHO, the United Nations Industrial Development Organization,
the United Nations Institute for Training and Research and the Organisation for Economic
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is to promote coordination of the policies and activities pursued by the Participating
Organizations, jointly or separately, to achieve the sound management of chemicals in
relation to human health and the environment.
WHO Library Cataloguing-in-Publication Data
Dermal absorption.
(Environmental health criteria ; 235)
1.Skin absorption. 2.Risk assessment. 3.Environmental exposure.
I.World Health Organization. II.International Programme on Chemical Safety.
III.Series.
ISBN 92 4 157235 3 (NLM classification: WR 102)
ISBN 978 92 4 157235 4
ISSN 0250-863X
World Health Organization 2006
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iii
CONTENTS
ENVIRONMENTAL HEALTH CRITERIA FOR
DERMAL ABSORPTION
PREAMBLE x
ACRONYMS AND ABBREVIATIONS xvii
1. SUMMARY 1
2. INTRODUCTION AND DEFINITIONS 6
2.1 Scope of the document 6
2.2 Definition of dermal absorption 8
2.3 Factors influencing dermal absorption 8
3. SKIN STRUCTURE AND FUNCTION 10
3.1 Functions of the skin 10
3.1.1 Barrier function 10
3.1.2 Temperature control 11
3.1.3 Defence and repair 11
3.2 Skin structure 12
3.2.1 Epidermis 12
3.2.2 Dermis 16
3.2.3 Skin appendages 16
3.3 The transport of chemicals through the skin 17
3.4 Variability in skin permeability 17
3.4.1 Species 17
3.4.2 Age, sex, and race 18
3.4.3 Anatomical site 19
3.4.4 Skin condition 19
3.4.5 Temperature and blood flow rate 19
3.4.6 Hydration 20
3.5 Reservoir effects 20
EHC 235: Dermal Absorption
iv
4. SKIN TRANSPORT MECHANISMS AND
THEORETICAL CONCEPTS 23
4.1 Transport through the skin 23
4.2 Theoretical aspects of diffusion 23
4.3 Physicochemical factors affecting skin permeation 26
4.3.1 Physical state 27
4.3.2 Molecular size/molecular weight 27
4.3.3 Maximum flux 28
4.3.4 Ionization 28
4.3.5 Binding properties 29
4.4 Concepts of finite and infinite dose 29
5. METABOLISM IN THE SKIN 32
5.1 The drug-metabolizing systems of the skin 33
5.2 Methodology for evaluating skin metabolism in
in vitro systems 35
5.3 Effects of skin metabolism 35
5.4 Importance of metabolism for percutaneous
absorption 36
6. IN VITRO TESTS FOR DERMAL ABSORPTION 38
6.1 Test guidelines 38
6.2 Principles of the standard in vitro tests using skin
samples 39
6.2.1 Test chambers 39
6.2.1.1 Static diffusion cells 40
6.2.1.2 Flow-through cells 40
6.2.1.3 Comparison of different in vitro cell
systems 42
6.2.2 Finite/infinite dosing 43
6.2.3 Skin preparations 44
6.2.3.1 Choice of skin 44
6.2.3.2 Preparation of tissue samples 45
6.2.3.3 Checking of barrier integrity 46
6.2.4 Application of test substance 47
6.2.4.1 Test substance 47
6.2.4.2 Vehicle 48
6.2.4.3 Receptor fluid 48
6.2.4.4 Application dose levels 50
v
6.2.5 Duration of exposure and sampling time 50
6.2.6 Evaluation of the results 50
6.2.6.1 Dermal absorption results after finite
dosing 51
6.2.6.2 Dermal absorption results after infinite
dosing 52
6.3 Other in vitro methods 52
6.3.1 Artificial skin 52
6.3.2 Tape-stripping technique in vitro 52
6.4 Examination of skin reservoir characteristics 53
6.5 Experimental factors affecting dermal absorption
in vitro 54
6.5.1 Species differences 54
6.5.2 Temperature 55
6.5.3 Occlusion 56
6.5.4 Thickness of skin 56
6.5.5 Further observations on application vehicle
effects 58
7. IN VIVO TESTS FOR DERMAL ABSORPTION 60
7.1 Laboratory animal studies 60
7.1.1 Test guidelines for laboratory animal studies 61
7.1.2 Principles of the standard in vivo tests 61
7.1.2.1 Preparation of the application site 62
7.1.2.2 Dose levels 62
7.1.2.3 Application of the test substance to the
skin 62
7.1.2.4 Duration of exposure 63
7.1.2.5 Sacrifice and time of termination 63
7.1.2.6 Evaluation of the results 64
7.2 Studies with human volunteers 65
7.2.1 Assessment using plasma, excreta, and breath
analysis 66
7.2.1.1 Methodology 66
7.2.1.2 Examples of in vivo human volunteer
7.2.1.3 Biomonitoring of occupational
exposure 67
7.2.2 Cutaneous microdialysis 68
7.2.3 Tape stripping 70
Contents
studies 66
vi
7.3 Other methods 73
7.3.1 Whole-body autoradiography 73
7.3.2 Skin biopsy 73
7.4 Factors affecting dermal absorption in vivo 74
7.4.1 Species, strain, and sex 74
7.4.2 Age 75
7.4.3 Anatomical site 75
7.4.4 Type of application and vehicle 77
7.4.5 Temperature and humidity conditions 78
8. COMPARATIVE STUDIES 79
8.1 Comparison between in vitro and in vivo skin
absorption results 79
8.2 Inter- and intralaboratory variation in in vitro
percutaneous absorption methodology 84
9. DATA COLLECTIONS 86
9.1 Data sets from homologous or closely related
9.2 Flynn data set 87
9.3 Expanded permeability coefficient data sets 88
9.4 EDETOX database 88
9.5 Maximum flux databases 89
10. ESTIMATION/PREDICTION OF DERMAL
PENETRATION 90
10.1 QSAR analysis 91
10.1.1 Prerequisites for QSPeR analysis 91
10.1.2 Historical overview 92
10.1.2.1 QSPeRs for skin permeability
prior to the 1990s 92
10.1.2.2 The Flynn (1990) data set and
subsequent analyses 93
10.1.2.3 Other data sets 97
10.1.3 Other approaches to QSPeR 97
10.1.4 Variability of data and its relevance
for QSPeRs 98
10.1.5 Statistical analysis (linear vs non-
linear) methods 98
molecules 86
vii
10.1.6 Selection of chemicals for further
tests on dermal penetration 98
10.1.7 Applicability domain for QSPeR 99
10.1.8 Maximum fluxes 99
10.1.9 Rules as an alternative to QSPeRs 100
10.2 Mathematical modelling 100
10.3 Mathematical pharmacokinetic models of
percutaneous penetration 103
11. USE OF DERMAL PENETRATION STUDIES IN RISK
ASSESSMENT 105
11.1 Decision-making process for setting dermal
absorption values 106
11.1.1 Default values 107
11.1.2 Measured values 108
11.1.3 Values from mathematical skin permeation
models (e.g. QSARs/QSPeRs) 110
11.2 Use of relative absorption values versus flux (and
their derived permeability coefficients) 110
11.3 Other topics related to risk assessment 111
12. CONTROVERSIAL TOPICS IN THE ASSESSMENT OF
DERMAL ABSORPTION 112
12.1
12.2 Reduction of intralaboratory/interlaboratory
variation 112
12.3 Consequences of reservoir effect for risk assessment 113
12.4 Relevance of percutaneous measurements to data
required by risk assessors: finite and infinite
exposures 114
12.5 Single- versus multiple-exposure regimes 114
12.6 Barrier integrity test for skin barrier function of
human skin in skin penetration tests 115
12.7 Dermal absorption in susceptible populations 115
12.8 Skin notation 117
12.8.1 Skin notation criteria in different countries 118
12.8.2 Quantitative approaches 119
12.8.3 New approaches 120
12.9 Dermal absorption of nanoparticles 121
Contents
QSARs/QSPeRs 112
viii
13. CONCLUSIONS AND RECOMMENDATIONS 124
REFERENCES 127
APPENDIX 1: GUIDELINES AND PROTOCOLS 163
APPENDIX 2: PAST AND PRESENT INITIATIVES ON
EXCHANGE OF INFORMATION AND HARMONIZATION
OF METHODOLOGY ON DERMAL ABSORPTION 170
RESUME 186
RESUMEN 192
ix
NOTE TO READERS OF THE CRITERIA MONOGRAPHS
Every effort has been made to present information in the criteria
monographs as accurately as possible without unduly delaying their
publication. In the interest of all users of the Environmental Health
Criteria monographs, readers are requested to communicate any
errors that may have occurred to the Director of the International
Programme on Chemical Safety, World Health Organization,
Geneva, Switzerland, in order that they may be included in corri-
genda.
x
Environmental Health Criteria
PREAMBLE
Objectives
In 1973, the WHO Environmental Health Criteria Programme
was initiated with the following objectives:
(i) to assess information on the relationship between exposure to
environmental pollutants and human health, and to provide
guidelines for setting exposure limits;
(ii) to identify new or potential pollutants;
(iii) to identify gaps in knowledge concerning the health effects of
pollutants;
(iv) to promote the harmonization of toxicological and epidemio-
logical methods in order to have internationally comparable
results.
The first Environmental Health Criteria (EHC) monograph, on
mercury, was published in 1976, and since that time an ever-
increasing number of assessments of chemicals and of physical
effects have been produced. In addition, many EHC monographs
have been devoted to evaluating toxicological methodology, e.g. for
genetic, neurotoxic, teratogenic, and nephrotoxic effects. Other
publications have been concerned with epidemiological guidelines,
evaluation of short-term tests for carcinogens, biomarkers, effects on
the elderly, and so forth.
Since its inauguration, the EHC Programme has widened its
scope, and the importance of environmental effects, in addition to
health effects, has been increasingly emphasized in the total
evaluation of chemicals.
The original impetus for the Programme came from World
Health Assembly resolutions and the recommendations of the 1972
UN Conference on the Human Environment. Subsequently, the work
became an integral part of the International Programme on Chemical
Safety (IPCS), a cooperative programme of WHO, ILO, and UNEP.
In this manner, with the strong support of the new partners, the
xi
importance of occupational health and environmental effects was
fully recognized. The EHC monographs have become widely estab-
lished, used, and recognized throughout the world.
The recommendations of the 1992 UN Conference on Environ-
ment and Development and the subsequent establishment of the
Intergovernmental Forum on Chemical Safety with the priorities for
action in the six programme areas of Chapter 19, Agenda 21, all
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chemicals.
Scope
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Preamble
xii
The EHC monographs are intended to assist national and inter-
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orities on risk assessment methodology.
Procedures
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distribution.
xiii
Commitment to draIt EHC
Document preparation initiated
DraIt sent to IPCS Responsible OIIicer (RO)
(+&35(3$5$7,21)/2:&+$57
Revision as
necessary
Possible meeting
oI a Iew experts
to resolve
controversial issues
First DraIt
Responsible OIIicer, Editor check Ior coherence oI text and
readability (not language editing)
International circulation to Contact Points (150)
Comments to IPCS (RO)
Review oI comments, reIerence cross-check;
preparation oI Task Group (TG) draIt
Task Group meeting
Insertion oI TG changes
Post-TG draIt; detailed reIerence cross-check
Editing
Word-processing
Camera-ready copy
Final editing
Approval by Director, IPCS
WHO Publication OIIice
Printer ProoIs Publication
Graphics
Library Ior
CIP Data
French/Spanish
translations oI Summary
Working group,
iI required
Editor
routine procedure
optional procedure
Preamble
xiv
The three cooperating organizations of IPCS recognize the
important role played by nongovernmental organizations. Represen-
tatives from relevant national and international associations may be
invited to join the Task Group as observers. Although observers may
provide a valuable contribution to the process, they can speak only
at the invitation of the Chairperson. Observers do not participate in
the final evaluation of the chemicals; this is the sole responsibility of
the Task Group members. When the Task Group considers it to be
appropriate, it may meet in camera.
All individuals who as authors, consultants, or advisers partici-
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dinator, IPCS, the monograph is submitted for printing.
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xv
WHO TASK GROUP ON ENVIRONMENTAL HEALTH
CRITERIA FOR DERMAL ABSORPTION
The first draft of the EHC monograph was prepared for IPCS by
the Fraunhofer Institute, Hanover, Germany, in 2005. It was widely
distributed by IPCS for international peer review for two months
from March to May 2005. A revised proposed draft document, tak-
ing into account comments received, was prepared by the Fraun-
hofer Institute. An EHC Task Group was convened from 28 June to
1 July 2005, in Hanover, Germany, to further develop and finalize
the document.
Ms C. Vickers was responsible for the production of the mono-
graph.
The efforts of all who helped in the preparation and finalization
of the monograph are gratefully acknowledged.
* * *
Risk assessment activities of IPCS are supported financially by
the Department of Health and Department for Environment, Food &
Rural Affairs, United Kingdom; Environmental Protection Agency,
Food and Drug Administration, and National Institute of Environ-
mental Health Sciences, USA; European Commission; German
Federal Ministry of Environment, Nature Conservation and Nuclear
Safety; Health Canada; Japanese Ministry of Health, Labour and
Welfare; and Swiss Agency for Environment, Forests and Land-
scape.
Task Group Members
Dr R. Bronaugh, Office of Cosmetics and Colors, Food and Drug
Administration, Laurel, MD, USA
Dr A.L. Bunge, Chemical Engineering Department, Colorado
School of Mines, Golden, CO, USA
Preamble
xvi
Dr J. Heylings, Central Toxicology Laboratory, Syngenta CTL,
Alderley Park, Macclesfield, Cheshire, United Kingdom
Dr S. Kezic, Coronel Institute of Occupational and Environmental
Health, Academic Medical Center, University of Amsterdam,
Amsterdam, The Netherlands
Dr J. Krse, Kinetox, Vleuten, The Netherlands
Dr U. Mueller, Office of Chemical Safety, Therapeutic Goods
Administration, Woden, ACT, Australia
Dr M. Roberts, Department of Medicine, University of Queensland,
Princess Alexandra Hospital, Buranda, Queensland, Australia
(Chairperson)
Dr J.J.M. van de Sandt, Food and Chemical Risk Analysis, TNO
Quality of Life, Zeist, The Netherlands
Dr K.A. Walters, Loughborough, United Kingdom
Professor F.M. Williams, Toxicology Unit and Environmental Med-
icine, The Medical School (School of Clinical Laboratory Sci-
ences) and Institute for Research on Environment and Sustainability,
University of Newcastle, Newcastle upon Tyne, United Kingdom
Secretariat
Dr J. Kielhorn, Department of Chemical Risk Assessment, Fraun-
hofer Institute of Toxicology and Experimental Medicine, Hanover,
Germany (Rapporteur)
Dr I. Mangelsdorf, Department of Chemical Risk Assessment,
Fraunhofer Institute of Toxicology and Experimental Medicine,
Hanover, Germany
Dr S. Melching-Kollmu, Department of Chemical Risk Assess-
ment, Fraunhofer Institute of Toxicology and Experimental Medi-
cine, Hanover, Germany
Ms C. Vickers, International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland
xvii
ACRONYMS AND ABBREVIATIONS
ACGIH American Conference of Governmental
Industrial Hygienists
ADME absorption, distribution, metabolism, and
excretion
AOEL acceptable operator exposure level
AUC area under the curve
CEFIC European Chemical Industry Council
CEFIC-LRI European Chemical Industry Council Long-
range Research Initiative
COLIPA European Cosmetic Toiletry and Perfumery
Association
DECOS Dutch Expert Committee on Occupational
Standards
DOEL dermal occupational exposure limit
DPK dermatopharmacokinetics
EC European Commission
ECETOC European Centre for Ecotoxicology &
Toxicology of Chemicals
ECVAM European Centre for the Validation of
Alternative Methods
EDETOX Evaluations and Predictions of Dermal
Absorption of Toxic Chemicals
EHC Environmental Health Criteria
EU European Union
EURO POEM European Predictive Operator Exposure
Model
GC gas chromatography
HPLC high-performance liquid chromatography
ILO International Labour Organization
IPCS International Programme on Chemical
Safety
xviii
J
ss
steady-state flux
K
m
vehicle/stratum corneum partition coefficient
K
ow
octanol/water partition coefficient
K
p
permeability coefficient
LC-MS liquid chromatography/mass spectrometry
LD
50
median lethal dose
MAK maximum allowable concentration
(Germany)
MAK Commission Senate Commission on the Investigation of
Health Hazards of Chemical Compounds in
the Work Area (Germany)
MW molecular weight
NOAEL no-observed-adverse-effect level
OECD Organisation for Economic Co-operation
and Development
OEL occupational exposure limit
OSHA Occupational Safety and Health
Administration (USA)
PAH polycyclic aromatic hydrocarbon
PPP Perspectives in Percutaneous Penetration
(formerly Prediction of Percutaneous
Penetration)
QSAR quantitative structureactivity relationship
QSPeR quantitative structurepermeability
relationship (also called QSPR)
REACH Registration, Evaluation, Authorisation and
Restriction of Chemicals
RO Responsible Officer
SCCNFP Scientific Committee on Cosmetic Products
and Non-Food Products Intended for
Consumers
SCOEL Scientific Committee for Occupational
Exposure Limits (European Union)
TLV threshold limit value
xix
TSCA Toxic Substances Control Act (USA)
TWA time-weighted average
UN United Nations
UNEP United Nations Environment Programme
USA United States of America
USEPA United States Environmental Protection
Agency
USFDA United States Food and Drug Administration
UV ultraviolet
WHO World Health Organization
Acronyms and Abbreviations
1
1. SUMMARY
Dermal (percutaneous, skin) absorption is a global term that
describes the transport of chemicals from the outer surface of the
skin both into the skin and into the systemic circulation. This
Environmental Health Criteria document presents an overview of
dermal absorption and its application to the risk assessment of
chemicals. In addition, it presents and discusses current topics of
interest in the field of dermal absorption. Dermal absorption can
occur from occupational, environmental, or consumer skin exposure
to chemicals, cosmetics, and pharmaceutical products.
The skin is a complex organ and a living membrane. The func-
tions of the skin include protection, regulation of body temperature
and water loss, and defence and repair. The skin is composed of an
outer region, the epidermis, and an inner region, the dermis. The
epidermis consists of various cell layers, the outermost layer, the
stratum corneum or horny layer, functioning as the main barrier to
the entry of extraneous chemicals. The viable epidermis can metabo-
lize chemicals that pass through the stratum corneum. The dermis
provides physiological support for the avascular epidermis and is the
locus of blood vessels, sensory nerves, and lymphatics in the skin.
The skin also contains appendages, such as hair follicles, sweat
glands, and sebaceous glands, which originate in the subpapillary
dermis.
There is considerable variability in the measurement of skin
permeability. There can be major differences in permeability
between species. Little is known about variation due to age,
although the skin structure does change with age; however, sex and
ethnic background do not seem to be sources of variation in permea-
bility. Percutaneous absorption is also dependent on the anatomical
site, on the skin condition, and on the hydration state of the skin.
Factors influencing percutaneous absorption through the skin
include 1) physicochemical properties of the test compound,
2) physicochemical and other properties of the vehicle in which the
test compound is dissolved, 3) interactions between the test com-
pound or vehicle and the skin, 4) skin properties and metabolism,
2
and 5) factors inherent to the test system used for measurement
for example, dose and volume of test substance, occlusion or non-
occlusion of test area, in vitro or in vivo test systems, and duration
of exposure.
Theoretical equations and models have been developed to
describe the transport of a diffusing chemical through the skin.
Typically, the steady-state flux (J
ss
) and the permeability coefficient
(K
p
) are the main parameters assessed from in vitro experiments in
which the donor concentration of the penetrant is maintained at
constant (infinite) dose conditions. The estimation of maximum flux,
time for maximum flux, lag time, residual (reservoir) amounts
retained in the stratum corneum, and mass balance under real
application conditions is now recognized to be of prime importance
in exposure estimations.
Measurement of metabolism of a chemical in contact with skin
may be important in both efficacy and safety evaluations. Some
chemicals can be significantly metabolized during dermal absorp-
tion, which may result in either inactive or active metabolites. The
measurement of this metabolism may therefore be important in an
appropriate safety evaluation. Toxic chemicals such as benzo[a]py-
rene have been shown to be activated in skin, whereas other chemi-
cals may undergo hydrolysis and/or conjugation reactions in the
skin, resulting in a decrease in the availability of those chemicals to
the body. In general, the viability of skin can be maintained in an in
vitro diffusion cell by using fresh skin and a physiological buffer. It
is recommended that this viability be verified through measuring the
activity of relevant metabolizing enzymes.
The permeability properties of the stratum corneum are, for the
most part, unchanged after its removal from the body. As a con-
sequence, a good correlation exists between measurements derived
from both in vivo and in vitro skin diffusion experiments with the
same chemicals (at least for hydrophilic compounds). In vitro exper-
iments are an appropriate surrogate for in vivo studies and offer a
number of advantages over whole-animal or human volunteer
experiments. In vitro methods measure the diffusion of chemicals
into and across skin to a fluid reservoir and can utilize non-viable
skin to measure diffusion only or fresh, metabolically active skin to
simultaneously measure diffusion and skin metabolism. Test Guide-
Summary
3
line 428 of the Organisation for Economic Co-operation and Devel-
opment (OECD) encourages harmonization of methodology. Experi-
mental factors affecting dermal absorption in vitro, in addition to
those mentioned above, include the thickness of skin sample, varia-
tions in temperature of the test system, and composition of the
receptor fluid. Static or flow-through in vitro diffusion cells can be
used. Additional techniques, requiring further refinements, include
tape stripping and the use of artificial or reconstituted skin.
In vivo methods allow the determination of the extent of cutane-
ous uptake as well as systemic absorption of the test substance. The
main advantage of performing an in vivo study rather than an in
vitro study is that it uses a physiologically and metabolically intact
system. In vivo dermal penetration studies are carried out in labora-
tory animals, usually rodents, and in human volunteers. In vivo
dermal penetration studies in human volunteers have been widely
used for human pharmaceuticals and, to a more limited extent, for
other chemicals. In vivo studies in humans are the gold standard.
The conduct of any in vivo study has ethical issues. The main dis-
advantage in the use of laboratory animals is that they have different
skin permeability and systemic disposition compared with humans.
The results of human volunteer studies have shown that occupa-
tional exposure to liquids (such as solvents) can result in consid-
erable dermal absorption. Skin uptake from vapours may be an
important contributor to the total uptake for some volatile sub-
stances, such as the glycol ethers.
In vitro dermal absorption studies are increasingly being sub-
mitted for registration purposes for industrial chemical, cosmetic,
and crop protection products. There are many published studies that
compare in vitro and in vivo results in laboratory animals and
humans. Properly conducted in vitro studies that follow the OECD
test guidelines have demonstrated that the in vitro approach can
provide good prediction of in vivo dermal absorption.
Over the decades, a large number of data have been generated
on the percutaneous penetration of a wide range of chemicals,
pesticides, cosmetics, and pharmaceuticals. Studies have included
work on human volunteers, in vivo studies using animal models, in
4
vitro studies on excised human, rodent, pig, guinea-pig, etc., skin,
and, more recently, in vitro studies on synthetic skin.
There have been many attempts over the last 50 years to predict
the rate and extent of dermal absorption and so reduce the need for
in vitro and in vivo testing. This need is even greater in response to
increasing ethical difficulties with respect to human and laboratory
animal experiments as well as the legislatively imposed economic
and time considerations particularly in the risk assessment of
industrial chemicals. Quantitative structurepermeability relation-
ships (QSPeRs) are statistically derived relationships between the
steady-state flux of a compound and various physicochemical
descriptors and/or structural properties of the molecule. Efficacy and
safety considerations also recognize quantitative structureactivity
relationships (QSARs) in irritation, skin sensitization, metabolism,
chemical effects, and clearance. QSARs are therefore involved at a
number of levels in chemical safety.
Mathematical models have been used to simulate the dynamics
of the partition, diffusion, metabolism, and other processes involved
in dermal absorption and can lead to the prediction of the extent and
rate of chemical permeation through the skin. Mathematical model-
ling plays a key role in linking permeability coefficient and flux data
obtained from tests under steady-state conditions (i.e. infinite dose)
to absorption estimates for finite dose applications that are more
typical of occupational exposure (i.e. non-steady-state conditions).
In risk assessment, the initial estimate for dermal absorption is
usually obtained by the use of a tiered approach, where the greatest
safety margin is defined by the worst case and more refined
estimates better define the real margin. Hence, as a first step, 100%
absorption is assumed when no data are available. In the second
step, a more realistic estimate of the extent of dermal absorption is
provided by a consideration of the physicochemical properties of the
chemical and the vehicle. The third step is a consideration of any
experimental in vitro and in vivo dermal absorption data. If, at the
end of these steps, an unacceptable risk is calculated, the risk
assessment is best refined by means of actual exposure data.
In the last few years, partly due to regulatory pressures, there
have been several initiatives to accelerate progress in the fields of
Summary
5
international harmonization of methodology and protocols, cul-
minating in the publication of the OECD test guidelines for skin
absorption studies in 2004. This international collaboration includes
projects such as an international validation study involving 18
laboratories, the European Evaluations and Predictions of Dermal
Absorption of Toxic Chemicals (EDETOX) project, and projects
sponsored by industry, as well as conferences, such as the Perspec-
tives in Percutaneous Penetration (formerly the Prediction of Percu-
taneous Penetration, or PPP) and Gordon Research conferences.
Available data on skin fluxes and permeability coefficients have
been collected into databases and analysed. Progress has been made
in further developing QSARs that link permeation data to the phys-
icochemical properties of chemicals. As a consequence, it is becom-
ing increasingly possible to more reliably predict penetration data
for a large number of chemicals. A possible outcome is a reduction
in the expensive and ethically demanding testing of chemicals using
laboratory animals and humans.
In spite of the successes in interdisciplinary international har-
monization to date, there are a number of aspects to be further
improved and which remain as topics of discussion. These include
the extent of intralaboratory and interlaboratory variation in in vitro
and in vivo studies; the acceptance of QSPeRs; the reservoir effect
of chemicals in the stratum corneum and its interpretation in risk
assessment; the relevance of dermal absorption measurements to
data required by risk assessors; and the use of the barrier integrity
test for skin barrier function. Other topics that must be considered
include dermal absorption in susceptible populations, the necessity
for harmonization of skin notation, and the dermal absorption of
nanoparticles.
Recommendations are made by the Task Group concerning the
benefits of using human skin over laboratory animal skin; study
design and harmonization of methodology; correlation of in vitro
and in vivo data and development of reliable prediction models;
encouraging support, maintainance, and update of databases; and
furthering the evaluation of QSARs for risk assessment purposes,
and preparation of guidance on their use.
6
2. INTRODUCTION AND DEFINITIONS
2.1 Scope of the document
Chemicals present in workplaces or the environment may come
into contact with the skin and be absorbed. Although interest in
dermal absorption for risk assessment purposes is comparatively
recent, research into the factors involved in the passage of com-
pounds through the skin has been conducted for other purposes.
Some scenarios where dermal absorption considerations are impor-
tant include:
the development of transdermal drug delivery systems (e.g. for
analgesia);
dermatological formulations for localized transport (e.g. psori-
asis);
safety assessment of cosmetics; and
risk assessment of occupational, environmental, or consumer
exposure.
Although these applications involve dermal absorption, they all
have different aims and approaches. For some drugs, it may be
important that the substance passes through the skin and into the
bloodstream. For cosmetics and sunscreen lotions, it may not be
necessary or desirable for the product to penetrate the skin; instead,
the product may simply remain in the upper skin layer.
In occupational and consumer scenarios, the skin absorption of
chemicals and pesticides needs to be minimized. Risk assessments
are usually performed to determine the extent to which exposure to a
particular substance is acceptable and therefore the extent to which
the substance is safe to use. For many chemicals, there is no infor-
mation on dermal absorption.
This Environmental Health Criteria document concentrates on
dermal absorption from occupational, environmental, or consumer
exposure, which may involve exposure to liquids, solids, or vapours.
The exposure to liquids is usually intermittent; volatile substances
Introduction and Definitions
7
may evaporate from the skin surface. Occupational exposure may be
single or repeated, thus requiring risk assessment and control.
The steps between the presence of a chemical in the environ-
ment and systemic exposure may be divided into two phases
(Johanson, 2003). The first phase is the dermal exposure to the
chemical (amount, area, duration). This is affected by a number of
factors, such as the properties of the chemical, the work process, the
individuals behaviour and work practices, type of clothing, type of
protective equipment, etc. This document does not cover these
aspects. It deals with the second phase, that from exposure of the
skin to systemic exposure the dermal absorption.
The purpose of this document is to present to the newcomer an
assessment of chemicals. It does not intend to be comprehensive. A
further aim is to present and discuss current topics of interest in the
field of percutaneous penetration. In the last few years, partly due to
regulatory pressures, there have been several initiatives to accelerate
progress in the fields of international harmonization of methodology
and protocols, culminating in the publication of the Organisation for
Economic Co-operation and Development (OECD) test guidelines
for skin absorption studies in 2004 (OECD, 2004a,b,c; see Appen-
dix 1) and the European Evaluations and Predictions of Dermal
Absorption of Toxic Chemicals (EDETOX) project (Appendix 2).
Further, available data on permeation have been collected into
quantitative structureactivity relationships (QSARs) linking phys-
icochemical properties to permeation data so that in the future it may
be possible to predict the data for a large number of chemicals rather
Appendix 2). In addition, projects have been initiated to investigate
interdisciplinary international harmonization, there are still points
Owing to the large amount of literature available, only some
specific studies are cited. In the respective chapters throughout the
measurement (chapters 68), in particular with regard to the risk
overview of dermal (percutaneous) absorption (chapters 35) and its
databases (see chapter 9), and progress has been made in developing
than undertake expensive testing of chemicals (see chapter 10 and
that are topics of discussion (chapter 12), and a way forward is
proposed (chapter 13).
risk assessment processes (chapter 11). In spite of these successes in
8
document, however, the reader is referred to reviews where more
information can be found.
2.2 Definition of dermal absorption
Dermal (percutaneous, skin) absorption is a global term that
describes the transport of chemicals from the outer surface of the
skin to the systemic circulation (OECD, 2004a). This is often
divided into:
penetration, which is the entry of a substance into a particular
layer or structure, such as the entrance of a compound into the
stratum corneum;
permeation, which is the penetration through one layer into a
second layer that is both functionally and structurally different
from the first layer; and
resorption, which is the uptake of a substance into the skin
lymph and local vascular system and in most cases will lead to
entry into the systemic circulation (systemic absorption).
These definitions will be used in this document. However, it
should be recognized that these terms may be interpreted differently
in the various product sectors.
2.3 Factors influencing dermal absorption
There are a number of factors that influence the dermal absorp-
and other factors are more fully discussed in the appropriate sections
of the document.
tion of a substance, and some of these are listed in Table 1. These
Introduction and Definitions
9
Table 1. Some important considerations in dermal absorption
System
components
Factors Discussion
in
Test
compound
Physical state
Molecular size
Lipid/water partition coefficient
Ionization
Local skin effects
Chapters 4
and 10
Skin Species
Anatomical site
Temperature
Hydration of stratum corneum
Damage to stratum corneum
Metabolism
Diseased skin
Desquamation
Blood and lymph flow
Chapters 3,
5, 6, and 7
Vehicle Solubility
Volatility
Distribution in stratum corneum
Excipients
Effect on the stratum corneum
pH
Chapters 6
and 7
Application
dose
Concentration
Finite and infinite dose
Skin area dose (film thickness,
concentration)
Total skin area in contact with vehicle
Duration of exposure
Chapters 6
and 7
Other factors Reservoir effect and its interpretation in risk
assessment
Chapters 3,
6, and 12
10
3. SKIN STRUCTURE AND FUNCTION
The skin is the largest organ in the body, with a surface area of
approximately 1.8 m
2
and a total weight estimated, for a typical
adult of 70 kg, to be 4 kg (Pannatier et al., 1978). In normothermic
conditions, the cutaneous circulation comprises 510% of the total
cardiac output (Johnson et al., 1986). For 70-kg human males, the
skin blood flow is approximately 4.64 cm
3
/s or 16 700 cm
3
/h
(Kasting & Robinson, 1993). Thus, the ratio of the total capillary
flow to the corresponding skin surface area is approximately
0.93 cm/h.
The skin is a heterogeneous organ, containing a number of
layers as well as appendages, such as sweat glands, hair follicles,
composition of the stratum corneum vary according to body region.
Until the beginning of the 20th century, the skin was thought to be
completely inert and impermeable to chemicals that might otherwise
enter the body. While the skin does act as a barrier, it is not a
complete barrier. Many chemicals do penetrate the skin, either
intentionally or unintentionally, and cutaneous metabolism does
occur. Because of its large surface area, the skin may be a major
route of entry into the body in some exposure situations.
Several authors have provided more extensive reviews of the
topics addressed in this section (e.g. Wiechers, 1989; Singh &
Singh, 1993; Schaefer & Redelmeier, 1996; Walters & Roberts,
2002; Madison, 2003; Monteiro-Riviere, 2004, 2005).
3.1 Functions of the skin
3.1.1 Barrier function
The skin provides a sturdy, flexible, and self-repairing barrier to
the exterior environment, protecting the internal body organs and
fluids from external influences. It prevents loss of endogenous water
and nutrients (humans are approximately 70% water) and protects
against many unwanted toxic substances and pathogenic micro-
and sebaceous glands (see Figure 1). The thickness of the skin and
Skin Structure and Function
11
organisms. The skin also responds to mechanical forces (elasticity
and cushioning).
Fig. 1. A schematic diagram of the structure of the skin.
3.1.2 Temperature control
The skin plays an essential role in the control of body tempera-
ture, which is regulated by sweating and arteriovenous thermo-
regulation. The skins resting blood flow of 250300 cm
3
/min can
increase to 60008000 cm
3
/min in response to an increase in internal
body temperature, with much of the flow going directly from
arterioles to venules (Cross & Roberts, 2005).
3.1.3 Defence and repair
The skin contains systems that provide the ability for defence
and repair, such as touch sensitivity (Merkel cells, nociceptors),
immunity (Langerhans cells), protection against ultraviolet (UV)
radiation (melanocytes), wound healing, and cutaneous metabolism.
Repair occurs automatically through the continuous turnover of the
skin, but this mechanism can be accelerated via cytokine release
following insult or injury.
Pore of
sweat gland duct
Epidermis
Hair shaft
Stratum Comeum
Nerve fibres
Nerve fibres
Blood vessels
Paccinian corpuscle
Stratum germinativum
Basement membrane
Dermis
Sweat gland
Subcutaneous
Layer
(Hypodermis)
12
3.2 Skin structure
Based on structure and embryonic origin, the cellular layers of
the skin are divided into two distinct regions. The outer region, the
epidermis, develops from the embryonic ectoderm and covers the
connective tissue; the dermis is derived from the mesoderm
(Maibach & Patrick, 2001; Monteiro-Riviere, 2005).
3.2.1 Epidermis
The epidermis comprises about 5% of full-thickness skin and is
divided into five or six layers, based on cellular characteristics (see
Figure 2). The majority of cells in the epidermis are called keratino-
cytes, which are formed by differentiation from one layer of mitotic
basal cells. The number of distinguishable layers is dependent upon
the anatomical site.
Fig. 2. The epidermis. All cell layers and locations of the two dendritic cell
types, melanocytes (A) and Langerhans cells (B), are shown (Maibach &
Patrick, 2001). [Copyright (2001) From Principles and Methods of Toxicology
by W.A. Hayes. Reproduced by permission of Routledge/Taylor & Francis
Group, LLC]
Stratum
corneum
Stratum lucidum
(palms and soles)
Stratum
granulosum
Stratum
spinosum
A
B
Basal
layer
Basal lamina
ntermediate
zone
13
Basal layer (stratum germinativum) keratinocytes are metabol-
ically active cells with the capacity to divide. Some of the resulting
daughter cells of the basal layer move outward and differentiate. The
cells adjacent to the basal layer start to produce lamellar granules,
intracellular organelles that later fuse with the cell membrane to
release the neutral lipids that are thought to form a barrier to pene-
tration through the epidermis (see next paragraph). The desmosomes
(adhesive junctions; Hatsell & Cowin, 2001) and other bridges
connecting the adjacent cells have the appearance of spines, giving
the name stratum spinosum to this three- to four-cell-thick layer
above the basal layer. The keratinocytes migrate outwards to the
third epidermal layer, called the stratum granulosum, which is char-
acterized by the presence of keratohyalin granules, polyribosomes,
large Golgi bodies, and rough endoplasmic reticulum. These are the
outermost viable cells of the epidermis.
The major barrier to permeation within the skin is the non-
viable stratum corneum, the outermost cornified layer, usually 15
20 cells thick and consisting of cells (corneocytes) that have lost
their nucleus and all capacity for metabolic activity. The dominant
constituent of these cells is keratin, a scleroprotein with chains
linked by disulfide and hydrogen bonds. The corneocytes are con-
nected by corneodesmosomes and surrounded by extracellular non-
polar lipids. The stratum corneum intercellular barrier lipids orig-
inate in the lamellar granules most prominent in the granular cell
layer of the epidermis (Madison, 2003). Each corneocyte is enclosed
within a protein-rich cornified cell envelope that provides covalent
linkage sites for the intercellular lipids.
The process of desquamation disrupts intercellular attachment,
and the outermost cells are sloughed from the surface. The turnover
rate for keratinocytes has been calculated to be between 17 and
71 days, depending upon anatomical site: e.g. 3236 days for the
human palm and 58 days for the anterior surface of the forearm
(Maibach & Patrick, 2001). Although the thickness of non-hydrated
stratum corneum is about 1050 m over most of the body, it may
be 10 times thicker on friction surfaces such as the hands and soles
of the feet (Rushmer et al., 1966), where the corneodesmosomes
also have a higher prevalence than in other skin regions. The stratum
corneum has a water content of 520%, compared with 70% in the
physiologically active basal layer.
14
Owing to the relative impermeability of the cornified envelope
to most compounds, the major route of penetration across the
stratum corneum has been identified as the tortuous pathway
between the corneocytes (see Figure 3), implying that stratum
corneum lipids play a key role in the skin barrier function (Michaels
et al., 1975; Elias, 1981; Grubauer et al., 1987; Mao-Qiang et al.,
1993; Bouwstra et al., 2001, 2003a; Ponec et al., 2003).
Fig. 3. Diagram of the stratum corneum membrane, showing two possible
routes for diffusion (Barry, 1991). [Copyright (1991) From In Vitro
Percutaneous Absorption: Principles, Fundamentals, and Applications by
R.L. Bronaugh and H.I. Maibach. Reproduced by permission of
Routledge/Taylor & Francis Group, LLC]
The hydrophobic lipids present in the intercellular spaces of the
stratum corneum are (all weight per cent) 4550% ceramides, 25%
cholesterol, 15% long-chain free fatty acids (mostly with chain
lengths C22 and C24), and 5% other lipids, the most important being
cholesterol sulfate, cholesterol esters, and glucosylceramides (Wertz
et al., 1987; Law et al., 1995; Madison, 2003). Nine subclasses of
ceramides have been identified in human stratum corneum (de Jager
et al., 2003; Ponec et al., 2003). The ceramides consist of a
ntercellular route
Plasma
membrane
ntercellular
space
Aqueous
Aqueous
Cholesterol
Glucosylceramide
Ceramide
Lipid?? Keratin
Lipid
Lipid
Fatty acid
Cell
cytoplasm
Transcellular route
15
sphingosine or a phytosphingosine base to which a non-hydroxy
fatty acid or an alpha-hydroxy fatty acid is chemically linked (see
Figure 4). The fatty acid chain is mainly C24 and C26. Cholesterol
sulfate, although present in only small amounts, has been shown to
be involved in the regulation of the desquamation process. The
importance of the relationship between lipid organization and com-
position has been shown from studies with diseased skin in which an
impaired barrier function paralled altered lipid composition and
organization (Bouwstra et al., 2001; Kalinin et al., 2002; Madison,
2003).
Fig. 4. Molecular structure of ceramides (CER) in human stratum corneum
(de Jager et al., 2003). [Reprinted from Chemistry and Physics of Lipids, Vol.
124, No. 2, M.W. de Jager et al., The phase behaviour of skin lipid mixtures
based on synthetic ceramides, pp. 123134, Copyright (2003), with
permission from Elsevier]
In addition to keratinocytes, the epidermis contains two den-
dritic cell types melanocytes and Langerhans cells. Melanocytes
lie directly adjacent to the basal layer and produce melanin, the
principal pigment of human skin, which is then transferred to basal
layer keratinocytes. Langerhans cells bear Ia (immune recognition)
o
o
o
oH
oH
H-N
CER1
o
o
o
oH
oH
oH
H-N
CER4
CER2
o
oH
oH
oH
oH
oH
H-N
o
H-N
CER3
CER5
o
oH
oH
oH
oH
oH
oH
H-N
o
H-N
CER6
o
o
o
oH
oH
oH
H-N
CER9
oH
CER7
o
oH
oH
oH
oH
oH
oH
oH
H-N
o
H-N
CER8
16
antigens and have surface receptors for the Fc portion of immuno-
globulin G and 3b, a component of complement C3 (Ahmed, 1979;
Romani et al., 2003). Langerhans cells lie in epidermal layers
containing enzymes that can metabolize exogenous chemicals.
3.2.2 Dermis
The dermis provides the nutritional support for the avascular
epidermis. The dermis is a 0.2- to 0.3-cm-thick tissue that comprises
a fibrous protein matrix, mainly collagen, elastin, and reticulum,
embedded in an amorphous colloidal ground substance. The physi-
cal behaviour of the dermis, including elasticity, is determined by
the fibre bundles and ground substance. The dermis is the locus of
blood vessels, sensory nerves (pressure, temperature, and pain), and
lymphatics. It contains the inner segments of the sweat glands and
pilosebaceous units. The dermis provides flexibility with strength,
serves as a barrier to infection, and functions as a water storage
organ (Singh & Singh, 1993).
3.2.3 Skin appendages
The skin appendages originate in the subpapillary dermis and
consist of eccrine sweat glands, apocrine sweat glands, sebaceous
glands, and hair follicles, with their associated erector muscles.
Appendages are found in most anatomical sites, although the number
of each varies significantly by site. An average human skin surface
contains 4070 hair follicles and 200250 sweat ducts per square
centimetre. Sebaceous glands are most numerous and largest on the
face, especially the forehead, in the ear, on the midline of the back,
and on anogenital surfaces. They secrete sebum, the principal com-
ponents being glycerides, free fatty acids, cholesterol, cholesterol
esters, wax esters, and squalene. Sebum acts as a skin lubricant and
a source of stratum corneum plasticizing lipid and maintains acidic
conditions (pH 5) on the skins outer surface.
The eccrine glands are epidermal structures that are simple
coiled tubes arising from a coiled ball of approximately 100 m in
diameter located in the lower dermis. These glands secrete a dilute
salt solution with a pH of about 5. This secretion is stimulated by
temperature-controlling determinants, such as exercise and high
environmental temperature, as well as emotional stress, through the
autonomic (sympathetic) nervous system. The apocrine glands are
17
limited to specific body regions (e.g. armpit, the breast areola, and
the perianal region) and are about 10 times larger than eccrine ducts.
The apocrine glands, present after puberty, are sweat ducts in
axillary skin.
3.3 The transport of chemicals through the skin
The transport of chemicals through the skin is a complex
process. The skin is a complex organ and also a living membrane.
The skin and the environment are in constant interaction.
There are three major mechanisms by which skin absorption
1) Transcellular absorption: The chemical is transferred through
the keratin-packed corneocytes by partitioning into and out of
the cell membrane.
2) Intercellular absorption: The chemical is transferred around the
corneocytes in the lipid-rich extracellular regions.
3) Appendageal absorption: The chemical bypasses the corneo-
cytes, entering the shunts provided by the hair follicles, sweat
glands, and sebaceous glands.
As the relative surface area of these shunts (appendages) is only
0.11.0% of the total area, they are not thought to play a decisive
role in the absorption of many chemicals in humans. However, the
relative surface area of the shunts may be of greater significance in
areas of the body such as the scalp, where the density and size of
hair follicles are much greater than in, for example, the skin of the
back. Further, these shunts may be important at early time points
following application of the penetrant. Sebaceous glands may act as
a drug reservoir for some materials (Scheuplein, 1967).
3.4 Variability in skin permeability
3.4.1 Species
There are significant differences between the dermal absorption
observed in laboratory animals and that in humans (see also sections
may occur (see also Figures 1 and 3):
18
6.5 and 7.4). For the majority of chemicals, laboratory animal skin is
considerably more permeable (ECETOC, 1993; Vecchia & Bunge,
2005). Differences in the lipid content, structure, and thickness of
the stratum corneum are important factors (Walters & Roberts,
1993). Laboratory animal skin has a much higher number of appen-
dageal openings per unit surface area than human skin, and this may
be an additional causative factor for compounds for which appenda-
geal transport may be significant. The skin of weanling pigs and
monkeys appears to be the most predictive model for human per-
7).
A review of permeability coefficient data for several animal
species (rats, mouse, snake, etc.) is provided by Vecchia & Bunge
(2005).
3.4.2 Age, sex, and race
Premature and low-birth-weight babies have a poorly developed
stratum corneum, but they develop a competent barrier within
4 weeks after birth (Kalia et al., 1996).
Changes that occur in ageing skin include 1) increased stratum
corneum dryness, 2) reduction in sebaceous gland activity, resulting
in a decrease in the amount of skin surface lipids, 3) flattening of the
dermalepidermal junction, and 4) atrophy of the skin capillary
network, resulting in a gradual attenuation of blood supply to the
viable epidermis. Some studies have shown that the barrier function
of the skin increases with increasing chronological age and that
relatively hydrophilic compounds are particularly sensitive. How-
ever, little is known about the influence of such age-related changes
on percutaneous absorption (Roskos et al., 1989).
In general, regardless of environmental conditions, sex, and
ethnic background, the barrier properties of the skin are reasonably
similar. No observable differences were found in the percutaneous
absorption of benzoic acid, caffeine, or acetylsalicylic acid between
Asian, black, and Caucasian subjects (Lotte et al., 1993). However,
it should be pointed out that all investigated compounds were hydro-
philic.
cutaneous penetration (Wester & Maibach, 1985; see also chapter
19
3.4.3 Anatomical site
The skin thickness of the eyelid is approximately 0.05 cm and
that of the palm and sole about 0.4 cm (Maibach & Patrick, 2001).
Percutaneous absorption varies depending on the site of the body
(Feldmann & Maibach, 1967; Wester & Maibach, 1999a; see also
within and between individuals (Southwell et al., 1984).
3.4.4 Skin condition
Skin condition can have a significant impact on the penetration
and permeation of chemicals, especially when the barrier function is
disrupted. The permeability of the skin can be increased by physical
(e.g. weather, sunlight, occlusion), chemical (e.g. solvents, deter-
gents, acids, alkalis), and pathological factors (e.g. mechanical
damage, disease state) (Wiechers, 1989). Mixtures of polar and non-
polar solvents delipidize the skin, resulting in a substantial reduction
of the barrier function.
Some of the most common skin diseases, such as psoriasis and
atopic eczema, are characterized by a virtual absence of the granular
layer in the viable epidermis. In psoriasis, the proliferation is exces-
sive, but keratinization is incomplete (Barry, 1983). There are
several genetic skin diseases with known defects in lipid metabolism
that have scaly or ichthyotic skin as part of the clinical picture (for
details, see Williams & Elias, 2000; Madison, 2003). However, in
diseased skin, the degree of barrier efficiency may vary widely and
depends on the precise pathological state of the stratum corneum
(see also section 12.7).
3.4.5 Temperature and blood flow rate
Skin temperature can have an impact on the rate of penetration
of chemicals in two different ways (Bunge & McDougal, 1999).
First, increasing the temperature of the skin has been shown to
increase the rate of penetration by a direct effect on the diffusion
within the skin (Scheuplein & Blank, 1971). Temperature can also
affect the structure of the stratum corneum, particularly the crystal-
line structure of the lipid bilayers (Pilgram et al., 1999; de Jager et
al., 2004), which can lead to higher permeability. Second, tempera-
chapter 7). There is also considerable variability at a given site and
20
ture may affect blood flow to the skin. However, this mainly affects
the amount of lipophilic chemicals absorbed, as their clearance by
the blood is often rate limiting (Cross & Roberts, 2005). Clearance
into the blood is also dependent on the physiology of the skin.
Warming the skin will increase cutaneous blood flow, and the
penetration rate through the skin may increase, but some of the
dermal blood is shunted through anastomoses to facilitate skin
cooling. Such flow is less effective than nutritive dermal blood flow
in clearing chemicals from the epidermis (Cross & Roberts, 2005).
In general, blood clearance is of importance for small, moderately
lipophilic chemicals that penetrate the skin rapidly but absorb into
the blood slowly (Siddiqui et al., 1989).
3.4.6 Hydration
The stratum corneum normally contains 520% water, but it can
contain up to 50% when hydrated, and this can affect the permea-
bility of the skin to chemicals (Scheuplein & Blank, 1971; Roberts
& Walker, 1993; Bouwstra et al., 2003b; Warner et al., 2003;
Rawlings & Matts, 2005). Idson (1971) claimed that increasing
hydration increases the absorption of all substances that penetrate
the skin. However, increasing hydration (due to occlusion) does not
always increase penetration rates. For example, Bucks et al. (1991)
reported that hydration diminished the penetration rate of hydro-
philic compounds like hydrocortisone (log octanol/water partition
coefficient, or log K
ow
, of 1.61). In contrast, Wurster & Kramer
(1961) observed that occlusive coverings that prevented water loss
increased the dermal absorption of some hydrophilic compounds.
Increased skin hydration has been cited as the probable cause of the
increase in absorption, although in most studies contributions from
potentially confounding effects such as increased temperature or
accumulation of sweat in the dressing cannot be dismissed (Wurster
& Kramer, 1961; Fluhr et al., 1999; Zhai & Maibach, 2001, 2002;
Schfer et al., 2002).
3.5 Reservoir effects
It has long been understood that a substance, instead of passing
entirely through the skin, can remain partly in the skin and can act as
a reservoir, being released (or not) at a later time (Vickers, 1972;
Roberts et al., 2004). This effect has been used in the topical
21
application of medications; for example, salicylic acid was found to
be excreted in the urine more slowly when applied topically than
when injected intradermally (Guillot, 1954). This is applicable for
the more slowly diffusing drugs (i.e. those with long lag times; see
also act as a depot for drugs (or chemicals). The release of the
substance can be rapid on appropriate provocation of the skin some
time later (e.g. this has been shown with steroids, where an occlusive
dressing was applied to the original steroid application site several
weeks after the original application) or alternatively using chemical
enhancement (Roberts et al., 2004). The duration of the reservoir
depends on the nature of the permeant, the vehicle used, the temper-
ature of the skin, and the relative humidity to which the skin is
exposed (Vickers, 1972).
Although most studies have emphasized the stratum corneum as
a reservoir, viable epidermis, dermis, and underlying tissues may
themselves act as reservoirs (Roberts et al., 2004).
Reservoir effects are well documented for several lipophilic
compounds (Miselnicky et al., 1988). The water-insoluble fragrance
musk xylol showed rapid and significant diffusion from the skin
within 72 h after application of a dermal dose (Hood et al., 1996).
However, the formation of a skin reservoir for a chemical during
percutaneous absorption is not limited to lipophilic chemicals but
also applies to polar and non-polar chemicals that bind to the skin
during the absorption process (Yourick et al., 2004). Nicotine, caf-
feine, cationic -blocking drugs, surfactants, testosterone, malathion,
hair dyes, vitamin E, and glycolic and lactic acids have all been
reported as forming a skin reservoir (Roberts et al., 2004). Amounts
of phenanthrene, benzo[a]pyrene, and di(2-ethylhexyl)phthalate
remaining in hairless guinea-pig skin after 24 h eventually became
available for systemic absorption (Chu et al., 1996). The catechol
reservoir formed in skin during a 24-h study with catechol (having
good water and lipid solubility) did not decrease (in vivo) or only
partially decreased (in vitro) in a 72-h extended study (Jung et al.,
2003).
In a set of 19 pesticides tested in the rat in vivo to determine the
fate of the skin reservoir, it was found that absorption from the
washed skin continued for 15 pesticides at all doses tested.
chapter 4). The reservoir function of the skin means that the skin can
22
However, only nine showed an increase in systemic concentrations
(Zendzian, 2003).
The potential for a chemical to form a skin reservoir can be at
least partially predicted by the extent of protein binding, the rate of
penetration through the skin, and the chemicals solubility properties
(Miselnicky et al., 1988).
Reservoir characteristics are discussed in section 6.4, and the
consequences of reservoir effects for risk assessment are given in
section 12.3.
23
4. SKIN TRANSPORT MECHANISMS AND
THEORETICAL CONCEPTS
4.1 Transport through the skin
Percutaneous absorption includes permeation through the epi-
dermis and uptake by the capillary network at the dermalepidermal
junction (see section 2.2). Percutaneous absorption occurs mainly
transepidermally (across the stratum corneum intracellularly and
intercellularly); for many chemicals, transport through appendages is
Permeation of a chemical through the stratum corneum is basi-
cally a diffusion process in which active transport plays no role. The
layer with the highest resistance to diffusion is the rate-limiting
membrane. For many compounds, the lipophilic stratum corneum is
the primary or rate-limiting barrier. However, diffusion through the
hydrophilic epidermis and dermis can be rate limiting for very lipo-
philic materials and/or when the stratum corneum is damaged or
affected by disease.
After the chemical diffuses in the mainly aqueous environment
(living epidermis and dermis), it is taken up into the cutaneous blood
and lymphatic system (resorption). However, if blood flow is insuf-
ficient, compounds can accumulate in the viable epidermis, in the
dermis, and in deeper tissues.
4.2 Theoretical aspects of diffusion
Diffusion of compounds across a membrane is described by
Ficks first law (Crank, 1975):
which states that the flux (rate of transfer per unit area) of a com-
pound (J, mass/cm
2
per second) at a given time and position is
proportional to the differential concentration change cC over a
differential distance cx (i.e. the concentration gradient cC/cx). The
C
J D
x
not usually important in humans (see chapter 3).

24
negative sign indicates that the net flux is in the direction of
decreasing thermodynamic activity, which can often be represented
by the concentration. Ficks second law describing concentration
within a membrane
is derived by combining a differential mass balance in a membrane
with Ficks first law and, when considering the skin, assuming that
the compound does not bind, the compound is not metabolized, and
its diffusion coefficient does not vary with position or composition
(Crank, 1975).
Ficks first law can be applied to describe the diffusion proc-
esses in the individual layers of the skin, which are treated as
pseudo-homogeneous membranes (Scheuplein & Blank, 1971;
Dugard, 1977).
For a membrane of thickness h, the flux at steady state (J
ss
) is
given by:
J
ss
= D (C
1
C
2
) / h [Equation 1]
where C
1
and C
2
are the concentrations of the chemical in the
membrane at the two faces (i.e. at x = 0 and x = h). When used to
describe heterogeneous membranes like the stratum corneum, D is
an effective diffusion coefficient.
Commonly, the stratum corneum controls dermal absorption, h
is the thickness of the stratum corneum, and the concentration at x =
h is zero or very small (i.e. C
2
- 0, which is sometimes called sink
conditions). Also, the concentration of chemical at x = 0 is in local
equilibrium with the vehicle (i.e. C
1
= K
m
C
v
, in which K
m
is the
pseudo-homogeneous partition, or distribution, coefficient between
the stratum corneum and the vehicle and C
v
is the vehicle concen-
tration). Under these conditions, Equation 1 becomes:
J
ss
= D K
m
C
v
/ h [Equation 2]
2
2
C C
D
t x

=

Skin Transport Mechanisms and Theoretical Concepts
25
The steady-state flux across the skin is sometimes written in
terms of the permeability coefficient (K
p
) as follows:
J
ss
= K
p
C
v
[Equation 3]
Comparing Equations 2 and 3,
K
p
= K
m
D / h [Equation 4]
Note that, although the partition coefficient K
m
is unitless, to be
consistent with its use in Ficks law, it is the ratio of concentrations
in the stratum corneum and vehicle in units of mass/volume (Cleek
& Bunge, 1993).
Typically, the steady-state flux J
ss
and the permeability coeffi-
cient K
p
are assessed from an in vitro experiment in which the donor
concentration of the penetrant is maintained (more or less) constant
(i.e. infinite dose conditions), while the receiver phase provides
sink conditions. Over time, the flux approaches a steady-state
value (J
ss
), and the cumulative amount penetrating the skin increases
The slope of the linear portion of the graph of the cumulative
amount penetrated as a function of time represents the steady-state
flux J
ss
(Scheuplein & Blank, 1971; Crank, 1975; Dugard, 1977). As
indicated by Equation 3, K
p
is the ratio of J
ss
and the vehicle
concentration C
v
. The lag time (t
lag
) is the time intercept of the linear
portion of the graph in Figure 5. The time required for the permea-
tion rate across a membrane to reach 95% of the steady-state value is
approximately 2.3 times the lag time (96% 2.4, 97% 2.5, 98%
2.8, 99% 3.2) (Barry, 1983; Bunge et al., 1995). Thus,
estimates of steady-state flux and permeability coefficients should
include data only from times greater than the time to reach steady
state. Including data for times before the steady state is established
will lead to a false estimate, usually underestimate, of the permea-
bility coefficient and lag time (Shah, 1993; Schaefer & Redelmeier,
1996; Geinoz et al., 2004). In reality, depletion of the donor phase,
the use of non-sink receptor conditions, and a deterioration of the
skin over time can occur and result in inaccuracies in steady-state
flux and lag time estimations.
linearly in time, as illustrated in Figure 5.
26
Fig. 5. Illustration of the relationship between the cumulative mass pene-
trating a membrane area (M
out
/A) and the steady-state flux, permeability
coefficient, and lag time (t
lag
).
The maximum flux (J
max,ss
) of a solute through a membrane
occurs for a pure solid or a saturated solution of a chemical in a
vehicle when C
2
- 0 (Higuchi, 1960; Roberts et al., 2002). At
equilibrium, a saturated solution of chemical in a vehicle will be in
equilibrium with the saturated concentration of solute in the stratum
corneum (S
sc
). The maximum flux J
max,ss
is therefore given by
Equation 5, which is derived from Equation 1. It is to be noted that
J
max,ss
is also related to the permeability coefficient of a solute in a
given vehicle K
p,v
and the solubility of the solute in that vehicle S
v
.
J
max,ss
= S
sc
D / h = K
p,v
S
v
[Equation 5]
In principle, higher than maximum fluxes can be observed in
intrinsically unstable systems, such as supersaturated solutions.
4.3 Physicochemical factors affecting skin permeation
The physical and chemical properties of a compound and its
vehicle have a decisive influence on its permeation through the skin.
time
M

t
u
o
/
A
t
lag
2.4 t
lag
Slope = -
VV
.
p
&
v
0
out
/ $ -
ss
(W W
lag
)
.
p
&
v
(W W
lag
)
27
4.3.1 Physical state
Thermodynamically, pure powders and saturated liquid solu-
tions of the same compounds have the same driving force for dermal
absorption (Higuchi, 1960; Roberts et al., 2002). However, avail-
ability to the skin surface may cause the absorption rates to be
different. Absorption from dry particulates can occur even without
surface moisture (Romonchuk & Bunge, 2003). However, dermal
absorption of chemicals in solutions may be more rapid than
absorption from particulates. Although data are limited, particle size
would be expected to have an effect, with slower absorption rates
from larger particles (see also section 12.9 on nanoparticles).
4.3.2 Molecular size/molecular weight
Molecular size is an important factor in membrane permeation
(Berner & Cooper, 1984; Cussler, 1997). Theoretically, molecular
volume should be a better predictor of flux and the permeability
coefficient K
p
. However, molecular weight is often used instead,
because it is more readily available and unambiguous (i.e. not
estimation method dependent) (Patel et al., 2002; Magnusson et al.,
2004a). The equations for estimating K
p
have been derived from
databases containing data on primarily hydrocarbons. For most
hydrocarbons, the ratio of molecular weight to molecular volume is
nearly constant; thus, K
p
estimates based on molecular weight are as
good as those based on molecular volume. For dense compounds
(e.g. halogenated molecules), molecular weight will be larger rela-
tive to molecular volume than for hydrocarbons, and K
p
values cal-
culated from equations using molecular weight will be system-
atically underestimated (Vecchia & Bunge, 2003a). There is good
evidence that permeation and maximum flux decrease exponentially
with molecular weight (Kasting et al., 1987; Potts & Guy, 1992;
Magnusson et al., 2004a). Thus, the extent of absorption of com-
pounds with a molecular weight over 500 daltons through normal
human skin is low (Box & Meinardi, 2000).
Using either low-frequency sonophoresis (Tezel et al., 2003) or
iontophoresis (Roberts et al., 1998), both of which increase skin per-
meability, it has been shown for a number of compounds that perme-
ability is also related to solute size and that the optimal pore size
utilized by solutes is related to their molecular radii. In low-
28
frequency sonophoresis, larger permeants experience a less tortuous
pathway compared with ions and smaller solutes (Tezel et al., 2003).
4.3.3 Maximum flux
The maximum flux of a solute through the skin defines the high-
est exposure risk for a chemical. Maximum flux, either measured or
estimated, is a better guage for dermal absorption than a given
physicochemical property of a solute. Hence, neither water solubility
nor the octanol/water partition coefficient alone is a reliable indica-
tor of the likelihood of significant dermal absorption. The maximum
flux of chemical permeation through skin should occur at the solu-
bility limit of the chemical (Roberts et al., 2002). Thus, one estimate
of the maximum flux of a chemical is the product of the chemicals
solubility limit in water and the skin permeability coefficient of the
chemical from water. Estimated values can also be derived using
that are completely miscible in water, density of the neat compound
can be used in lieu of the water solubility for making preliminary
estimates of maximum flux. Generally, dermal absorption would be
no larger than the maximum flux, except when the compound or
vehicle can damage or alter the skin or in the unusual situation of
supersaturated solutions (Davis & Hadgraft, 1991).
For purposes of risk assessment, decisions about whether pene-
tration is important or not must be linked to toxicological potency.
For highly potent compounds, the potential for toxic or therapeutic
activity might be significant even when maximum flux values are
low.
4.3.4 Ionization
Ionized species do not penetrate the skin very well. The stratum
corneum permeability coefficients for non-ionized compounds are
frequently 12 orders of magnitude larger than permeability coeffi-
cients for ionized forms of the same compound (Vecchia & Bunge,
2003a). However, ionization effects are less evident in maximum
flux estimations (Magnusson et al., 2004a). The exact relationship
between the non-ionized and ionized forms would be expected to
depend upon the compound and the lipophilicity of the non-ionized
chemical, in particular, but also on the vehicle and salt form of the
chemical used, as ion pairing may also facilitate ionized drug
QSAR equations, as discussed further in chapter 10. For compounds
29
transport (Hadgraft & Valenta, 2000). Intuitively, the penetration
rates for non-ionized and ionized forms of the same chemical should
be more similar when the non-ionized species is hydrophilic and less
similar when it is lipophilic.
Abraham & Martins (2004) claim that ionization effects are
different for acids and bases. They argue that K
p
for the neutral form
[K
p
(N)] of an acid is very much larger than K
p
for the ionized form
[K
p
(I)]. For proton bases, for four different bases studied by two
different sets of workers, the K
p
(N)/K
p
(I) ratios averaged 17.5
(Abraham & Martins, 2004). This conclusion for two of the basic
compounds (fentanil and sufentanil) is not accepted by all (e.g.
Vecchia & Bunge, 2003a).
4.3.5 Binding properties
Permeation through the stratum corneum can be slower than
expected for some compounds due to binding. Examples include
certain metal ions (particularly Ag
+
, Cd
2+
, Be
2+
, and Hg
2+
), acrylates,
quaternary ammonium ions, heterocyclic ammonium ions, and sulfo-
nium salts. Other potential agents that may have slower than
expected permeation possibly due to binding include diethanolamine
(Kraeling et al., 2004; Brain et al., 2005), quinines, alkyl sulfides,
acid chlorides, halotriazines, and dinitro- or trinitrobenzenes (EC,
2003).
4.4 Concepts of finite and infinite dose
An infinite dose is defined as the amount of test preparation
applied alone or in a vehicle to the skin such that a maximum rate of
absorption of the test substance (per unit area of skin) is achieved
and maintained (OECD, 2004a). In principle, the application volume
should be large enough that the concentration of the chemical is not
depleted. However, an apparent depletion near the skin can occur
when the vehicle is sufficiently viscous to cause a diffusional limita-
tion. The infinite dose does not reflect the situation for many occu-
pational exposure scenarios. However, infinite dose exposure can
occur in humans. For example, exposure to chemicals such as bio-
cides in swimming pools or bathing water is whole-body infinite
exposure. As stated above, the main skin absorption parameters
30
determined in an infinite dose study are the steady-state flux and the
permeability coefficient.
Under the conditions of a finite dose, the maximum absorption
rate may be reached for some of the time, but it is not maintained, or
it may not be achieved (OECD, 2004a). The concentration of the
chemical in the donor fluid changes due to uptake of the chemical
into the skin or evaporation, and it may also change (increase) due to
evaporation of the donor fluid. This situation happens in the in vitro
cell when it is not occluded, which may be more consistent with
most occupational exposures. The finite dose study enables the esti-
mation of both maximal absorption rate (maximal exposure) and
total absorption (overall exposure). Simulated examples of the skin
permeation rate, cumulative absorption, and vehicle concentration
are shown in Figure 6 for infinite, semi-infinite, and finite doses.
The semi-infinite dose is simulated as an intermediate case between
the true infinite dose (no decrease in concentration in the vehicle
during the exposure experiment) and a finite dose (in which the
concentration in the vehicle is reduced to less than 10% of the initial
concentration after about 2024 h of exposure).
Fig. 6. The effect of finite and infinite doses in dermal permeation. Simula-
tions of a dermal permeation study with a compound having a water solubility
of 25 g/ml and a stratum corneum/water partition coefficient of 200. The
compound is dissolved in a water vehicle. a) The skin permeation rate
(g/cm
2
per hour) as a function of time under infinite and finite dose
conditions. (continued on next page)
0
Time (hours)
0.0
0.1
0.2
0.3
0.4
0.5
S
k
i
n

p
e
r
m
e
a
t
i
o
n

r
a
t
e

/

f
l
u
x

(
g
/
c
m
2
/
h
)
nfinite dose
Semi-infinite dose
Finite dose
a
24 18 12 6
31
Fig. 6 (contd). b) The cumulative amount (g/cm
2
) absorbed through the skin
as a function of time under infinite and finite dose conditions; c) The
concentration (g/cm
3
) in the vehicle (donor compartment) as a function of
time under infinite and finite dose conditions (J. Krse, personal
communication, 2005).
Time (hours)
0.0
2.0
4.0
6.0
8.0
10.0
nfinite dose
Semi-infinite dose
Finite dose
E
0 24 18 12 6
C
u
m
u
l
a
t
i
v
e

a
b
s
o
r
p
t
i
o
n

(
g
/
c
m
2
)
Time (hours)
0.0
5.0
10.0
15.0
20.0
25.0
30.0
nfinite dose
Semi-infinite dose
Finite dose
c
0 24 18 12 6
C
o
n
c
e
n
t
r
a
t
i
o
n

i
n

v
e
h
i
c
l
e

(
g
/
c
m
3
)
32
5. METABOLISM IN THE SKIN
The skin is a metabolically active organ and contains enzymes
that are able to catalyse not only endogenous chemicals such as
hormones, steroids, and inflammatory mediators but also xeno-
biotics, including drugs, pesticides, and industrial and environmental
chemicals. Although the metabolism of xenobiotic compounds in the
skin is intended to detoxify potentially reactive chemicals by con-
verting lipophilic compounds into polar, water-soluble compounds
that are readily excreted into the bile and urine, in some cases a
compound may be activated, leading to enhanced local and/or sys-
temic toxicity (Hotchkiss, 1998). Cutaneous activation and detoxifi-
cation can be a critical determinant of systemic exposure in humans
following dermal absorption.
If transport through the viable epidermis is rate limiting and the
metabolite is less hydrophobic than the parent compound, then
percutaneous absorption of the metabolized compound could be
faster than (different from) that of the parent compound. Ben-
zo[a]pyrene was found to penetrate hairless guinea-pig skin more
readily through viable than through non-viable skin, presumably
because the more polar metabolites could diffuse more readily
through the aqueous viable tissue. Total dermal absorption of
benzo[a]pyrene through viable skin (67.4% of applied dose) agreed
closely with in vivo absorption data (Ng et al., 1992).
It is often assumed that the skin has no first-pass effect, but this
is not true for many compounds. Examples include benzo[a]pyrene
(Ng et al., 1992), nitroglycerine (Wester et al., 1983), herbicide
esters (Clark et al., 1993; Hewitt et al., 2000), methyl salicylate
(Cross et al., 1997), and parabens (Cross & Roberts, 2000). These
effects can not only reduce systemic availability, but also increase
localized activity of the metabolites.
Some compounds are designed to be absorbed across the
stratum corneum and then metabolized in the skin to the active
moiety. Examples of these skin prodrugs and codrugs include retinyl
palmitate (Boehnlein et al., 1994), aminolevulinic acid (Donnelly et
Metabolism in the Skin
33
al., 2005), corticosteroid esters (Hotchkiss, 1998), and the prodrug
5-fluorouracil-triamcinolone-acetonide (Cabral-Lilly & Walters,
2004). Aspects of skin prodrug absorption and metabolism have
been reviewed by Hotchkiss (1998) and Benson (2005).
5.1 The drug-metabolizing systems of the skin
The skin contains enzymes that catalyse Phase 1 (e.g. oxidation,
reduction, hydrolysis) and Phase 2 (conjugation) reactions (see
lism in the liver and other tissues have been identified in skin
(Pannatier et al., 1978). The specific activities of cutaneous xeno-
biotic-metabolizing enzymes in the skin, measured in subcellular
fractions, are lower than those of their counterparts in the liver
(generally 0.128% of the activities in the liver for Phase 1; 0.6
50% for Phase 2; see reviews by Kao & Carver, 1990; Hotchkiss,
1998; Hewitt et al., 2000). However, although the basal activities
may be relatively low, if the surface area of the skin exposed to the
contaminant is very large, the metabolism in the skin is likely to
make a contribution to the overall metabolism of the compound
following dermal exposure. However, while the skin is a large
organ, the only place that metabolism will be important for most
substances will be the actual area that is exposed to the chemical.
The extent to which topically applied chemicals will be metab-
olized depends on the chemical and the enzymes involved. Some
chemical groups, such as esters, primary amines, alcohols, and acids,
are particularly susceptible to metabolism in the skin (Bronaugh,
2004b).
Esterases are particularly active in the skin, so esters may be
extensively, if not completely, hydrolysed to their parent alcohol and
acid molecules during skin penetration. Examples of this are the
fragrance chemicals such as benzyl acetate as well as the phthalate
esters dimethyl-, diethyl-, and dibutylphthalates (Hotchkiss, 1998),
retinyl palmitate (Boehnlein et al., 1994), herbicide esters (Clark et
al., 1993; Hewitt et al., 2000), and methyl salicylate (Cross et al.,
1997). Primary amines are frequently acetylated during percuta-
neous absorption through the skin (Nathan et al., 1990; Kraeling et
al., 1996; Yourick & Bronaugh, 2000). Oxidation/reduction of
alcohols and acids and glycine conjugation of acids also commonly
Table 2). All of the major enzymes important for systemic metabo-
34
occur in the skin (Nathan et al., 1990; Boehnlein et al., 1994). Con-
jugation with glutathione (Jewell et al., 2000), sulfate (Yourick &
Bronaugh, 2000), and glucuronic acid (Moss et al., 2000) also
occurs.
Table 2. Some cutaneous metabolizing pathways
Phase 1 reactions Phase 2 reactions
Oxidation
Hydroxylation
Deamination
Dealkylation
Epoxidation
Aldehyde oxidation
Alcohol oxidation
Glucuronidation
Sulfation
Glutathione conjugation
Acetylation
Amino acid conjugation
Methylation
Reduction
Azo reduction
Nitroxide reduction
Quinone reduction
Hydrolysis
Carboxylester hydrolysis
Sulfate ester hydrolysis
Phosphate ester hydrolysis
Peptide hydrolysis
Epoxide hydrolation
a
From Sartorelli et al. (2000).
Studies in human and rodent skin have shown that cytochrome
P450 (CYP1A1/A2) and Phase 2 metabolizing enzymes (e.g. gluta-
thione S-transferase) in the skin are localized within specific cell
types, particularly in the epidermis and epidermal-derived tissues
namely, sebaceous glands and hair follicles (Pendlington et al.,
1994; Hotchkiss, 1998). Lipase, protease, phosphatase, sulfatase,
and glycosidase activities have been identified in the stratum cor-
neum; these activities have been linked with the maturation and
desquamation processes (Howes et al., 1996). Alcohol dehydrogen-
ase and aldehyde dehydrogenase have also been shown to be present
in epidermal basal layers (Haselbeck et al., 1997; Lockley et al.,
2004).
35
5.2 Methodology for evaluating skin metabolism in
in vitro systems
The extent of cutaneous metabolism of a chemical applied to
the skin is difficult to differentiate in vivo from systemic metab-
olism, mainly in the liver, using blood and excreta samples. In vitro
studies isolate the skin from the metabolic activity in the rest of the
body. The use of viable skin is essential. An in vitro flow-through
diffusion cell system (Bronaugh & Stewart, 1985; Collier et al.,
Hanks balanced salt solution as receptor fluid has proved to be an
effective method for studying the effect of metabolism of chemicals
during percutaneous absorption. Skin metabolism studies have also
been conducted in flow-through cells using minimum essential
medium to maintain skin viability (Clark et al., 1993) and in an
organ culture model (van de Sandt et al., 1993). Additional
information can be obtained from studies on the cytosolic fraction of
whole and dermatomed skin, such as studies on glycol ethers
(Lockley et al., 2004). Skin equivalents have the capacity to metabo-
lize chemicals during dermal absorption, but absorption was also
shown to be much faster through skin equivalents than through
human skin (Eppler et al., 2005).
Caution should be applied to any in vitro study using skin tissue
that claims the tissue is metabolically active. Some enzymes and
cofactors, particularly cytochrome P450s and glutathione, deteri-
orate rapidly ex vivo (in minutes), even in fresh specimens (Jewell et
al., 2000). Just because the tissue is used quickly does not validate
the experiment. Studies would need to show the conversion rate of
known standard substrates for the class of enzyme being investi-
gated. Skin penetration is a passive process that can be studied ex
vivo. Information on metabolism from the in vitro approach needs to
be considered in an in vivo context, and additional in vivo studies
may be needed for mechanistic and kinetic understanding.
5.3 Effects of skin metabolism
Cutaneous metabolism may result in:
activation of inert compounds to toxicologically active species
e.g. polycyclic aromatic hydrocarbons (PAHs), such as
1989; Bronaugh, 2004a,b; see chapter 6) using a HEPES-buffered
36
benzo[a]pyrene (Ng et al., 1992) and 3-methylcholanthrene
(Cooper et al., 1980);
detoxification of toxicologically active chemicals to inactive
metabolites e.g. organophosphorus pesticides, such as diiso-
propylfluorophosphate, paraoxon (Sartorelli et al., 2000), and
propoxur (van de Sandt et al., 1993), and azo dyes (Collier et
al., 1993);
conversion of active chemicals to active metabolites e.g.
drugs such as testosterone and estradiol (Collier et al., 1989);
and
activation of prodrugs (Davaran et al., 2003; Valiveti et al.,
2005).
An overview of skin metabolism is given in Hotchkiss (1995,
1998), and metabolism in skin in in vitro absorption studies is
described in Bronaugh (2004a). Examples of these studies include
azo dyes (Collier et al., 1993), 2-nitro-p-phenylenediamine (a dye
used in semipermanent and permanent hair dye formulations), where
extensive metabolism was found on absorption (Yourick &
Bronaugh, 2000), and 2-ethoxyethanol, where no metabolism was
observed (Lockley et al., 2002).
5.4 Importance of metabolism for percutaneous
absorption
That skin can metabolize compounds before they enter the
bloodstream is important for risk assessment purposes and for drug
delivery (Howes et al., 1996). However, there are conflicting views
concerning the influence of local skin metabolism on percutaneous
absorption. Metabolism may not be rate limiting if the compound is
stable in the stratum corneum and the stratum corneum limits percu-
taneous absorption. If the viable tissue is a significant barrier,
metabolism of a lipophilic compound in the stratum corneum to a
more polar compound might result in enhanced permeation.
Although skin contains enzymes that have the capacity to
metabolize glycol ethers localized in the basal layer of the epider-
mis, the physicochemical properties of the penetrants that result in
rapid penetration significantly reduce the potential for first-pass
dermal metabolism during percutaneous penetration (Lockley et al.,
2004).
37
However, for compounds that either bind in the skin or, due to
their physicochemical properties, stay longer in the skin, the metab-
olism may be of importance. This may be the case, for example, for
PAHs. Seven per cent of the absorbed dose of phenanthrene was
biotransformed to the three diol metabolites (trans-9,10-dihydrodiol,
trans-1,2-dihydrodiol, trans-3,4-dihydrodiol) in the skin (Ng et al.,
1991). These metabolites are not carcinogenic, which correlates with
the lack of tumorigenicity of phenanthrene in rodents. In contrast, in
skin metabolism studies with benzo[a]pyrene, the metabolite identi-
fied in the receptor fluid was benzo[a]pyrene-7,8,9,10-tetrahydro-
tetrol, which is the hydrolysis product of the ultimate carcinogen,
7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrotetrol (Ng et al.,
1992). These results agree with the formation of skin tumours fol-
lowing topical administration of benzo[a]pyrene. Studies in mouse
skin had previously shown that induction of cutaneous drug-
metabolizing enzymes can result in a 2- to 3-fold increase in the in
vitro permeation of topical benzo[a]pyrene (Kao et al., 1985).
The hair dye 2-nitro-p-phenylenediamine penetrates both human
and rat skin. Under conditions that simulate normal consumer use,
approximately 510% of the 2-nitro-p-phenylenediamine that con-
tacts the skin would be expected to be absorbed. There was exten-
sive metabolism of 2-nitro-p-phenylenediamine upon absorption.
The extent of metabolism and the metabolic profile depended on
species (human, rat) and dosing vehicle (ethanol or hair dye
formulation) and also tissue (when compared with results with
intestinal tissue) (Yourick & Bronaugh, 2000).
38
6. IN VITRO TESTS FOR DERMAL ABSORPTION
In vitro methods are designed to measure the penetration of
chemicals into and subsequent permeation across the skin into a
fluid reservoir and can utilize non-viable skin to measure penetration
and permeation only or fresh, metabolically active skin to simul-
taneously measure permeation and skin metabolism (OECD, 2004a).
Permeation across the non-living outer layer of skin, the stratum
corneum, is often the rate-limiting step for percutaneous absorption
(Dugard & Scott, 1984; Dugard et al., 1984). Permeation of a chem-
ical through the stratum corneum is believed to depend upon
permeant-specific factors, such as molecular weight, water and lipid
meability properties of the stratum corneum are unchanged after
excision from the body, and a good agreement between in vivo and
in vitro experiments with the same chemicals has been observed (see
human dermal penetration. Furthermore, such experiments offer a
number of advantages over whole-animal or human volunteer exper-
iments, including a saving in time and costs, better reproducibility of
results, and less restricted parameter variations (van Ravenzwaay &
Leibold, 2004).
In the first part of this chapter (sections 6.16.4), various
aspects of the methodology of in vitro tests are presented. A dis-
cussion of experimental factors that cause variability in results is
given in section 6.5.
6.1 Test guidelines
It is only recently that a test guideline for in vitro dermal
penetration has been adopted (OECD, 2004a,c). The following
descriptions are based on the OECD guideline. A variety of proto-
cols and experimental conditions have been used, and these were
later developed into protocols for testing specific substances (e.g.
certain regulated chemical substances [USEPA, 2004a]; cosmetics
[SCCNFP, 2003b]; see also Appendix 1). The number of different
guidelines and protocols have led, in part, to the present problems of
comparability of results.
solubility, polarity, and state of ionization (see chapter 4). The per-
chapter 8). Thus, in vitro experiments are appropriate to predict
In Vitro Tests for Dermal Absorption
39
Although OECD (2004a,c) allows the use of various protocols,
the guidelines list the issues that investigators must consider in
designing their protocols and data analysis, and this should reduce
data variation.
6.2 Principles of the standard in vitro tests using skin
samples
The test substance, which may be radiolabelled, is applied to the
surface of a skin sample separating the two chambers of a diffusion
cell (OECD, 2004a). Most common methods for the evaluation of in
vitro skin penetration and permeation use diffusion cells, which
range in complexity from a simple two-compartment static cell
(Franz, 1975) to multijacketed flow-through cells (Bronaugh &
Stewart, 1985). In all cases, excised skin is mounted as a barrier
between the donor compartment and the receptor compartment, and
the amount of test chemical and/or its metabolites permeating from
the donor to the receptor side is determined as a function of time.
The chemical remains on the skin for a specified time under speci-
fied conditions, before removal by an appropriate cleansing proce-
dure (OECD, 2004a). Efficient mixing of the receptor phase (and
sometimes the donor phase) is essential (Brain et al., 1998a; OECD,
2004a).
6.2.1 Test chambers
Diffusion cells are of the upright/vertical or side-by-side type,
with receptor chamber volumes of about 0.510 ml and surface
areas of exposed membranes of about 0.22 cm
2
(Brain et al.,
1998a). Both receptor chamber volume and the exposed surface area
should be accurately measured and recorded for individual diffusion
cells. Vertical cells are useful for studying absorption from semi-
solid formulations spread on the membrane surface and are optimal
for simulating in vivo performance. The donor compartments can be
capped, to provide occlusive conditions, or left open, according to
the objectives of the particular study (Brain et al., 1998a). Side-by-
side-type (horizontal) diffusion cells are useful for studying mech-
anisms of diffusion through skin (Bronaugh, 2004b), as the permea-
tion from one stirred solution through the membrane into another
stirred solution is measured (Brain et al., 1998a). The chambers in
side-by-side diffusion cells are often, but do not need to be, of equal
40
volume. To evaluate topical products intended for human use,
standard infinite dose cells in a side-by-side configuration are of
limited use; nonetheless, they have been used for this purpose by
several authors (Feldmann & Maibach, 1969; Michaels et al., 1975;
Bronaugh & Maibach, 1985; Sartorelli et al., 2000; Moss et al.,
2002).
Both the vertical and side-by-side cells can be operated in static
(i.e. no flow) or flow-through mode.
6.2.1.1 Static diffusion cells
The Franz diffusion cell is one of the most widely used systems
for in vitro skin permeation studies (Friend, 1992). Franz-type
diffusion cell systems are relatively simple in design; the receptor
fluid beneath the skin is manually sampled by removing aliquots
periodically for analysis (Bronaugh, 2004b). These cell systems may
be run as static or as flow-through cells (ECETOC, 1993). With this
type of apparatus, any type or any amount of vehicle (within the
capacity of the donor chamber) may be applied to the skin. For more
realistic exposure scenarios, either 510 l of a liquid vehicle
containing the test compound is applied per square centimetre of
skin or 25 mg of a non-liquid vehicle containing the test compound
is applied per square centimetre. An important factor that has to be
considered, especially in static diffusion systems, is the solubility of
the test compound in the receptor fluid. This may affect the sink
capacity and would have an influence on the receptor chamber
dimensions or sampling frequency (Brain et al., 1998a). A typical
6.2.1.2 Flow-through cells
Flow-through cells are characterized by a continuously replaced
receptor fluid, which represents, more or less, in vivo conditions.
This method can be useful when a permeant has a very low sol-
ubility in the receptor medium. Sink conditions are maximized, as
the fluid is continually replaced using a suitable pump (at a rate of
system has been developed by Bronaugh & Stewart (1985) (see
static diffusion cell is shown in Figure 7.
about 1.5 ml/h) (Brain et al., 1998a). A flow-through diffusion cell
Figure 8).
41
Fig. 7. Design of a typical static diffusion cell for in vitro dermal absorption.
HPLC = high-performance liquid chromatography (from OECD, 2004c).
[Reprinted with permission]
Fig. 8. Flow-through cell and holding block. A Cross-section of diffusion
cell; B Aluminium holding block used to position cells over vials in a
fraction collector and to maintain the cells at a physiological temperature
(Bronaugh, 1991). [Copyright (1991) From In Vitro Percutaneous Absorption:
Principles, Fundamentals, and Applications by R.L. Bronaugh and H.I.
Maibach. Reproduced by permission of Routledge/Taylor & Francis Group,
LLC]
AUTOSAMPLER
Programmed to sample
specific time-course
Autopipette/syringe sampling into
scintillation or HPLC vials
Volume maintained by replacement
of fresh receptor fluid
Activated charcoal filter
(for volatile penetrants)
Cell donor chamber
Skin membrane (2.54cm
2
)
Support grid
Glass diffusion cell
Magnetic stirrer bar
Receptor chamber/ fluid maintained at 32C1C in a
temperature-controlled water bath
B
A
o
42
The cells were constructed from Teflon and contained a glass
window in the receptor chamber for viewing the receptor contents.
Several flow-through diffusion cell systems that are comparable to
the Bronaugh cell exist (e.g. Clowes et al., 1994; Tanojo et al.,
1997).
The receptor fluid and/or the flow rate are selected on the basis
of solubility considerations and the volume of the receptor chamber.
The criteria that establish a suitable flow rate are 1) that the concen-
tration in the receptor fluid is not greater than about 10% of the
solubility limit (as for static diffusion cells) but is large enough to be
detected and 2) that there is adequate mixing in the receptor
chamber (Skelly et al., 1987). It is important to appreciate that there
is a system lag time associated with flow-through cells due to the
volume of the receptor chamber and outlet tubing and typically low
flow rate used. Either this lag time needs to be corrected for or
conditions need to be altered to minimize this effect (Anissimov &
Roberts, 2001). One way to minimize the lag effect is by using an
adequate flow rate to flush the receptor chamber and outlet tubing
completely a number of times each hour (Bronaugh & Stewart,
1985). Automated flow-through systems can allow unattended sam-
pling, and commercial systems are available for example, the
automated in vitro dermal absorption method, which employs tube-
shaped skin permeation cells that fit directly into standard 2-ml glass
autosampler vials (Moody, 1997, 2000). This procedure permits a
quicker sample analysis and minimizes the loss of permeant through
evaporation, hydrolysis, and degradation. Good agreement between
the automated in vitro dermal absorption method and the Bronaugh
procedure was demonstrated using an N,N-diethyl-m-toluamide-
based mosquito repellent and permeation through rat skin (Moody,
1997).
6.2.1.3 Comparison of different in vitro cell systems
Static cells are simpler in design than flow-through cells, the
costs are lower, and they are available in a wide range of surface
areas for skin exposure (Bronaugh, 2004b). It also needs to be rec-
ognized that adsorption of the test chemical onto the apparatus can
occur in the donor and receptor chambers and, where present, the
tubing for both types of cell. From a practical point of view,
therefore, a further advantage for static cells over flow-through cells
43
is a higher skin:apparatus ratio, which improves the absorption
signal and the mass balance distribution assessment. Also, pump
problems, connection difficulties, and potential binding of permeant
to tubing do not occur using static cells. In contrast to the static cell
types, flow-through cells provide the continuous replacement of a
nutrient medium necessary to maintain physiological conditions and
are therefore recommended for metabolism studies (Bronaugh,
2004a,b).
In several comparative studies, no differences in skin permea-
bility measurements using static and flow-through cells were found
(Bronaugh & Maibach, 1985; Bronaugh & Stewart, 1985; Hughes et
al., 1993; Clowes et al., 1994). In a multicentre comparison study of
in vitro predictions of skin absorption of caffeine, testosterone, and
benzoic acid, no differences between static and flow-through diffu-
sion cell systems were observed (van de Sandt, 2004). In another
international multicentre study using a standard silicone rubber
membrane, there was no consistent pattern of differences between
static and flow-through cells (Chilcott et al., 2005).
6.2.2 Finite/infinite dosing
There are two dosing regimens: infinite and finite dose (see
required to be more representative of the in-use scenario; the dose
solution is applied in a volume sufficient to cover the skin and nor-
mally remains unoccluded. Using this regimen, dose depletion is
likely to occur. Finite dose experiments may be performed with all
types of vertical/upright cells.
In the infinite dose procedure, the dose solution is applied in
excess and can be occluded for the duration of the study (Sartorelli
et al., 2000; OECD, 2004c). Dose depletion is unlikely to occur.
The dosing regimen and test substance preparation are dictated
by the type of experiment being performed. For example, in the
assessment of the absorption of a cosmetic ingredient for the pur-
poses of risk assessment, a finite dose with an in-use application
protocol and with the test compound in the formulation of interest
would be preferred. If a permeability constant is being measured, an
chapter 4). The finite dose regimen is used where the application is
44
infinite dose would be applied for a period sufficient to reach and
maintain the steady-state situation.
6.2.3 Skin preparations
6.2.3.1 Choice of skin
The choice of skin depends on the purpose of the test and the
availability of skin samples. For risk assessment purposes, human
skin is preferred. As stated above, the epidermis is the major barrier
for the compound, because once the permeant has transferred across
the epidermis, it has access to the cutaneous circulation. In the in
vivo situation, the dermis normally has little effect on the dermal
absorption of compounds whose rate of absorption is limited by the
stratum corneum.
Skin from human and laboratory animal sources can be used.
Although the use of human skin samples gives data more appropriate
to human in vivo conditions, human skin samples are not always
readily available. Further, the use of human skin is subject to
national and international ethical considerations (ECETOC, 1993).
Typical human in vitro experiments with viable skin involve the use
of female abdominal and/or breast skin obtained at autopsy or from
cosmetic surgery (Dressler, 1999). Non-viable skin from several
anatomical sites of male and female cadavers has also been used.
Rat skin is often used where in vivo toxicological studies have
been performed in rats. It is also required for special protocols, such
as that for pesticide testing in the USA (USEPA, 1998; Zendzian,
2000). According to the Scientific Committee on Cosmetic Products
and Non-Food Products Intended for Consumers (SCCNFP), human
skin (abdomen or breast) and pig skin are recommended for cos-
metic testing (Steiling et al., 2001; SCCNFP, 2003b). The potential
for hydration damage (Bond & Barry, 1988a) and exaggerated
effects of enhancers (Bond & Barry, 1988b) with animal skin needs
to be recognized. Indeed, Rigg & Barry (1990) concluded: Wher-
ever possible, human skin should be used in absorption studies and
not hairless mouse or snake skin; otherwise, misleading results may
be obtained.
45
Although viable skin (Bronaugh, 2004b) is preferred, especially
for metabolism studies, non-viable skin can also be used for certain
tests.
OECD Test Guideline 428 (OECD, 2004c) allows a number of
skin types and methods of skin preparation for the measurement of
dermal absorption in vitro. Indeed, some species are used in in vitro
models, either as surrogates for human skin (e.g. pig skin) or to
compare absorption between species (e.g. rat and human skin).
use human skin as the gold standard when the dermal absorption
potential of a chemical is required for human health risk assessment
purposes. Much of the validation type of work in this area has
focused on developing in vitro methods to predict dermal absorp-
tion in rodents. The emphasis of such work should be to develop
consistent and well controlled studies with human skin in order to
predict dermal absorption in humans.
6.2.3.2 Preparation of tissue samples
Different methods can be used to prepare skin samples (Brain et
al., 1998a; OECD, 2004c):
1) Full-thickness skin, incorporating the stratum corneum, viable
epidermis, and dermis: Other than for mouse skin, full-thickness
skin is normally used for mechanistic studies and should be
used in other studies only when justified (OECD, 2004c).
2) Dermatomed skin, in which the lower dermis has been
removed: Split-thickness skin is produced by dermatoming to
obtain skin samples of uniform shape and thickness (Steiling et
al., 2001). A dermatome is a surgical instrument that is used to
cut skin grafts. Electric and manual dermatomes are generally
used. Dermatoming can be carried out from either side of the
skin. The trimming and thickness reduction of pig skin are done
from the dermis side, in order to generate skin samples with
intact stratum corneum and epidermis (Steiling et al., 2001).
Skin thickness (usually 0.81.0 mm) should be measured by an
appropriate method (e.g. using a micrometer gauge; Kenyon et
al., 2004a). The skin samples should be prepared and trimmed
to fit the diffusion cell (SCCNFP, 2003b).
However, the Task Groups recommendation (see chapter 13) is to
46
3) Epidermal membranes, comprising the viable epidermis and the
stratum corneum: Preparation of an epidermal layer by separa-
tion of the epidermis from the dermis using heat is effective for
non-hairy skin. (Full-thickness skin is submerged in 60 C water
for approximately 45 s, and the epidermal and dermal layers can
be pulled apart with forceps, but the metabolic viability of the
skin is destroyed [Bronaugh, 2004b; USEPA, 2004a].) Other
methods used to separate the dermis from the epidermis include
chemical and enzyme treatment. The use of epidermal mem-
branes may in some cases overestimate human in vivo skin
absorption (van de Sandt et al., 2000; SCCNFP, 2003b).
According to Bronaugh (2004b), chemical separation tech-
niques and enzyme methods for epidermal separation are
limited to use in young rats; according to OECD (2004c),
however, they may be used for skin preparation.
4) Stratum corneum alone: This tissue is prepared from epidermal
membranes by enzyme treatment with trypsin. Stratum corneum
membranes are primarily used for mechanistic studies and
partition coefficient determination.
The various guidelines indicate that the skin samples that may
be used during in vitro studies are split-thickness (200400/500 m)
(OECD, 2004c; USEPA, 2004a) or, when justified, full-thickness
(5001000 m) skin preparations (OECD, 2004c). Hairy animal
skin should be shaved and the subcutaneous fat removed.
Provided the samples are in their normal state of hydration
when cooled, animal and human skin can be stored for up to 1 year
at 20 C (Bronaugh et al., 1986; Steiling et al., 2001). Frozen
stored skin may not be suitable for some metabolism studies. Barrier
integrity should be evaluated after storage.
6.2.3.3 Checking of barrier integrity
It has been recommended that before and in some cases after the
experiment, the barrier integrity of the skin should be checked by
physical methods, such as transepidermal water loss or trans-
cutaneous electrical resistance (Davies et al., 2004; OECD, 2004c;
USEPA, 2004a). The barrier integrity of the skin samples may also
be checked using the tritium method, where the permeation of
47
tritiated water through the skin is determined and compared with
standard values (Ursin et al., 1995; OECD, 2004a,c; USEPA,
2004a). Furthermore, post-study data analysis can be used to identi-
fy damaged skin preparations by comparison with the mean absorp-
tion data for the other cells (OECD, 2004c).
Typically, skin samples exhibiting a permeability coefficient for
tritiated water above 2.5 10
3
cm/h are rejected as being out of the
normal range. In a study to investigate the usefulness of this form
of barrier integrity check, K
p
values from 1110 human skin samples
were found to follow a non-Gaussian distribution (Roper et al.,
2004). A rejection criterion of K
p
above 2.5 10
3
cm/h resulted in
rejection of 230 (21%) of these samples. It is likely that many of
these rejected samples were atypical rather than damaged,
resulting in an underestimation of absorption in such an individual.
There is currently discussion as to whether this rejection criterion is
6.2.4 Application of test substance
6.2.4.1 Test substance
For practical purposes, the test substance ideally should be
radiolabelled (preferably with carbon-14 at a metabolically stable
position). However, radiolabelled scintillation counting does not dis-
tinguish between metabolites, and further analysis may be necessary.
It is essential to determine radiochemical purity (pre- and post-
permeation), and the possibility of tritium exchange, where appro-
priate, should be examined. If radiolabelling is not possible, suitable
validated assay procedures must be established for the respective
chemicals and metabolites (OECD, 2004c). Current sensitive tech-
niques, such as high-performance liquid chromatography (HPLC),
gas chromatography (GC), and liquid chromatography/mass spec-
trometry (LC-MS), will be useful if a reproducible, validated anal-
ytical method to detect the test compounds is available.
Before application of the test substance, various factors that
may have an influence on permeation should be considered. These
include the nature of the vehicle and the dermal dose (which is
dependent on the concentration of the test substance in the vehicle
and the applied amount per square centimetre of skin). Other factors
justified (see chapter 12).
48
include the dosage regimen (finite or infinite) and post-dose condi-
tions, such as occlusion. The test substance application and post-
application regimen are dictated by the type of experiment being
performed. For example, in the case of the assessment of the perme-
ation of a cosmetic ingredient for risk assessment purposes, a finite
dose, with an in-use application time, in the formulation of interest
must be used. If a permeability coefficient is being measured, an
infinite dose would be applied for sufficient time to reach and
maintain steady-state permeation.
6.2.4.2 Vehicle
The type of application vehicle will almost certainly have an
influence on penetration and permeation (see section 6.5.5). In
principle, as shown in Equation 5 (section 4.2), the maximum flux of
a solute from saturated solutions across a membrane should be
identical and independent of the vehicle, providing the vehicle does
not affect the membrane and supersaturation does not occur (Roberts
et al., 2002). Hence, in comparing solute fluxes from a given con-
centration in different vehicles, the vehicle effect may also be seen
as a modulation of the vehicle/stratum corneum partition coefficient
K
m
, which is an important factor in determining the rate of pene-
tration of a chemical into the stratum corneum (Scheuplein & Blank,
1971) and may also be the result of a potential direct effect of the
vehicle on the stratum corneum. The vehicle/stratum corneum parti-
tion coefficient describes the relative affinity of a chemical for the
vehicle in which it is applied and the stratum corneum (Suskind,
1977). The more soluble the penetrant in the vehicle, the more likely
it is to be retained within the vehicle (Baker, 1986). If the vehicle
contains components that directly interact with stratum corneum
intercellular lipids, then it is likely that the interaction will cause
enhancement or retardation of permeation (Davis et al., 2002).
Ideally, the test substance application should be identical to that to
which humans or other target species may be exposed. The applica-
tion may be neat, diluted (ideally with water), or otherwise formu-
lated, as appropriate (OECD, 2004a,c).
6.2.4.3 Receptor fluid
The receptor fluid should not act as a rate-limiting step in the
permeation process due to limited solubility of the test compound
within the medium (i.e. partitioning from the skin to the receptor
49
fluid should be unhindered). In addition, the receptor fluid should
not affect skin barrier integrity (OECD, 2004a). For water-soluble
compounds, the use of normal saline or an isotonic buffered saline
solution is sufficient. For lipophilic test substances, the receptor
fluid may contain solvent mixtures such as ethanol:water (although
50% aqueous ethanol may in some cases overestimate permeation),
<6% (dependent on permeant) polyoxyethylene[20] oleyl ether in
water, or 5% bovine serum albumin (Sartorelli et al., 2000;
Bronaugh, 2004b; OECD, 2004a,c). Although 6% polyoxyethyl-
ene[20] oleyl ether may have been optimal for the permeants used in
the original Bronaugh & Stewart (1984) study, it may not be optimal
for all compounds. The surfactant is present to solubilize permeating
compounds, and it is possible that lower concentrations may be just
as effective at this task for many permeants. When fresh skin is used,
the viability of skin can be maintained with either a tissue culture
medium or a HEPES-buffered Hanks balanced salt solution. Care
must be taken with surfactants and organic solvents, as the skin
barrier can be damaged, particularly when split-thickness skin prep-
arations are used (Bronaugh et al., 1999). It was reported that 1%
aqueous solutions of sodium laurate, sodium lauryl sulfate, and other
surfactants were found to increase the permeability of water through
human epidermis (Sartorelli et al., 2000). However, the enhance-
ment of permeation through addition of permeation-enhancing
materials to the receptor phase, although generating incorrect data,
will lead to an erroneous but conservative risk assessment.
Ramsey et al. (1994) tested fluazifop-butyl in static diffusion
cells with rat and human epidermal membranes using three different
receptor fluids: 1) 50% aqueous ethanol, 2) 6% polyoxyethylene[20]
oleyl ether in saline, or 3) tissue culture medium. In a comparison
with in vivo experiments in rat and human volunteer experiments,
use of both 6% polyoxyethylene[20] oleyl ether in saline and tissue
culture medium significantly underestimated the in vivo absorption,
whereas the use of 50% aqueous ethanol as receptor fluid was ade-
quately predictive of in vivo absorption. Concern has, however,
been expressed about ethanol in the receptor phase increasing epi-
dermal permeability (Saunders & Pugh, 2002).
50
6.2.4.4 Application dose levels
For finite dose experiments, an appropriate quantity (15
mg/cm
2
or 10 l/cm
2
) of the test preparation should be spread on the
skin surface (OECD, 2004c). For infinite dose experiments, typical
doses of >10 mg/cm
2
or >100 l/cm
2
are required to obtain steady-
state conditions from which the steady-state flux or absorption rate
and the permeability coefficient K
p
can be calculated (OECD,
2004c).
6.2.5 Duration of exposure and sampling time
The exposure time should reflect in-use conditions. The expo-
sure time may therefore vary between a few minutes for a rinse-off
product up to a maximum of 24 h for a leave-on product and 68 h
for industrial products (OECD, 2004c). For finite dose experiments,
the wash-off from the skin should be performed with an aqueous
soap solution, and the time of the wash-off determines the exposure
(OECD, 2004c). It is important to sample the receptor fluid for at
least a 24-h period. Increased exposure times are appropriate only in
cases of long lag phases or for infinite applications in order to
achieve a steady-state flux (OECD, 2004c). Periods of 24 or 48 h
should be adequate to study dermal absorption. Unless adequately
preserved, the skin can deteriorate beyond this time. Standard
exposure times for test substances in finite dosing experiments are
up to 24 h and shorter in the case of rinse-off products (although the
measurement of penetration of material continues for at least 24 h).
6.2.6 Evaluation of the results
The terminal procedures of an in vitro dermal absorption study
are slightly different following infinite and finite dosing experi-
ments. After finite dosing, the mean maximum amount of dermally
absorbed material is determined, which requires complete recovery
of the test substance (90110% [OECD, 2004c] or 85115%
[SCCNFP, 2003b]). The quantity washed from the skin, the quantity
associated with the skin (and in the different skin layers, if
analysed), and the amount present in the receptor fluid should be
determined. For infinite dose applications, the steady-state flux is
determined and the permeability coefficient K
p
calculated; the
recovery determination is not relevant to the calculation of K
p
,
51
because the only important end-point is the appearance of the test
substance in the receptor fluid (OECD, 2004c).
6.2.6.1 Dermal absorption results after finite dosing
The quantity of the test compound and/or its metabolites should
be determined in (OECD, 2004a,c):
applicator (spreader, glass rod, loop, etc.);
donor chamber (amount removed following rinse procedures);
dislodgeable dose from the skin surface (terminal washing,
sponges, etc.);
the stratum corneum, when sampled (tape strips, etc.);
remaining skin sample;
receptor fluid and receptor chamber; and
volatile material trap (if present).
For radiolabelled test substances, scintillation counting is
performed. For non-radiolabelled substances, HPLC, LC-MS, or GC
analysis may be appropriate. According to the OECD, an adequate
mean recovery is in the range of 100 10% (100 20% may be
acceptable for volatile substances where no trap is present, as lower
recovery rates are expected; Bronaugh et al., 1999). Reasons for
discrepancies in recovery rates should be explained (OECD, 2004c).
According to OECD Test Guideline 428 (OECD, 2004c), the
test substance remaining in the skin should be considered as
absorbed unless it can be demonstrated that absorption can be deter-
mined from receptor fluid alone. However, when the test substance
remains in the skin at the end of the study, it may need to be
included in the total amount absorbed (OECD, 2004c).
Cosmetic guidelines suggest that dermal absorption should be
expressed as an absolute amount (g/cm
2
of skin surface) and as a
percentage of the amount of test substance contained in the intended
dose applied per square centimetre of skin surface (Bronaugh et al.,
1999; SCCNFP, 2003b). For cosmetic testing, amounts that are
retained by the stratum corneum at the time of sampling are not con-
sidered to be dermally absorbed, and thus they do not contribute to
the systemic dose (SCCNFP, 2003b). However, for other risk
52
assessments, this amount may be considered as a possible reservoir
for systemic dose.
6.2.6.2 Dermal absorption results after infinite dosing
The permeability coefficient K
p
is calculated (in units of cm/h or
cm/s) by dividing the steady-state flux (measured in g/h per cm
2
)
by the concentration of the test substance (measured in g/cm
3
)
applied to the skin (Bronaugh et al., 1999; USEPA, 2004a).
6.3 Other in vitro methods
6.3.1 Artificial skin
Several researchers have developed artificial skin equivalents.
Such materials generally attempt to produce membranes that exhibit
both hydrophilic and hydrophobic regions and therefore mimic the
stratum corneum. Living skin equivalent models have also been
employed to assess percutaneous absorption. They consist of skin
membranes, including, for example, reconstituted epidermis, grown
in tissue culture and used as alternatives to animal tissues (e.g.
EpiDerm) (Wagner et al., 2001; Moss et al., 2002). The use of arti-
ficial skin is not yet recommended for in vitro testing because of
differences in barrier function compared with natural skin (Coquette
et al., 2000). Permeation measurements from artificial skin have
been inconsistent, making a correlation with either in vitro or in vivo
measurements impossible (Heylings et al., 2001; Ponec et al., 2001).
6.3.2 Tape-stripping technique in vitro
The methodology of tape stripping is discussed in section 7.2.3.
Several studies have evaluated the use of tape stripping in vitro (e.g.
for metabolism experiments, see Clark et al., 1993; for dermato-
pharmacology, see Surber et al., 1999; for establishing a model for
in vivo and in vitro experiments, see Trebilcock et al., 1994). In
cosmetic testing, it is commonplace to remove the stratum corneum
after in vitro testing for mass balance and other purposes.
hair dyes, where the amount of test substance identified in the tape-
stripped stratum corneum is separated from the amount detected in
the deeper skin layers. The test chemicals found in each compart-
Table 3 shows the results after in vitro penetration studies with
53
ment are shown, and these are related to the amounts recovered as
rinses, adsorbed, absorbed, and penetrated (Steiling et al., 2001).
Table 3. Results of percutaneous absorption studies of hair dyes
a
% applied dose
Hair dye I
without
developer
Hair dye I
with
developer
Hair dye II
with
developer
Rinsings (skin, equipment) 89.5 85.2 88.4
Adsorption (on tape strips)
b
3.3 2.6 3.7
Absorption (in residual skin) 0.91 0.39 0.63
Penetration (in the receptor) 0.11 0.01 0.30
Bioavailability
c
1.02 0.40 0.93
Per cent recovery 94.7 89.1 93.0
a
Adapted from Steiling et al. (2001).
b
It should be noted that for cosmetic products, the amount remaining in the
stratum corneum that can be removed by tape stripping is not considered
as systemically available.
c
Bioavailability: Absorption (in residual skin) + penetration (in the receptor
fluid).
6.4 Examination of skin reservoir characteristics
Skin reservoir effects are well documented for steroids (Misel-
nicky et al., 1988). The reservoir can exist in the stratum corneum,
in the viable avascular tissue (viable epidermis), and in the dermis
(Roberts et al., 2004) (see also section 12.3).
In order to investigate the potential for the skin to act as a
storage depot for a specific substance, a flow-through diffusion cell
with either laboratory animal or human full- or split-thickness skin
samples may be used. The radiolabelled substance is applied to the
skin surface for 5 h. After this time, any remaining test substance is
removed by wiping, and the experiment is then left to run for
another 15 h, during which time any radioactivity already absorbed
into the skin could continue to diffuse across and into the receptor
fluid. This receptor fluid is collected every hour (2 ml) for a total of
20 consecutive hours until the end of the experiment. Radioactivity
still remaining in the upper stratum corneum may be estimated by
tape stripping.
54
For example, in a study investigating the fate of dihydroxy-
acetone, 7-(2H-naphtho[1,2-d]triazol-2-yl)-3-phenylcoumarin, and
disperse blue 1, the skin penetration and permeation through human
and fuzzy rat skin were determined over 24 or 72 h in flow-through
diffusion cells (Yourick et al., 2004). Of the three compounds stud-
ied, two (dihydroxyacetone and disperse blue 1) formed epidermal
reservoirs, and the authors concluded that the amount remaining in
the skin should not be considered as absorbed material. On the other
hand, the data for 7-(2H-naphtho[1,2-d]triazol-2-yl)-3-phenylcou-
marin indicated that this material was spread throughout the epi-
dermis and dermis and, as such, could not be justifiably excluded
from the total absorbed dose without further experimentation. These
data demonstrate the importance of determining the fate of chemi-
cals remaining in skin.
6.5 Experimental factors affecting dermal absorption in
vitro
6.5.1 Species differences
The skins of many species have been evaluated as models for
permeation through human skin. Although rat, mouse, and rabbit
skins are more permeable than human skin, they have been used to
provide a conservative estimate of skin penetration for safety assess-
ments (Feldmann & Maibach, 1969, 1970; Bartek et al., 1972; Scott
et al., 1986; ECETOC, 1993; Bronaugh et al., 1999; van Ravenz-
waay & Leibold, 2004).
Rat skin was more permeable than human skin to all of a range
of tested substances (organic compounds: molecular weight = 231
466 g/mol; aqueous solubility = 0.057600 000 mg/l; log K
ow
= 0.7
4.5), with a mean difference of 10.9-fold (van Ravenzwaay &
Leibold, 2004). Lipophilic compounds showed the highest penetra-
tion rates through rat skin in vitro (van Ravenzwaay & Leibold,
2004). However, the mean value of 10.9-fold should be treated with
caution because of the differences in the experimental protocols
between in vitro human and in vivo rat evaluations.
polar molecule) and
14
C-labelled paraquat dichloride (a fully ionized
divalent cation) through human and various laboratory animal skin
Table 4 shows the in vitro absorption of tritiated water (a small
55
samples (Scott et al., 1986). In another study, rat skin was shown to
be 2.9- to 6.3-fold more permeable than human skin in vitro to
trimethylamine (log K
ow
= 0.16; aqueous solubility = 8.9 10
5
mg/l)
(Kenyon et al., 2004a).
Table 4. In vitro absorption of tritiated water and
14
C-labelled paraquat
dichloride through human and animal skin
a
Permeability coefficient (K
p
)
(cm/h 10
5
)
Species Strain
Water Paraquat
Human 93 0.7
Rat Wistar Alpk/AP 103 27
Hairless rat 103 36
Nude rat 152 35
Mouse Alpk/AP 144 97
Hairless mouse 350 1066
Rabbit New Zealand White 253 80
Guinea-pig Dunkin-Hartley 442 196
a
Adapted from Scott et al. (1986).
In their review of rat permeability coefficients for compounds
from water, Vecchia & Bunge (2005) found the average ratio of the
permeability coefficient for rat skin to the permeability coefficient
for human skin to be about 2. This study also examined hairless
mouse, hairless rat, and snake skin permeability coefficient data.
They reported that, on average, hairless mouse skin is 3.1 times
more permeable than human skin, apparently independent of K
ow
,
but perhaps weakly dependent on molecular size.
6.5.2 Temperature
When carrying out in vitro experiments, the skin must be main-
tained at the physiological temperature of 32 1 C (OECD,
2004a,c; USEPA, 2004a). Since diffusion is a temperature-
dependent process, alteration in skin temperature will affect the
absorption process (OECD, 2004a). In the in vitro human skin data-
base evaluated by Vecchia & Bunge (2003a), a statistically discern-
ible difference between the permeability coefficients measured at
different temperatures was observed. It has also been observed that,
56
for the same heating bath temperature (in this case, it was 35 C),
the average temperature of the diffusion cells (average of receptor
and donor solutions) was different for the configurations of the flow-
through and static cells examined, which led to a measurable differ-
ence in the permeability across a silicone rubber membrane (Romon-
chuk & Bunge, 2004). The authors recommended that such experi-
ments be conducted isothermally.
6.5.3 Occlusion
The normal exposure situation is most accurately simulated by
open unoccluded conditions, because in most instances human skin
may be protected but not occluded during exposure. In certain cases,
occlusion will be more representative of occupational exposure (e.g.
protective gloves, etc.). Unoccluded conditions can avoid the skin
barrier modulation caused by excessive hydration, which, in most
cases, increases the penetration rate (Kligman, 1983; Bronaugh &
Stewart, 1985; Baker, 1986; ECETOC, 1993). A 5- to 10-fold
increase in permeability of the stratum corneum was noted when the
skin was occluded (Sartorelli et al., 2000). However, volatile sub-
stances may evaporate during unoccluded testing, and, because of
the dosage regimen, infinite dosing experiments can be done only
under occluded conditions (Bronaugh, 2004b; OECD, 2004c). In a
dermal absorption study with catechol applied in ethanol, occlusion
resulted in 78% of the applied dose permeating into the receptor
fluid, compared with 55% in dermal samples that were not occluded
(Jung et al., 2003).
6.5.4 Thickness of skin
The thickness of the skin sample used in in vitro studies may
have an influence on the permeation of the test chemical. Cnubben
et al. (2002) found that permeation through viable full-thickness skin
membranes was much less than permeation through only the epi-
dermis. In this investigation, the authors studied the in vitro skin
penetration of [
14
C]ortho-phenylphenol (log K
ow
= 3.28) through
human and rat viable skin, human and rat epidermal membranes, and
perfused pig ears (four to six skin membranes were used per experi-
mental group). Human skin was obtained directly after abdominal
surgery, and rat skin was obtained from male albino Wistar Outbred
rats. Rat (from Sprague-Dawley rats with clipped dorsal and flank
skin) and human epidermal membranes were prepared by overnight
57
immersion of the skin in a solution of 2 mol of sodium bromide per
litre containing 0.01% sodium azide. Human and rat epidermis
showed a marked higher cumulative permeation (g/cm
2
) than
human and rat full-thickness viable skin (Figure 9).
Fig. 9. In vitro skin penetration expressed as cumulative amount reaching the
receptor fluid (from Cnubben et al., 2002). [Reprinted from Regulatory
Toxicology and Pharmacology, Vol. 35, No. 1, N.H. Cnubben et al., Com-
parative in vitroin vivo percutaneous penetration of the fungicide ortho-
phenylphenol, Pages 198208, Copyright (2002), with permission from
Elsevier]
The influence of skin thickness (0.51.3 mm) on percutaneous
penetration has been studied using caffeine, testosterone, butoxy-
ethanol, and propoxur by Wilkinson et al. (2004) (EDETOX, 2004).
Some differences in the maximum flux and the cumulative doses in
receptor fluid, dependent on skin thickness, were seen. This was
because the relationship between skin thickness and permeant phys-
icochemical properties is complex (Wilkinson et al., 2004, 2006).
Vecchia & Bunge (2003a) also examined whether there were any
statistically significant detectable differences between experiments
that used different skin layers. Their conclusion was consistent with
the observations of Wilkinson et al. (2004, 2006).
Rat epidermis
Human epidermis
Human full skin
Rat full skin
Perfused pig ear
Time (h)
0
0
30
60
90
120
150
8 16 24 32 40 48
C
u
m
u
l
a
t
i
v
e

p
e
n
e
t
r
a
t
i
o
n

(
g
/
c
m
2
)
58
6.5.5 Further observations on application vehicle effects
Because the solubility of a permeating chemical is usually
different in different vehicles, the flux of a compound presented to
the skin at the same concentration in different vehicles should be
different. If the compound is instead presented to the skin at the
same saturation ratio, the flux will be the same, provided the vehicle
itself does not alter the skin barrier significantly. As a result, the
permeability coefficient K
p
, which is the ratio of the flux to the
concentration, can be quite different for different vehicles, even if
the vehicle does not change the skin, simply because the solubility is
different. So, it is not surprising that testosterone was found to have
a higher K
p
when applied in water, in which it has low solubility,
than when applied in petrolatum jelly or ethylene glycol, in which it
has higher solubility (Bronaugh & Franz, 1986). The in vitro skin
permeability values of caffeine were similar for petrolatum jelly and
the water vehicle, but lower for ethylene glycol.
In a recent investigation with a flow-through system, the per-
meability of [
3
H]ricinoleic acid a machine cutting fluid ingredient
through silastic membranes and porcine skin was significantly
reduced when it was applied in polyethylene glycol ethers with
several additives (2% triazine, 5% triethanolamine, 5% linear alkyl-
benzene sulfate) compared with several mineral oil mixtures. This is
surprising, as these additives are dermal irritants and should theor-
etically enhance solute permeability. The fact that the opposite was
observed suggests that the mixture interaction is more physico-
chemical in nature and probably not related to the chemical-induced
changes in the biological membrane when skin was exposed to these
cutting fluid mixtures (Baynes & Riviere, 2004).
The cumulative percutaneous penetration of a
14
C-labelled sun-
screen, octyl salicylate, in two different formulations (hydro-
alcoholic lotion and oil/water emulsion), determined using human
epidermal membranes prepared from skin obtained from autopsy,
was very similar in each case (1.58 g/cm
2
over 48 h). However, the
amount of applied material remaining in the epidermal membranes
at 48 h was markedly higher for the hydroalcoholic solution
(32.77%) than for the oil/water emulsion (17.18%) (Walters et al.,
1999).
59
Use of isopropyl myristate may be necessary for lipophilic
compounds (USEPA, 2004a), but difficulty in extrapolation to test
results from exposure conditions involving other vehicles should be
acknowledged. USEPA (1992) discussed theoretical relationships
that could be used to translate permeability coefficients measured
from one vehicle to another vehicle. The approach is to multiply the
measured K
p
by the ratio of the solubility of the chemical in the
vehicle of concern to the solubility of the chemical in the test
vehicle.
60
7. IN VIVO TESTS FOR DERMAL ABSORPTION
This chapter is an overview of the methodology for in vivo tests
for dermal absorption. Although some examples are given, this is
only a small part of the literature available on this subject. For a
compilation of available studies, the reader is referred to databases
such as that of EDETOX (undated).
The in vivo methods allow the determination of the extent of
cutaneous uptake as well as systemic absorption of the test sub-
stance. The main advantage in performing an in vivo study rather
than an in vitro study is that the in vivo study uses a physiologically
and metabolically intact system (OECD, 2004a,b). In vivo dermal
penetration studies are carried out in laboratory animals, usually
rodents, and in human volunteers. The latter has been widely used
for human pharmaceuticals and, to a more limited extent, for other
chemicals. In vivo studies in humans are the gold standard. The
conduct of any in vivo study has ethical issues. The main dis-
advantage of using laboratory animals is that they have different skin
permeability and systemic disposition compared with humans. Fur-
ther, in animals, the dosing site must be protected from grooming
behaviour (leading to the disappearance of topical applied com-
pound and, in some cases, oral intake of the test compound) or from
contact with the cage. Care needs to be taken to avoid discomfort of
the animals.
7.1 Laboratory animal studies
The rat is the most commonly used species for animal in vivo
studies, having the advantage that information from other toxicity
and toxicokinetic studies is mostly obtained from this species. The
male rat is the species and sex required for dermal absorption
studies according to the USEPA (1998); however, OECD (2004b)
states that other animal species can be used when they have been
shown to have skin absorption more similar to that of humans than
the rat. It is known that data from rat studies generally overestimate
human skin absorption (ECETOC, 1993). Monkeys and pigs show
dermal absorption similar to that of humans, but the disadvantages
of employing these two animal models are the relatively high main-
In Vivo Tests for Dermal Absorption
61
tenance costs and the possible handling difficulties. Other animal
models include hairless rats (Simonsen et al., 2002) and mice and
fuzzy rats (Klain & Reifenrath, 1991).
There are three classes of in vivo animal studies: 1) in which a
compound is measured in blood or excreta, 2) in which material is
measured in the skin by biopsy or some other method, and 3) in
which the compound is measured in all tissues (residue analysis).
Determination of dermal absorption should be based on mass
balance considerations.
7.1.1 Test guidelines for laboratory animal studies
It is only recently that the OECD adopted the guideline for in
vivo dermal absorption (OECD, 2004a,b). This is similar to the
guideline published by the USEPA (1998) for pesticides; however,
the standard OECD protocol requires considerably fewer animals,
since the number of exposure intervals and the number of dose
levels are lower than in the USEPA (1998) protocol. Both protocols
are being used for testing industrial chemicals and pesticides.
Zendzian (2000) reported that over 160 pesticides have been evalu-
ated on the basis of the USEPA (1998) protocol.
7.1.2 Principles of the standard in vivo tests
The test chemical is applied to a designated area of skin in an
appropriate format (e.g. in solvent or formulation for a defined
period). Body fluids, tissue, or excreta are collected at predefined
intervals, and the quantity of chemical and/or metabolite in the
samples is measured by a suitable analytical procedure (Wester &
Maibach, 1999b). The collection of expired air should be considerd
when there is information that the chemical and/or its metabolite are
excreted by this route. The analytical method of choice must be
appropriately sensitive, since percutaneous absorption is often low
(ECETOC, 1993). In vivo tests are sometimes required for regula-
tory purposes for registration of pharmaceuticals and pesticides (e.g.
USEPA, 1998).
62
7.1.2.1 Preparation of the application site
At least 24 h before treatment, the application site should be
prepared (OECD, 2004b). (USEPA [1998] specifies 24 h for mea-
suring dermal absorption from pesticides.) In the rat, for example,
the hair on the shoulders and the back is removed with animal hair
clippers; shaving of the application site should be avoided, in order
to avoid abrasions, which will artificially increase dermal penetra-
tion (OECD, 2004a,b). If the skin is washed prior to application
(with water or a mild detergent), the possibility of skin or barrier
property modifications has to be carefully considered (OECD,
2004c).
7.1.2.2 Dose levels
Skin absorption of chemicals applied in a finite dose formula-
tion is generally not linearly related to dose, the flux being lower at
the higher dose per unit area. It is therefore important that the
selected doses span the range of doses expected in field exposure.
These should be determined on the basis of quantity per unit area of
exposed skin (mg/cm
2
). The USEPA (1998) recommended that four,
but at least three, doses should be used, which should be at log
intervals. OECD Test Guideline 427 (OECD, 2004b) mentions that
the test substance preparation should be the same (or a realistic
surrogate) as that to which humans may be exposed. For pesticides,
most often the concentrate formulation and one or two application
dilutions are tested.
7.1.2.3 Application of the test substance to the skin
The test substance preparation, which ideally is radiolabelled in
a metabolically stable position, is applied to 510% of the surface of
the skin (for rats with body weight 200250 g: 10 cm
2
) (USEPA,
1998). The OECD (2004b) guidance specifies application of 10
l/cm
2
for a liquid substance and 15 mg/cm
2
for a solid. This speci-
fication of the volume is based on an unconstrained application (i.e.
to prevent the solution from running over a larger area) if one were
trying to simulate a skin splash. Other volumes per area might be
used if the area is constrained by a ring or other means that prevents
any spreading of the chemical outside the defined area and prevents
the animal from interfering with the application site. A common
procedure uses a ring of an inert material (rubber, polytetrafluoro-
63
ethylene), which is glued to the skin over the clipped area, before
application of the test formulation, using cyanoacrylate adhesive to
confine the treated area (OECD, 2004b). The application system
must be covered with, for example, a nylon gauze, which may be
glued over the ring to protect the test preparation against uninten-
tional removal or spreading. Some laboratories use a collar or rubber
tubing placed behind the forelegs or neck of the animals. The use of
a jacket with a plastic shield to cover the site is also acceptable. As
long as one can prevent the animal from grooming the treatment site
and prevent excessive loss of material from the site into the cage,
there is no restriction (within ethical limits) as to what can be used.
7.1.2.4 Duration of exposure
In general, the extent of absorption is directly related to the
duration of application (Roberts & Walters, 1998). The duration of
exposure is the time between application and removal of the skin by
skin washing. OECD Test Guideline 427 (OECD, 2004b) requires
an exposure period relevant to the occupational situation (typically 6
or 24 h). The USEPA (1998) guideline requires evaluation with time
(in the standard protocol: 0.5, 1, 2, 4, 10, and 24 h). The animal
should be observed for clinical signs of toxicity throughout the
duration of the study, and the treated skin should be observed for
visible signs of irritation (OECD, 2004b).
After the exposure period, the test preparation is removed by an
appropriate cleansing procedure. In the USEPA (1998) protocol, the
animals are sacrificed at this time point. In the OECD (2004b)
protocol, the animals are housed individually in metabolism cages
after the skin is washed. Excreta and (if appropriate) exhaled air are
collected until sacrifice of the animals.
7.1.2.5 Sacrifice and time of termination
OECD Test Guideline 427 (OECD, 2004b) mentions that
animals will be sacrificed at different time intervals for example,
at the end of the exposure period (typically 6 or 24 h) and at
subsequent occasions (e.g. 48 and 72 h). The USEPA (1998)
protocol requires that the animals be sacrificed at the exposure inter-
vals, which, in the standard study, are 0.5, 1, 2, 4, 10, and 24 h. In
64
both guidelines, blood and various other samples are collected for
analysis.
7.1.2.6 Evaluation of the results
The quantity of non-pharmaceutical chemical or its metabolites
must be determined in (OECD, 2004c):
dislodgeable dose from the skin surface (washing water);
skin from the treated and non-exposed site (stratum corneum,
epidermis, and dermis);
dislodgeable dose and desquamated skin (from protective dress-
ing post-exposure);
urine, faeces, and cage washing;
expired air, if applicable (>5% volatile metabolites of applied
dose);
blood and remaining carcass; and
solvent washing of contaminated material and application sys-
tem.
An adequate mean recovery is in the range of 100 10%,
although this recovery is higher than is often observed. Recoveries
outside the given range should be justified.
The dermal absorption is usually given as a percentage of the
dose applied and is obtained by addition of amounts recovered from
urine, faeces, and cage washing, expired gas, blood and remaining
carcass, and dose skin. Tape stripping can be performed in order to
obtain information on the test chemical deposition (Trebilcock et al.,
1994).
If the animals are not sacrificed, then an indirect measure of
absorption (by monitoring urine, faeces, or blood) is required,
including some sort of adjustment to account for material that is
absorbed but not measured in the fluid that is monitored. Where
possible, pharmacokinetic analyses are undertaken to estimate the
rate and the extent of absorption. The overall extent of absorption
can be defined in terms of the absolute bioavailability i.e. the
fraction F of the dose reaching the systemic circulation intact after
extravascular administration. This bioavailability is best determined
by comparison with plasma/blood or urine levels of unchanged
65
solute achieved with the intravenous administration of the solute.
Normally, either the area under the plasma concentrationtime
profile (AUC) or the amount excreted unchanged in the urine (A
u
)
from the time of administration to infinite time is used to define F
(Roberts & Walters, 1998):
F = [A
u,topical
/dose
topical
] / [A
u,iv
/dose
iv
]
= [AUC
topical
/dose
topical
] / [AUC
iv
/dose
iv
]
The percentage of dermal absorption can therefore be calculated
from:
Percentage dermally absorbed =
total % excreted after topical dose
100
total % excreted after intravenous dose
In the absence of intravenous studies, the percentage absorbed
has to be estimated by mass balance, which is given by the amount
excreted divided by the amount applied (Roberts & Walters, 1998).
7.2 Studies with human volunteers
Studies with human volunteers provide definitive data for the
assessment of the absorption of chemicals through human skin; for
technical and ethical reasons, however, their use is limited and their
conduct is closely regulated (Declaration of Helsinki, 1964 and
updates: World Medical Association, 2004; and the International
Conference on Harmonization Guideline for Good Clinical Practice:
ICH, 1996). The use of radiolabelled compounds for human studies
is subject to further regulation by the International Commission on
vivo can be assessed using different approaches, such as plasma and
excreta assessments, breath analysis, tape stripping of the stratum
encounters specific problems, which will be discussed in more detail
in the following sections.
corneum, microdialysis, and radiography. Each of these approaches
Radiological Protection (http://www.icrp.org). Dermal absorption in
66
7.2.1 Assessment using plasma, excreta, and breath analysis
7.2.1.1 Methodology
In vivo experiments are often used as a gold standard for alter-
native methods, such as in vitro studies and predictive modelling. It
is therefore important that for the purpose of comparison, in vivo
experiments deliver the same parameters e.g. absorption rate,
p
, etc. Deconvolution of pharmacokinetic
data can be used to estimate the absorption rate profiles of topically
applied chemicals using plasma data and comparison with intra-
venous plasma data. Several methods of performing pharmaco-
kinetic deconvolution are available (e.g. Yu et al., 1996; Levitt,
2003). The extent of absorption can be defined by the equations
given in section 7.1.2.6. However, in most in vivo studies for chem-
icals, the intravenous data are not usually available, and a preferred
approach is the use of mass balance or data from another admin-
istration route. In the case of mass balance, where the amount recov-
ered (in the stratum corneum and in urine) is compared with the
amount applied, only the average absorption rate can be estimated
(Roberts & Walters, 1998). By use of another route of admin-
istration, the relative extent of absorption can be compared (Mrz &
Nohov, 1992; Brooke et al., 1998), or the average absorption rate
can be estimated if the amount administered by the other route is
known. However, a preferable method would be to estimate dermal
absorption rate profiles in a similar manner as described for intra-
venous dosing e.g. by using inhalation with a known input rate.
This method has been widely used for the determination of dermal
absorption of liquids (Kezic et al., 2001; Jakasa et al., 2004b), solids
(including those dissolved in an appropriate vehicle), and vapours
(Kezic et al., 2000, 2004).
The chemical is usually applied on the forearm or back skin.
For vapours, however, whole-body exposure has also been used.
Pharmaceutical preparations would be applied on the site of
intended use.
7.2.1.2 Examples of in vivo human volunteer studies
Some examples of compounds applied in liquids include hor-
mones (e.g. Feldmann & Maibach, 1969), drugs (e.g. Dehghanyar et
al., 2004; Martin et al., 2004), hair dyes (e.g. Wolfram & Maibach,
67
1985; Dressler, 1999), disinfectants (e.g. Turner et al., 2004),
fragrances (e.g. Ford et al., 2001; Hawkins et al., 2002), industrial
chemicals (e.g. Hanke et al., 1961, 2000; Dutkiewicz & Tyras, 1967,
1968; Feldmann & Maibach, 1970; Dutkiewicz et al., 2000; Kalnas
& Teitelbaum, 2000; Kezic et al., 2000), and pesticides and
herbicides (e.g. Feldmann & Maibach, 1974; Dick et al., 1997a,b;
Garfitt et al., 2002a,b,c; Meuling et al., 2005; Ross et al., 2005).
In contrast to liquids, there are fewer studies on dermal absorp-
tion of vapours in humans. A number of early studies were con-
ducted in the former Soviet Bloc and nearby countries, including
studies on benzene (Hanke et al., 1961, 2000), aniline (Dutkiewicz
& Piotrowski, 1961), phenol (Piotrowski, 1971), m-xylene, styrene,
toluene, 1,1,1-trichloroethane, and tetrachloroethene (Riihimaki &
Pfaffli, 1978). More recent compounds studied include xylene,
toluene, 2-butoxyethanol, tetrahydrofuran, methyl ethyl ketone, 1-
methoxypropan-2-ol, N,N-dimethylformamide, and N,N-dimethyl-
acetamide (Johanson & Boman, 1991; Mrz & Nohov, 1992; Kezic
et al., 1997, 2004; Brooke et al., 1998; Nomiyama et al., 2000,
2001).
The results of these studies have shown that for some sub-
stances, such as the glycol ethers, skin uptake from vapours may be
an important contributor to the total uptake; for example, a 510%
contribution to the total body burden was seen for 1-methoxy-
propan-2-ol (Brooke et al., 1998). Also, other studies demonstrate
good dermal absorption of various glycol ether vapours (Johanson &
Boman, 1991; Kezic et al., 1997; Jones et al., 2003; Jakasa et al.,
2004b) or, to a lesser extent, of xylene (Kezic et al., 2004). About
40% contribution to the total body burden was reported for N,N-
dimethylformamide and N,N-dimethylacetamide (Mrz & Nohov,
1992; Nomiyama et al., 2000, 2001).
7.2.1.3 Biomonitoring of occupational exposure
Biomonitoring gives an indication of the internal dose of a
chemical by monitoring levels of chemicals and metabolites in the
blood, urine, or breath. For practical and ethical reasons, usually
urine samples are monitored (IPCS, 2001). However, the amount of
exposure by the dermal route may not be distinguishable from that
by the inhalation or ingestion route. Human volunteer data in which
68
exposure is controlled will allow establishment of the relationship
between levels in the biological fluids and the dermally applied dose
(e.g. Jakasa et al., 2004b).
Unless a worker is breathing clean air, it may not be possible to
separate dermal exposure from inhalation exposure. Observation of
the worker and work practices is important for defining the routes of
exposure.
Biological monitoring of urinary metabolites generally reflects
metabolism in the liver rather than skin metabolism, even for a
dermally absorbed dose. Dermal metabolism could potentially be
distinguished if the dermal metabolic profile differs from the liver
metabolic profile, as suggested for cypermethrin by Woollen et al.
(1992), or, if dermal absorption is extensive, by comparison with the
urinary excretion of an intravenous dose. Generally, only one urine
sample is available for analysis, taken at the end of shift or later for
chemicals with a long half-life. Theoretically, the urinary profile of a
chemical absorbed through the skin would be more delayed than the
urinary profile of the chemical taken up by inhalation due to the lag
time in dermal absorption; thus, if two or more samples are available
and an excretion profile were obtained, it might be possible to
deduce whether most of the exposure was by the dermal route. How-
ever, this approach has not been fully investigated.
Interindividual variability in liver metabolism, which may be up
to 10-fold, may contribute to variability in urinary parent and
metabolite levels. Knowledge of the chemical half-life, metabolism
profile, clearance, and the enzymes involved is required to interpret
biological monitoring data.
7.2.2 Cutaneous microdialysis
Cutaneous microdialysis is an in vivo sampling technique used
for the measurement of endogenous and exogenous substances in the
extracellular space beneath the skin using perfused dialysis. This
technique has been used in human volunteers (Anderson et al., 1998;
Benfeldt, 1999; Schnetz & Fartasch, 2001; Korinth et al., 2004;
Joukhadar & Muller, 2005) and in laboratory animal experiments
(Benfeldt, 1999; El Marbouh et al., 2000; Mathy et al., 2004). The
microdialysis system consists of microinjection pumps and micro-
69
dialysis probes with polyurethane (semipermeable) membranes (El
Marbouh et al., 2000), which may be implanted into blood vessels,
the dermis, or the subcutaneous tissue. The blood flow in a vessel
underneath the skin surface is mimicked by continuously passing a
receptor fluid through the microdialysis tubing and collecting it in a
collector. Two methods are commonly used: 1) one requiring two
puncture sites and referred to as a push-through method (Figure 10),
and 2) a single-puncture system with inlet and outlet of the same
site. Using these methods, it is possible to measure the local
concentrations of a test compound in the dermis and to monitor
percutaneous absorption of various substances (El Marbouh et al.,
2000).
Fig. 10. Scheme of the microdialysis system (from Schnetz & Fartasch,
2001). [Reprinted from European Journal of Pharmaceutical Sciences, Vol.
12, No. 3, E. Schnetz & M. Fartasch, Microdialysis for the evaluation of
penetration through the human skin barrier a promising tool for future
research?, Pages 165174, Copyright (1991), with permission from Elsevier]
Dermal absorption kinetics and dermal metabolism may be
studied using this technique. The results are usually expressed in
terms of relative recovery (Leveque et al., 2004), as the quantity of a
compound recovered across the microdialysis tubing is only a
fraction of the quantity present in the tissue.
Microdialysis has been used to investigate the influence of
penetration enhancers, vehicles, or iontophoresis on percutaneous
absorption in vivo in rats. The experiments in volunteers are limited
to 68 h, and most studied compounds are fast penetrating. One
problem with microdialysis is the calibration of the technique (e.g.
the assessment of the recovery). This can be determined using a
parallel in vitro approach. The position of the tubing should be
Microdialysis pump
Stratum Corneum
Viable Epidermis
Dermis
Perfusate
Application site
Membrane
= solute
Diffusion of solutes into the perfusate
Dialysate
70
determined for standardization using ultrasound (Muller et al., 1997;
Anderson et al., 1998) or related techniques. Microdialysis normally
uses an aqueous perfusate and is used to estimate water-soluble
substances. Dialysates containing albumin have been used in esti-
mating the dermal absorption of other more lipophilic solutes. A
second problem is the variability associated with dermal micro-
dialysis (McCleverty et al., 2006). Care must be taken with study
design, using, where possible, each subject as his or her own control
twice to remove subject-to-subject variability in sample size estima-
tions and to provide an estimate of the intrasubject variability.
7.2.3 Tape stripping
Tape stripping of human stratum corneum has frequently been
used for investigation of skin penetration, barrier function, and the
factors involved in skin pathologies (Bashir et al., 2001; Choi et al.,
2004; Loffler et al., 2004). The method is simple, inexpensive, and
minimally invasive and can be used in both humans and laboratory
animals.
In a tape-stripping experiment, a predetermined area of skin is
exposed to a chemical for a fixed application period. After exposure,
stratum corneum from the exposed skin site is removed sequentially
by successive application of adhesive tape. The amount of the
substance recovered by the tape is then analysed using an appro-
priate analytical method. A comparison of various tape-stripping
methods is given in Choi et al. (2004), which shows the variance in
the methods used. The number of tape strips needed to remove a
given fraction of the stratum corneum varies with the type of tape
(Bashir et al., 2001), experimental factors such as the pressure
applied during application, peeling force for removal, and anatomi-
cal site, as well as age, sex, and possibly ethnicity of the subject
(Palenske & Morhenn, 1999; Loffler et al., 2004). It has also been
shown that the adhesive properties of the tape as well as the cohe-
sion of the corneocytes may be influenced by the vehicle (Surber et
al., 1999), and this will also affect the number of strips required to
completely remove the stratum corneum.
Despite these complications, some protocols have specified the
number of tape strips that should be collected. Rougier et al. (1999)
recommended that the capacity of the stratum corneum reservoir for
each compound be defined as the sum of the amounts found in the
71
first six strippings. The USFDA (1998) guidance specifies that 12
tape strips should be collected, and OECD (2004c) recommends that
1525 strips be used for human studies. The amount of stratum
corneum on each tape strip can be obtained by weighing (Pirot et al.,
1998; Weigmann et al., 2003). Indirectly, it can be determined by
measuring the amount of proteins on the tape (Chao & Nylander-
French, 2004), by monitoring of transepidermal water loss (Weig-
mann et al., 2005), or spectrophotometrically by measuring light
absorption of the proteins on the tape (Weigmann et al., 2003).
There are three different approaches in the methods that utilize
the tape-stripping technique. The United States Food and Drug
Administration (USFDA, 1998) has proposed a guideline for the
determination of bioequivalence of topically applied products using
a tape-stripping technique. The method is based on the determina-
tion of the total amount of drug in the collected strips in the stratum
corneum during uptake and elimination phases. For drug uptake, the
product is applied at multiple sites, and the stratum corneum
samples are removed from each site just after removal of the applied
drug at sequentially increasing times. To assess drug elimination, the
drug is applied to multiple sites and maintained for a period of time.
All the application sites are then cleaned, and the stratum corneum is
removed at different times after cleaning. Bioequivalence is assessed
based on the AUC. The results are plotted as the amount/surface
area against time. The method was shown to be useful in deter-
mining the rate and extent of drug penetration into the stratum
corneum (Shah et al., 1998; Weigmann et al., 2001; Pershing et al.,
2002a,b, 2003). However, this method has not yet been sufficiently
validated, and there has been some criticism of the method concern-
ing its robustness, reliability, and reproducibility for bioequivalence
testing. The USFDA withdrew the dermatopharmacokinetics (DPK)
guidance in 2002, citing two principal concerns: 1) the adequacy of
the method to assess the bioequivalence of topical dermatological
drug products, and 2) the reproducibility of the method between
laboratories (USFDA, 2001).
Another approach using the skin-stripping technique is based on
the determination of the concentration profile of a chemical in the
stratum corneum in relation to the relative stratum corneum depth.
The depth of the stratum corneum for each strip can be estimated
from the mass of the stratum corneum stripped off from each
72
consecutive tape. The data on concentrationstratum corneum depth
profile were fitted into a solution of Ficks second law of diffusion.
From this, the partition coefficient of a chemical between the
stratum corneum and a vehicle (K
m
) and diffusivity (D) were
derived, enabling deduction of the chemical permeability coefficient
K
p
(e.g. Pirot et al., 1998; Alberti et al., 2001; Reddy et al., 2002).
However, this analysis assumes a profile consistent with a homo-
geneous membrane, and this is not always observed (Mueller et al.,
2003).
Finally, tape-stripping studies also follow an approach in which
the chemical is applied for a single exposure period and then the
amount of chemical in collected tape strips is determined (e.g.
Dupuis et al., 1984; Rougier et al., 1999; Chao & Nylander-French,
2004; Mattorano et al., 2004).
There are several problems encountered in the studies per-
forming tape stripping. Some authors do not measure the actual
depth of the stratum corneum but instead rely on the number of
consecutive strips used, assuming regular stripping of stratum
corneum. However, it has been shown that even in the case of very
strict standardized protocols, the amount of stratum corneum on
each tape strip varies considerably (Weigmann et al., 1999; Dickel
et al., 2004). Furthermore, it has been shown that due to furrows in
the stratum corneum, the amount of chemical measured in a par-
ticular strip comes from different layers, which might lead to mis-
interpretation of the results (van der Molen et al., 1997). For rapidly
penetrating compounds, results from tape-stripping experiments can
be affected significantly by chemical diffusion into the stratum
corneum during the time required to apply and remove all of the tape
strips (Reddy et al., 2002).
Because it can be used to test absorption in humans in vivo,
tape stripping offers opportunities for studies that cannot be
conducted readily in vitro or in laboratory animals. For example,
diseased compared with healthy skin can be studied, as described by
Jakasa et al. (2004a). In a tape-stripping study, the skin site of
atopics that was not affected by disease showed higher stratum
corneum penetration compared with healthy subjects. Furthermore,
tape stripping can be used as a means of comparing human in vivo
73
and in vitro data (e.g. Trebilcock et al., 1994; Pirot et al., 1998;
Reddy et al., 2002).
7.3 Other methods
7.3.1 Whole-body autoradiography
Whole-body autoradiography is a useful technique used to
determine the distribution of radiolabelled compounds in frozen
cross-sections of the entire animal. This method has been shown to
be a very useful tool for visualization of the radiolabelled chemical
in all body tissues. By using microautoradiography, the distribution
of a topically applied radiolabelled compound in specific areas of
the skin can be determined (Lockley et al., 2004). In a study by
Saleh et al. (1997), the distribution of [
14
C]malathion, an organo-
phosphorus pesticide, was investigated using electronic autoradiog-
raphy of whole-body sections of treated rats after intravenous, oral,
and dermal administration. Whereas the liver and kidney accumu-
lated high levels of the chemical after intravenous administration, it
was shown that the skin may act as a reservoir for the compound
after dermal application.
The whole-body autoradiography technique is becoming more
commonplace, although there are no guidelines to cover it.
7.3.2 Skin biopsy
A number of studies undertaken by Schaefer and colleagues
(Kammerau et al., 1975; Zesch & Schaefer, 1975; Schaefer &
Stuttgen, 1976; Kranz et al., 1977; Schaefer et al., 1977, 1978) have
measured the dermal levels of various drugs in humans in vivo and
in vitro. Much lower levels were found in vitro, demonstrating the
role of blood flow in dermal absorption. Singh & Roberts (1994)
used similar techniques in rats to show direct topical penetration of
drugs into dermal and deeper tissue levels at early times after
dosing. They also found that vasoconstriction increased dermal
levels (Singh & Roberts, 1994). A number of authors have described
the dermal concentration gradient by a dermal tissue diffusion
dermal blood flow clearance model (Gupta et al., 1995; Cross &
Roberts, 1999; Kretsos et al., 2004; Kretsos & Kasting, 2005).
74
7.4 Factors affecting dermal absorption in vivo
7.4.1 Species, strain, and sex
The skin of rats, guinea-pigs, and rabbits is more permeable
than that of humans, whereas the skin permeability of pigs and
monkeys is more similar to that of humans (OECD, 2004b). Other
possible animal models include the athymic (nude) rat skin flap
model, hairless rats, hairless mice, and fuzzy rats (Klain &
Reifenrath, 1991). In a comparative study involving several species,
radiolabelled haloprogin, N-acetylcysteine, cortisone, testosterone,
caffeine, and butter yellow dissolved in acetone were applied to the
skin of rats, rabbits, minipigs, and humans (only haloprogin and N-
acetylcysteine). The dose was 4 g/cm
2
skin surface applied using a
non-occlusive foam pad. The amount of radioactivity excreted in
urine for 5 days following application of the test compound was
used for quantifying skin absorption. The results obtained in this
study indicated that dermal absorption decreases in the following
order: rabbit, rat, pig, and human (see Figure 11) (Bartek et al.,
1972).
Fig. 11. Total absorption of different compounds in different species (adapted
from Bartek et al., 1972).
HaIoprogin
120
100
80
60
40
20
0
N-AcetyIcysteine
T
o
t
a
I

a
b
s
o
r
p
t
i
o
n

(
%

o
f

d
o
s
e
)
Testosterone
Cortisone
Caffeine
Butter yeIIow
Rabbit
Pig
Human
Rat
75
7.4.2 Age
In a comparative study with human volunteers, Roskos et al.
(1989) demonstrated that age can affect dermal absorption. Permea-
tion of hydrocortisone, benzoic acid, acetylsalicylic acid, and caf-
feine was significantly lower in aged subjects, whereas the absorp-
tion of testosterone and estradiol was similar in young and aged
subjects. An explanation might be that the diminished surface lipid
content and reduced hydration of old skin imply a diminished
dissolution medium for compounds administered topically, which
reduces the uptake of somewhat hydrophilic compounds especially.
In contrast, highly lipid-soluble chemicals such as testosterone and
estrogen may still be able to dissolve readily into the stratum
corneum, even when the available lipid medium is reduced (Roskos
et al., 1989).
7.4.3 Anatomical site
The percutaneous absorption of acetylsalicylic acid, benzoic
acid, caffeine, and benzoic acid sodium salt (radiolabelled) was
measured in male Caucasians on four body sites (arm, abdomen,
postauricular, forehead), using the tape-stripping method. Skin
penetration, as indicated by the amount of chemical in the stratum
corneum, was ranked as follows: arm _ abdomen _ postauricular _
forehead. It is noteworthy that whatever the compound applied, the
forehead was about twice as permeable as the arm or the abdomen
(Rougier et al., 1987, 1999).
Absorption of radiolabelled cortisol through various anatomical
sites after topical application of hydrocortisone in acetone to male
volunteers (13 cm
2
skin surface) was studied by analysing the urine
for a total of 5 days. The absorption through the forearm of each
number of other studies are summarized in Roberts & Walters
(1998).
These and similar data on hydrocortisone and pesticides were
combined to construct penetration indices for five anatomical sites,
as shown in Table 5 (Wester & Maibach, 1999a).
volunteer served as control. The results are shown in Figure 12. A
76
Fig. 12. Hydrocortisone absorption effect of anatomical region (adapted
from Feldmann & Maibach, 1967).
Table 5. Penetration indices for different anatomical sites
Penetration index based on Site
Hydrocortisone data Pesticide data
Genitals 40 12
Arms 1 1
Legs 0.5 1
Trunk 2.5 3
Head 5 4
Although all authors agree on the importance of anatomical
location in percutaneous absorption, the reviews on this topic give
contradictory explanations. Factors that influence regional differ-
ences in skin permeability include different thicknesses and com-
position of the stratum corneum in different anatomical locations,
size and number of follicles, sebum composition, etc. (Scheuplein &
Blank, 1971; Idson, 1975; Roberts et al., 1982; Barry, 1983). A
further explanation may be the closeness of capillaries to the surface
of the skin in certain regions of the body. This effect may promote
x 42
x 0.83
x 13
x 6
x 3.6
x 3.5
x 1.7
x 0.42
x 0.14
x 1.1
x 1.0 Forearm (ventraI)
Forearm (dorsaI)
Foot arch (pIantar)
AnkIe (IateraI)
PaIm
Back
ScaIp
AxiIIa
Forehead
Jaw angIe
Scrotum
77
resorption and may explain high percutaneous penetration in the
postauricular area found for benzoic acid, benzoic acid sodium salt,
and acetylsalicylic acid (Rougier et al., 1999).
7.4.4 Type of application and vehicle
Applied vehicles have the potential to either increase or
decrease the solubility and/or diffusion of a compound in the horny
layer and, thereby, to increase or decrease penetration.
The penetration of benzoic acid is enhanced by increasing the
water content of the vehicles, whatever the organic phase (Rougier
et al., 1999). The influence of nine vehicles on the in vivo percu-
taneous absorption of radiolabelled benzoic acid was studied in the
hairless rat, by assessing total percutaneous absorption and stratum
corneum reservoir (stripping method) (Rougier et al., 1999). Each
vehicle (propylene glycol/triton X-100 90:10, glycerol/triton X-100
90:10, ethylene glycol/triton X-100 90:10, ethylene glycol/triton X-
100 90:10/water 40:60, propylene glycol/triton X-100 90:10/water
40:60, ethanol/water 95:5, methanol/water 40:60, ethanol/water
60:40, ethanol/water 40:60), containing 200 nmol of benzoic acid,
was applied to 1 cm
2
of dorsal skin during 30 min. Although the
vehicles used were simple in composition, the total amount of ben-
zoic acid that penetrated over 4 days varied by a factor of 50. This
influence of the presence of water on the dermal absorption was also
reported in volunteers using 2-butoxyethanol. The percutaneous
absorption of the test substance from aqueous solution increased
markedly when compared with neat 2-butoxyethanol. As little as
10% water in 2-butoxyethanol produced approximately a 4-fold
increase in the permeation rates (Jakasa et al., 2004b). Similar
results were obtained in rats (EDETOX, 2004; see Appendix 2).
Contrary to this, dermal penetration of neat chloroform was higher
than that of aqueous solutions containing chloroform (Islam et al.,
1999); however, this was probably due to damage of the skin caused
by neat solvent.
White spirit enhanced the penetration of lindane compared with
an acetone vehicle (Dick et al., 1997a). For studies on the influence
of vehicles in in vitro studies, see section 6.5.5.
78
7.4.5 Temperature and humidity conditions
Increased percutaneous absorption rates were seen for 2-
butoxyethanol vapours with raised temperature and humidity con-
ditions (Johanson & Boman, 1991; Jones et al., 2003). The mean
baseline (25 C, 40% relative humidity, shorts and T-shirts) per-
centage of dermal absorption was 11% (range 914%) of the
whole-body burden. For each subsequent exposure, a single
parameter was changed: humidity (60%, 65%), temperature (20 C,
30 C), or clothing (minimal or overalls). At 30 C, the percentage
of dermal absorption was significantly increased, with a mean of
14% (range 1215%). Increasing the humidity increased the percen-
tage of dermal absorption, although not significantly. Clothing also
had little effect on the percentage of dermal absorption. In an
industrial scenario (30 C, 6065% relative humidity, overalls)
that might be encountered in a tank cleaning or similar operation,
skin absorption was significantly increased (mean of 39%) when
compared with the baseline dermal study (Jones et al., 2003).
The absorption of transdermally delivered nicotine to human
volunteers increased after they stayed in a sauna bath (mean tem-
perature 82 C, 28% humidity) for three 10-min periods separated
by two 5-min breaks, compared with measurements taken in a
previous control session (without sauna) (Vanakoski et al., 1996).
79
8. COMPARATIVE STUDIES
8.1 Comparison between in vitro and in vivo skin
absorption results
Many studies have compared in vitro and in vivo percutaneous
absorption (Table 6). In general, they verify the premise that prop-
erly conducted in vitro measurements can be used to predict in vivo
absorption. The basis of OECD Test Guideline 428 (OECD, 2004c)
is that in vitro studies can predict in vivo absorption when the
correct methodology for both tests is used.
Table 6. Some comparative studies on dermal absorption in vitro and in vivo
Compounds Species Lipophilicity/polarity References
12 organic
compounds
Human Mixed polarity Franz (1975,
1978)
Benzoic acid,
acetylsalicylic
acid, urea, and
caffeine
Rat Generally polar Bronaugh et al.
(1982)
Nitroaromatic
compounds
Monkey,
human
Mixed polarity Bronaugh &
Maibach (1985)
Benzoic acid,
caffeine, and
testosterone
Human Mixed polarity Bronaugh & Franz
(1986)
Anthracene Rat Lipophilic Yang et al.
(1986a)
Benzo[a]pyrene Rat Lipophilic Yang et al.
(1986b)
Cypermethrin
(pesticide)
Rat Lipophilic Scott & Ramsey
(1987)
5 pesticides Mouse Lipophilic and
hydrophilic
Grissom et al.
(1987)
Phenoxy
herbicides 2,4-
D, 2,4-D amine
Rat, rabbit,
rhesus
monkey,
human
Mixed polarity Moody et al.
(1990)
Benzyl acetate Rat Lipophilic Hotchkiss et al.
(1990)
Phenanthrene Guinea-pig Lipophilic Ng et al. (1991)
80
Table 6 (Contd)
Compounds Species Lipophilicity/polarity References
Tritiated water,
ethanol,
mannitol,
paraquat
Rat,
marmoset,
human
Hydrophilic Scott et al.
(1991a)
8 pesticides
(identity not
given)
Rat Mixed polarity Scott et al. (1992)
Pyrene,
benzo[a]pyrene,
di(2-ethylhexyl)-
phthalate
Guinea-pig Lipophilic Ng et al. (1992)
Fluazifop-butyl Rat, human Lipophilic Ramsey et al.
(1992, 1994)
Nitroglycerine Human Lipophilic Hadgraft et al.
(1993)
Hair dyes Rat, pig Mixed polarity Beck et al. (1994)
Vanilloids Rat Lipophilic Kasting et al.
(1997)
Lindane Human Lipophilic Dick et al.
(1997a,b)
Methyl salicylate Human Lipophilic Cross et al.
(1998)
Propoxur Rat, human Hydrophilic van de Sandt et
al. (2000)
Ortho-Phenyl-
phenol
Rat, human Lipophilic Cnubben et al.
(2002)
2-Butoxyethanol Human Hydrophilic Jakasa et al.
(2004b)
14 pesticides Rat Mixed polarity van Ravenzwaay
& Leibold (2004)
There are some exceptions; for example, for radiolabelled 2-
nitro-p-phenylenediamine, 4-amino-2-nitrophenol, nitrobenzene, p-
nitroaniline, and 2,4-dinitrochlorobenzene (4 g/cm
2
, acetone, 24 h),
the absorption in monkeys was slightly less in vitro than in vivo, but
the values were not significantly different (Bronaugh & Maibach,
1985). Another example was the volatile compound nitrobenzene,
where human data indicated significantly higher percutaneous
absorption in vivo compared with in vitro (Bronaugh & Maibach,
1985).
Comparative Studies
81
A comparison of the in vitro and in vivo percutaneous absorp-
tion of caffeine and testosterone in three vehicles (petrolatum,
ethylene glycol gel, and water gel) revealed no significant differ-
ences between most values. However, there was a trend towards
lower penetration in the in vitro system (Bronaugh & Franz, 1986).
In vitro absorption utilizing flow-through diffusion methodology
with human cadaver skin and human plasma receptor fluid gave a
per cent dose absorbed similar to that in in vivo human volunteer
studies for the pesticide isofenphos (Wester et al., 1992).
The in vitro and in vivo absorption and metabolism of pyrene,
benzo[a]pyrene, and di(2-ethylhexyl)phthalate were investigated in
hairless guinea-pig. The in vitro method, which involved the use of
flow-through diffusion cells and HEPES-buffered Hanks balanced
salt solution containing 4% bovine serum albumin as perfusate, was
shown to be a suitable system for predicting in vivo absorption of
these lipophilic compounds (Ng et al., 1992).
The dermal absorption of [
14
C]benzo[a]pyrene was studied in
vitro in different species (rat, hairless guinea-pig, 50- and 32-year-
old human volunteers) and in vivo in rat and hairless guinea-pig.
The total percentage of [
14
C]benzo[a]pyrene absorbed in vitro was
consistent with that absorbed in vivo, both demonstrating that ben-
zo[a]pyrene was well absorbed through skin (Moody et al., 1995).
Many of the in vitro/in vivo (human) comparisons have been
made with pesticides, including cypermethrin (Scott & Ramsey,
1987) and fluazifop-butyl (Ramsey et al., 1994). In addition, com-
parative studies in rats have shown the predictive ability of the in
vitro method (Scott et al., 1992; van Ravenzwaay & Leibold, 2004).
Beck et al. (1994) compared in vivo and in vitro data on dermal
absorption of hair dyes in studies with rats and pigs. The data were
from two laboratories; the studies had been performed under similar
conditions, but in general were spaced by several years, were per-
formed by different persons, and used different chamber systems.
The authors found that, despite these differences, the comparisons
were good between in vivo and in vitro results. Dick et al. (1997a,b)
investigated the dermal absorption of lindane from acetone and a
white spiritbased formulation in in vitro and in vivo studies using
human volunteers. The in vitro studies predicted the 40-fold
82
difference in absorption between the two applications observed in
the volunteers.
Jung et al. (2003) carried out in vitro and in vivo percutaneous
studies with catechol. Total in vitro skin penetration was 1.4-fold
higher than in vivo skin penetration. However, there were differ-
ences in conditions (e.g. the dorsal skin temperature of fuzzy rat was
35 C, and the in vitro studies were carried out at 32 C). There
were low recovery rates in the in vivo study compared with the in
vitro study.
In a study on the pesticide propoxur, the experimental condi-
tions were standardized with respect to dose, vehicle, and exposure
duration (van de Sandt et al., 2000). In vivo studies were performed
in rats and human volunteers, while in vitro experiments were
carried out in static diffusion cells using viable skin membranes (rat
and human), non-viable epidermal membranes (rat and human), and
a perfused pig ear model. In vivo and in vitro absorption were
compared on the basis of the absorbed dose after 4 and 24 h, the
maximum flux, the lag time, and the potential absorbed dose.
In human volunteers, it was found that approximately 6% of the
applied dose (150 g/cm
2
) was excreted via the urine (as the metab-
olite 2-isopropoxyphenol) after 24 h, while the potentially absorbed
dose (amount applied minus amount washed off) was 23 g/cm
2
. In
rats, these values were 21% and 88 g/cm
2
, respectively. Data
obtained in vitro were almost always higher than those obtained in
human volunteers. The potentially absorbed dose seemed to give the
best prediction of human in vivo percutaneous absorption. The
absorbed dose and the maximum flux in viable full-thickness skin
membranes corresponded well with the human in vivo situation
(maximum overestimation 3-fold); epidermal membranes overesti-
mated the human in vivo data by up to a factor of 8 (van de Sandt et
al., 2000).
The same group carried out a second study (Cnubben et al.,
2002) assessing the percutaneous penetration of the fungicide ortho-
phenylphenol using various in vitro approaches static diffusion
cells with viable full-thickness skin membranes of rats and humans,
non-viable epidermal membranes of rats and humans, and a perfused
pig ear model and compared the results with respective in vivo
Comparative Studies
83
rat and human volunteer data using standardized conditions (Figure
13). In viable full-thickness skin membranes, the amount system-
ically available and the potentially absorbed dose correlated reason-
ably well with the human in vivo situation. In contrast, the maximum
flux considerably underestimated the human in vivo situation. The
results obtained with epidermal membranes again overestimated
human in vivo absorption data (Cnubben et al., 2002).
Fig. 13. Factor of difference; compared with human volunteer data between
in vitro and in vivo skin absorption of [
14
C]ortho-phenylphenol based on the
systemically available amount (SA) at 4, 8, 24, and 48 h after a 4-h exposure
period of 120 g/cm
2
, the permeability coefficient (K
p
), and the potentially
absorbed dose (PA) (from Cnubben et al., 2002). [Reprinted from Regulatory
Toxicology and Pharmacology, Vol. 35, No. 1, N.H. Cnubben et al.,
Comparative in vitroin vivo percutaneous penetration of the fungicide ortho-
phenylphenol, Pages 198208, Copyright (2002), with permission from
Elsevier]
The extent of skin first-pass metabolism (Roberts & Walters,
5). In general, the skin first-pass metabolism is more extensive in
vivo than in vitro, as illustrated by methyl salicylate (Cross et al.,
1998). Hence, failure to recognize this first-pass effect in in vitro
studies can lead to an overestimate of the systemic exposure after
topical application in vivo.
In the EDETOX (undated) project, dermal absorption studies in
human volunteers were conducted in parallel with in vitro predic-
tions with human skin using the same dose, vehicle, and application
time for 50% 2-butoxyethanol/water (Wilkinson & Williams, 2002;
2
1
0
-1
-2
SA (4h)
F
a
c
t
o
r

o
f

d
i
f
f
e
r
e
n
c
e

(
l
o
g
)
SA (8h) SA (24 h) SA (48 h) Kp PA
Rat full skin
Rat epidermis
Human full skin
Human epidermis
Perfused pig ear
Rat
1998) can also vary between in vitro and in vivo studies (see chapter
84
Jakasa et al., 2004b). There was a close relationship between
absorption rate determined in vitro and that determined from in vivo
data (2.0 g/cm
2
per hour in vitro with dermatomed skin compared
with 2.7 g/cm
2
per hour determined from in vivo data).
There was also a good relationship between in vivo and in vitro
data for 50% butoxyethanol in the rat (2.0 g/cm
2
per hour com-
pared with 1.3 g/cm
2
per hour). A similar relationship was also
observed for the rat for di(2-ethylhexyl)phthalate, pyrene, and
benzo[a]pyrene, although the in vivo data were slightly underesti-
mated.
Although the above studies show more the exceptions to the
observation of a good correlation between in vitro and in vivo data,
there are many examples of studies in which in vitro and in vivo data
correlated well. The reason for the lack of correlation may be that
the experimental conditions may not have been correctly controlled
in the above examples, as they were conducted prior to the adoption
of OECD Test Guideline 428 (OECD, 2004c).
8.2 Inter- and intralaboratory variation in in vitro
percutaneous absorption methodology
As in vitro measurements of skin absorption are an increasingly
important aspect of regulatory studies, potential sources of inter- and
intralaboratory variations have to be investigated. Much effort has
been expended in comparing the different diffusion cells used to
measure percutaneous penetration, and it is generally accepted that
most diffusion cell systems provide comparable measurements of
skin absorption rates for a range of penetrants. It is well documented
that there is a large (natural) variation in skin permeability (South-
well et al., 1984). However, in addition to the known factors
which have been discussed in other parts of this document, there is a
likelihood of inter- and intralaboratory variation. A few studies have
investigated this.
The in vitro absorption of benzoic acid, caffeine, and testos-
terone representing a range of different physicochemical prop-
erties through human skin (nine laboratories) and rat skin (one
laboratory) was determined (van de Sandt et al., 2004). All
influencing percutaneous penetration (see Table 1 in chapter 2),
Comparative Studies
85
laboratories performed their studies according to detailed protocols
(dose, exposure time, vehicle, receptor fluid, preparation of mem-
branes, analysis), and each laboratory performed at least three
independent experiments (containing at least 57 replicates per
experiment) for each test chemical. The ranking of dermal absorp-
tion of all chemicals was the same for all participating laboratories.
There was variability between laboratories with respect to the total
absorption data (benzoic acid 22.5%; caffeine 40.9%; testosterone
64.3%; all percentages are coefficients of variation over six labora-
tories), most likely due, to a large extent, to interindividual variabil-
ity in absorption between samples of human skin and differences in
skin site and source. The intralaboratory variation was influenced by
the skin itself. Skin thickness only slightly influenced the absorption
of benzoic acid and caffeine; however, the maximum absorption rate
of the most lipophilic compound, testosterone, was clearly higher in
the laboratories using thin, dermatomed skin membranes (van de
Sandt et al., 2004).
In an international multicentre study involving 18 laboratories,
interlaboratory and intralaboratory variations in diffusion cell mea-
surements were determined using a silicon rubber membrane,
thereby excluding skin variability (Chilcott et al., 2005). The study
was performed using a standard penetrant, methyl paraben, and an
appropriate standard protocol. Standardized calculations of methyl
paraben flux were determined by a central laboratory from the data
submitted by each laboratory. The coefficient of variation between
laboratories was approximately 35%. There was a 4-fold difference
between the lowest and highest average flux values and a 6-fold
difference between the lowest and highest individual flux values.
Intralaboratory variation was lower, averaging 10% for five individ-
uals using the same equipment within a single laboratory.
Vecchia & Bunge (2003a) compared quality-controlled mea-
surements for human skin permeability coefficients from multiple
laboratories. The variation was between 1 and 2 orders of magni-
tude. A similar degree of variability has been shown for maximum
flux (Magnusson et al., 2004a).
These variations reinforce the need to conduct carefully con-
trolled in vitro and in vivo dermal absorption studies.
86
9. DATA COLLECTIONS
Over the decades, a large number of data have been generated
on the percutaneous penetration of a wide range of chemicals,
including pesticides, cosmetics, and pharmaceuticals. Studies have
included work on human volunteers and in vivo studies using animal
rodent, pig, guinea-pig, and, more recently, synthetic skin (see
agreed standard procedure for measuring dermal absorption. There
are numerous factors that can influence dermal penetration values,
such as species variation, application site, dosing regime, occlusion,
sex, and age, as well as interlaboratory and intralaboratory variations
While many of these studies on percutaneous penetration are
unpublished (being company or governmental property), there are a
number of studies in the open literature. There have been many
different compilations of data (e.g. Flynn, 1985; Johnson et al.,
1995; Wilschut et al., 1995; Patel et al., 2002; Vecchia & Bunge,
2003a; USEPA, 2004b). Under the auspices of the EDETOX
project, a database has been compiled giving details of studies on
percutaneous penetration. This database is freely available on the
EDETOX web site. Therefore, in the present document, it was not
attempted to mention every study, as these can be found easily in
9.1 Data sets from homologous or closely related
molecules
As well as studies on single compounds, several investigators
determined permeability data from homologous or closely related
modelling skin permeability in these series (Marzulli et al., 1965;
Scheuplein & Blank, 1971; Idson, 1975; Roberts et al., 1977;
Wester & Maibach, 1985; Idson & Behl, 1987; Ridout & Guy,
1988; Ridout et al., 1992). An overview of several of the combined
data sets is provided by Vecchia & Bunge (2003a,c). There has been
discussion about the quality of the steroid data from Scheuplein et
al. (1969) (Moss & Cronin, 2002; Moss et al., 2002).
this EDETOX database (http://www.ncl.ac.uk/edetox).
models (see chapter 7) and in vitro studies on excised human,
chapter 6). There has, until recently (OECD, 2004a,b,c), been no
(see Table 1 in chapter 2).
series of molecules (see Table 7). These data have been used for
Data Collections
87
Table 7. Permeability data sets for some homologous or closely related
series of molecules
Molecules References
n-Alkanols Scheuplein & Blank (1971, 1973); Flynn
& Yalkowsky (1972); Wiechers (1989)
Ethyl ether, 2-butanone, 1-
butanol, 2-ethoxyethanol, 2,3-
butanediol
Blank et al. (1967)
Steroids Scheuplein et al. (1969)
Hydrocortisone-21 esters (and
5'-vidarabine esters)
Flynn (1985)
Phenolic compounds Roberts et al. (1977)
Glycol ethers Dugard et al. (1984)
8 salicylates and 10 non-
steroidal anti-inflammatory drugs
in humans
Yano et al. (1986)
Hair dyes Bronaugh & Congdon (1984)
Metals Hostynek (2003)
Aromatic amines Levillain et al. (1998)
Para-substituted phenols Hinz et al. (1991)
PAHs Roy et al. (1998)
9.2 Flynn data set
A milestone in the development of percutaneous absorption
prediction was the publication of what is now known as the Flynn
data set. This is a publication of 97 permeability coefficients for 94
compounds in vitro through human skin and for toluene, ethyl ben-
zene, and styrene in vivo in humans (Flynn, 1990) and was for over
a decade the largest database of skin permeability values. However,
this is a compilation of 15 different literature sources (some of these
are given in Table 7), with the inherent disadvantage of having a
high degree of variability due to interlaboratory and intralaboratory
error as well as variation due to the skin being from different sources
and locations in the body (Moss et al., 2002). Based on this data set,
Flynn and several others have proposed a number of algorithms to
predict skin permeability (see chapter 10 on QSARs).
88
9.3 Expanded permeability coefficient data sets
For modelling studies, a number of data sets were compiled
from various earlier publications. For example, in the study by
Wilschut et al. (1995), data were given on 123 measured permea-
bility coefficients of 99 different chemicals; Vecchia & Bunge
(2003a) presented a sizeable and diverse data set of 170 measure-
ments for 127 compounds covering relative molecular weights from
18 to 584 and log K
ow
values from 3.1 to 4.6; Patel et al. (2002)
collected a comprehensive data set containing 186 permeability
coefficients for some 158 structurally diverse compounds (from
Further, there are repositories of data kept by industry and regu-
latory bodies (e.g. pesticide data sets, held by the United States
Environmental Protection Agency [USEPA], of almost 300 dermal
absorption studies of more than 160 different pesticides; Reddy &
Bunge, 2002; Jones et al., 2004).
9.4 EDETOX database
The EDETOX database was generated within the EDETOX
project, a multipartner project funded under the European Unions
(EU) Framework V Programme (EDETOX, 2004, undated; see also
section A2.3 in Appendix A). The purpose of the database was to
bring together in vivo and in vitro percutaneous absorption and dis-
tribution data from all available sources, together with the physico-
chemical data for each chemical of interest (Soyei & Williams,
2004). The database contains over 4800 studies for 320 chemicals.
Studies entering the database were assessed as to whether they
satisfied the following EDETOX criteria:
chemical concentration (of chemical applied);
dose volume (volume of chemical applied to skin);
loading (amount of chemical added per unit area);
area (area of skin to which the chemical was applied);
vehicle (application medium);
species (species of animal used in the study);
exposure time (length of time the chemical was left on the skin);
human in vitro skin data) (see also chapter 10).
Data Collections
89
analytical method (method by which the results were deter-
mined);
receptor fluid (medium that bathes the underside of the skin);
and
temperature (temperature of the receptor fluid/skin/water bath
during in vitro experiments).
Initially, it was intended to include only papers that satisfied the
EDETOX criteria. However, very few did. Therefore, all papers
were entered, but those not fitting the criteria (well over 50%) were
highlighted as such (Soyei & Williams, 2004). The database can be
9.5 Maximum flux databases
There appear to be only a limited number of maximum flux
databases for solutes in human skin. Early databases for alcohols
and phenolic compounds were described by Scheuplein & Blank
(1973) and Roberts et al. (1977). Perhaps the first database for a
range of heterogeneous compounds from one vehicle (propylene
glycol) was described by Kasting et al. (1987). A maximum flux
database for 278 compounds has recently been described and is
available as supplemental material from the Journal of Investigative
suppinfo/5602287s1.html?url=/jid/journal/v122/n4/full/5602287a.ht
ml. The criteria for inclusion of these compounds are included in the
accompanying paper (Magnusson et al., 2004a).
accessed at http://www.ncl.ac.uk/edetox (EDETOX, undated).
Dermatology (online) at http://www.nature.com/jid/journal/v122/n4/
90
10. ESTIMATION/PREDICTION OF DERMAL
PENETRATION
There has been much interest in the potential to predict dermal
absorption and avoid unnecessary and costly in vitro and in vivo
testing. This is partly due to ethical difficulties with respect to
human and laboratory animal experiments and partly due to econ-
omic and time considerations as a result of increasing legislation in
the risk assessment of industrial chemicals (e.g. proposed new
European chemicals strategy: Registration, Evaluation, Authorisa-
tion and Restriction of Chemicals, or REACH) (see also Jones et al.,
2004).
QSARs are generally used to relate properties of chemicals to
biological effects or transport properties and are an observation of
the association between an outcome and the properties likely to
affect that outcome. A QSAR provides predictions of coefficients
needed to estimate absorption for untested chemicals. It is not an
expression of theoretical physical relationships (e.g. mass balance)
and is therefore complementary to mathematical transport models
describing dermal absorption. Mathematical models simulate the
sequence of partition and transport processes involved in dermal
chemical permeation through the skin (Fitzpatrick et al., 2004).
Mathematical modelling plays a key role in linking permeability
coefficient data obtained from tests under steady-state conditions
(i.e. infinite dose) to absorption estimates for finite dose applications
that are more typical of occupational exposure (i.e. non-steady-state
conditions) (Jones et al., 2004). However, it should be noted that, in
the simplest case, a parameter additional to the permeability
coefficient K
p
(i.e. the partition coefficient K
m
) is needed to model
absorption from finite doses (Scheuplein & Ross, 1974). In reality,
the nature of the finite dose, interfacial resistances, and the receptor
sampling conditions will also affect model estimations (Anissimov
& Roberts, 2001).
absorption (see chapter 4) and can predict the extent and rate of
Estimation/Prediction of Dermal Penetration
91
10.1 QSAR analysis
QSARs, when applied to estimating dermal permeability coeffi-
cients, are sometimes known as quantitative structurepermeability
relationships (QSPeRs or QSPRs). Recent overviews of QSPeRs for
permeation into human skin from water are given by Moss et al.
(2002), Vecchia & Bunge (2003a,b), Walker et al. (2003), Fitzpat-
rick et al. (2004), and Geinoz et al. (2004).
10.1.1 Prerequisites for QSPeR analysis
Ideally, the QSPeRs are related to the mechanism of dermal
3). Considering the passage of a chemical through the skin from
water, the stratum corneum is essentially a tightly bound lipophilic
layer, which interfaces with an aqueous vehicle. Therefore, descrip-
tors of hydrophobicity, molecular size, and possibly hydrogen
bonding (which may describe non-covalent interactions with skin
proteins) are of importance for the development of QSARs (Moss et
al., 2002).
QSPeRs are statistically derived linear and non-linear relation-
ships between the steady-state permeability of a compound, usually
measured from water, and various physicochemical descriptors
and/or structural properties of the molecule. QSPeRs often are
derived using parameters that themselves are calculated from
QSARs. For example, the octanol/water partition coefficient K
ow
has
often been used as an input parameter to QSPeR equations. While
K
ow
has been measured for some chemicals, for others a QSAR pre-
diction is used, and these values will reflect the predictive limita-
tions of the QSAR, particularly when it is used outside its predictive
domain. Various software programs can provide differing estimates
for K
ow
and solute solubility.
The statistical nature of such QSARs means that the more data
used to derive a relationship, the more reliable the QSAR is likely to
be for predictive purposes, assuming that the relationship is accept-
able from a statistical point of view and that the data themselves are
deemed to be sufficiently varied and of satisfactory quality. For a
QSPeR, the data (observed permeation) should be consistent, pro-
duced from standardized experimental procedures, and obtained for
penetration, which depends on the structure of the skin (see chapter
92
a set of chemicals that cover the domain of relevant chemical prop-
erties (Jones et al., 2004).
The dermal absorption measurement that has been most widely
used in QSAR modelling is the permeability coefficient K
p
, because
it characterizes the steady-state permeation rate of a chemical from a
specific vehicle through a given membrane. Although K
p
is not
directly suitable for application in risk assessment, it can be used in
conjunction with measured (or estimated) solubility in the same
vehicle as was used for the K
p
measurement (usually water) to
predict a maximum flux through the skin (Jones et al., 2004). Also,
as stated above, it can be combined in mathematical models with
partition coefficient values for the skin (either from measurements or
from QSAR predictions) to estimate non-steady-state or finite dose
absorption.
The permeability coefficient K
p
of a substance through the
stratum corneum is given by:
K
p
= K
m
D / h
where h is the apparent thickness of the stratum corneum, D is the
effective permeant diffusivity in the stratum corneum, and K
m
is the
partition coefficient between the stratum corneum and the vehicle.
The partition coefficient from an aqueous vehicle, K
m
, has been
shown to be related to K
ow
(Cleek & Bunge, 1993; Roberts et al.,
1996; Vecchia & Bunge, 2003c). In reality, the stratum corneum is
heterogeneous, and the differential partitioning into protein and lipid
components (Raykar et al., 1988) should be recognized, as this
better determines solute permeation through the stratum corneum
(Anderson & Raykar, 1989; Nitsche et al., 2006; Wang et al., 2006).
10.1.2 Historical overview
10.1.2.1 QSPeRs for skin permeability prior to the 1990s
The majority of these older studies were based on the analysis
of homologous, or closely related, series of molecules, and often
only a relatively small number of compounds were assessed (see
increased in a linear relationship with hydrophobicity (Scheuplein &
Blank, 1971; Roberts et al., 1977). Some studies also reported a
[Equation 4 in chapter 4]
chapter 9). Many of these studies revealed that skin permeability
93
parabolic relationship with hydrophobicity, particularly if there was
a selection of compounds that included those that were highly
hydrophobic (Scheuplein & Blank, 1971). However, each model
existed in isolation for a particular class or series of compounds.
Further, as a consequence, there was co-linearity between the
descriptors for a congeneric series for example, hydrophobicity
and molecular size so that it is not possible to distinguish
between the effects of these two factors on the permeability of large
hydrophobic molecules (Moss et al., 2002).
10.1.2.2 The Flynn (1990) data set and subsequent analyses
Flynn (1990) proposed a number of algorithms to predict K
p
,
which stated that very hydrophilic and very hydrophobic compounds
had low and high skin permeability, respectively, and that different
K
ow
-dependent QSARs could be used to predict skin permeability
for high and low molecular weight compounds. Potts & Guy (1992)
demonstrated the use of log K
ow
in combination with either molec-
ular weight (MW) or molecular volume to predict K
p
data (in units
of cm/h) collected by Flynn (1990):
Log K
p
= 0.71 log K
ow
0.0061 MW 2.44 [Equation 6]
n = 93; r
2
= 0.67; s not reported; F not reported
where n is the number of observations or number of compounds, r is
the correlation coefficient, s is the standard error of the estimate, and
F is Fishers statistic.
Potts & Guy (1992) did not perform a full statistical analysis on
the data set. Although both descriptors used in the above equation
are statistically significant, the statistical fit to Equation 6 is compar-
atively poor (Moss et al., 2002). Potts & Guy (1992) did observe
that up to a 30% variability in the experimental data was to be
expected; however, they did not investigate the relationship for out-
liers or other statistical anomalies.
The publication of Flynns (1990) large heterogeneous data set
was a significant milestone and was subsequently the basis for
sion in Moss et al., 2002).
several analyses and publications (see Table 8 and detailed discus-
94
Table 8. A selection of recent QSPeR models illustrating sample size
(number of compounds), correlation coefficient (r), and the source of the
data used
a
Model n r Experimental data source
Flynn (1990) 95 Flynn (1990)
Potts & Guy (1992) 93 0.67 Flynn (1990)
Lien & Gao (1995) 22 0.96 Flynn (1990)
Barratt (1995a) 60 0.90 Flynn (1990)
Potts & Guy (1995) 37 0.94 Flynn (1990)
Abraham et al. (1995) 46 0.96 Flynn (1990)
Kirchner et al. (1997) 114 0.32 Flynn (1990) + Health
Canada
Hostynek & Magee (1997) 20 0.80 Miscellaneous
Roy et al. (1998) 60 0.64 Roy et al. (1998)
Abraham et al. (1999) 53 0.96 Flynn (1990)
Gute et al. (1999) 60 0.67 Roy et al. (1998)
Cronin et al. (1999) 107 0.86 Flynn (1990) + Health
Canada
Dearden et al. (2000) 91 0.83 Flynn (1990)
Patel et al. (2002) 143 0.90 Flynn (1990), Wilschut et
al. (1995)
Vecchia & Bunge (2003a) 127 0.55 Flynn (1990) +
miscellaneous
a
From Fitzpatrick et al. (2004) and Vecchia & Bunge (2003a).
Reviews of modelling the skin permeability of homologous, or
closely related, compounds are given by Scheuplein & Blank
(1971), Idson (1975), Wester & Maibach (1985), Idson & Behl
(1987), Ridout & Guy (1988), and Ridout et al. (1992). USEPA
(1992) and USEPA (2004b) present a slightly modified version of
the Potts & Guy (1992) correlation, which includes an adjustment
for the transport resistance through the viable epidermis, as pro-
posed by Cleek & Bunge (1993). USEPA (2004b) also presents the
effective prediction domain for this correlation i.e. the range of
K
ow
values and molecular weights for which this equation is valid
(see Exhibit A-1 of that document).
A larger database of 114 skin permeability values was prepared
by Kirchner et al. (1997) from the Flynn (1990) data set (51
chemicals), together with additional data from regulatory reports
95
from Health Canada. However, data for 56 of the additional 63
chemicals were calculated using Equation 6 and were not experi-
mental data (Poda et al., 2001; Frasch & Landsittel, 2002; Walker et
al., 2003). Not realizing this, Cronin et al. (1999) analysed the
Kirchner et al. (1997) database. After removing seven significant
outliers (large compounds, such as estriol, atropine, hydrocortisone,
etorphine, and digitoxin, as well as compounds with more than 10
sites to accept or donate a hydrogen bond), they analysed the
database against a wide variety of QSAR parameters, including
those for hydrogen bonding and other molecular properties. Not
surprisingly, given the origins of nearly half of the data, the equation
they derived, shown below, was similar to Equation 4:
Log K
p
= 0.77 log K
ow
0.0103 MW 2.33 [Equation 7]
n = 107; r
2
= 0.86; s = 0.39; F = 317
Because it is based on a number of calculated rather than experi-
mental data, Equation 7 should not be used (Frasch & Landsittel,
2002).
The problem of identifying and dealing with outliers is a contro-
versial issue. From the various studies (Pugh & Hadgraft, 1994;
Barratt, 1995a; Cronin et al., 1999), some compounds are found
consistently as outliers. Reinvestigation of some of the values by
Moss et al. (2002) and Moss & Cronin (2002) e.g. permeability
coefficients for steroids from Scheuplein et al. (1969) compared
with measurements from Johnson et al. (1995); and permeability
coefficients for diclofenac, naproxen, atropine, and nicotine com-
pared with measurements from Degim et al. (1998) showed that
at least some of the data in the Flynn (1990) data set may have
considerable error associated with them. This finding justified the
exclusion of these compounds, for example, by Cronin et al. (1999),
to produce a statistically valid model. This issue, in particular con-
cerning steroid permeabilities, is extensively discussed in Moss et al.
(2002).
Patel et al. (2002) collected a comprehensive data set containing
186 permeability coefficients for some 158 structurally diverse
compounds from human in vitro skin data from Flynn (1990) and
Wilschut et al. (1995). They removed some compounds (atropine,
diclofenac, naproxen, nicotine) that were considered as outliers from
96
the data set and developed a QSPeR that gave a value of r
2
= 0.90
for the remaining 143 compounds:
Log K
p
= 0.652 log K
ow
0.00603 MW 6.23 ABSQon
0.313 SsssCH 2.30 [Equation 8]
where ABSQon is the sum of absolute charges on oxygen and
nitrogen atoms and SsssCH is the sum of E-state indices for all
methyl groups.
The authors also fitted these data with a QSPeR of the general
form:
Log K
p
= a log K
ow
b(molecular size) + c [Equation 9]
Although calculated estimates of molecular volume have pre-
viously been shown to be better than molecular weight for the
prediction of skin permeation (Barratt, 1995a; Potts & Guy, 1995),
Patel et al. (2002) found molecular weight to be a better predictive
model; it also has the advantage that it is an easier descriptor to
obtain and apply. However, it must be recognized that the data set
they examined is dominated by hydrocarbons with relatively con-
stant values in the ratio of molecular volume to molecular weight.
For compounds with ratios of molecular volume to molecular weight
that deviate from the average of this data set, equations based upon
molecular volume should provide better estimates.
Fitzpatrick et al. (2004) reanalysed the same data set as that of
Patel et al. (2002) and discussed variability in the database. In
addition to problems associated with the K
p
data themselves, they
asserted that there may be problems in using partition coefficient
values for ionizable compounds determined consistent with guide-
lines (e.g. OECD Test Guideline 117; OECD 2004d) that require pH
adjustment to prevent ionization, which may require conditions that
are outside the normal physiological range encountered in trans-
dermal transport (see also ONeill & Fitzpatrick, 2004). As long as
permeation of the ionized species is much smaller than that of the
non-ionized species, then permeation will be primarily by the non-
ionized species (Vecchia & Bunge, 2003a). This topic of ionizable
compounds is also discussed in chapter 4.
97
Details of all the recent QSPeR models are beyond the scope of
the sources of data used. A more comprehensive listing is given in
Vecchia & Bunge (2003a), which includes a comparison of several
10.1.2.3 Other data sets
A few QSAR studies have been performed on smaller data sets
that were not part of the Flynn (1990) data set. Several of these are
based on congeneric series of compounds, typically drugs, and
assess their permeation rates. As the emphasis of this document is on
chemical risk assessment, these smaller studies are not described in
detail here.
Several studies have investigated the percutaneous absorption
of PAHs. That of van Rooij et al. (1995) was based on 10 PAHs. A
more extensive study (Roy et al., 1998) included 60 PAHs, all of
which were extremely hydrophobic (lowest K
ow
being 4.00). A
negative correlation was found between the percentage of applied
dose that penetrated the rat skin in vitro after 24 h and hydro-
phobicity. The data were reanalysed by Gute et al. (1999), who
modelled molecular weight alone rather than K
ow
against percuta-
neous absorption of PAHs, probably because molecular weight and
K
ow
are highly correlated for these compounds.
10.1.3 Other approaches to QSPeR
Hostynek & Magee (1997) correlated human skin absorption
data determined in vivo under different experimental conditions for
a set of 28 diverse chemical compounds with experimental permea-
bility coefficients obtained for 8 compounds in vitro on human skin
and with permeability coefficients calculated according to a modi-
fied PottsGuy algorithm. They suggested that both the vehicle in
which the chemical is delivered and the degree of occlusion of the
device were important. The most significant correlation was seen for
a subset of 10 semi-occluded applications of different compounds.
additional QSPeR models with data.
this document. Table 8 gives an overview of some of the studies and
98
10.1.4 Variability of data and its relevance for QSPeRs
Taking the values for estradiol reviewed by Johnson et al.
(1995) excluding that from Scheuplein et al. (1969), which has
been questioned (Moss & Cronin, 2002; Moss et al., 2002) there
is a variability of about 25% in permeability coefficients, which, due
to the inherent variability of the tissue and interlaboratory differ-
ences (and temperature differences), is tolerated by those working in
the field. For deriving QSPeRs, however, such variance is detri-
mental, not just to the development of a robust and biologically
relevant model, but also to its credibility and value in the question of
predictability of skin permeability for other compounds (Moss et al.,
2002). Permeability coefficients will differ from one solvent to
another, depending upon the solubility of the penetrant and also the
effect of the solvent on the skin. Nearly all of the published QSPeRs
are for compounds in water, chiefly because these are the data that
are available to model. As a result, the nature of drugvehicle inter-
actions and the choice of solvent are not considered by the existing
QSPeRs (Walters & Brain, 2000). Strategies for making vehicle-to-
vehicle adjustments have been proposed (e.g. USEPA, 1992), but
more experimental studies supporting these methods are needed.
10.1.5 Statistical analysis (linear vs non-linear) methods
For many QSARs, including QSPeRs, regression analysis is the
statistical method of choice, being simple, transparent, and highly
portable (Cronin & Schultz, 2001). However, there are a number of
disadvantages to using this method: in particular, firstly, the linear
technique, and secondly, that it is adversely affected by collinearity
between independent variables (e.g. log K
ow
and molecular weight).
It is not clear whether regression analysis is a suitable technique for
the development of QSPeRs; nor is it clear whether linearity is
appropriate for modelling of highly hydrophilic and hydrophobic
molecules. Other possibilities would be partial least squares and
artificial neural network modelling (Degim et al., 2003).
10.1.6 Selection of chemicals for further tests on dermal penetration
Ideally, for development of QSARs, the process of selection of
chemicals should include a chemometric analysis to ensure that
those compounds provide the maximum possible information. If the
physicochemical descriptors for the QSPeR are restricted to log K
ow
99
and molecular weight, then selection of chemicals is easier than for a
multivariant system. The current database must be critically ana-
lysed to decide how representative it is and what deficiencies it has
(Jones et al., 2004). The European Chemical Industry Council
(CEFIC) Working Group (and others before them) noted that there
may not be a linear relationship between permeability coefficient
and hydrophobicity for the complete range of log K
ow
values. Polar
compounds with low K
ow
values may penetrate skin by pathways that
are not represented by K
ow
. At high K
ow
values, the viable epidermis
will restrict permeation. This is part of the rationale behind recom-
mendations of EC (2004) that a reduced default absorption be
assumed when log K
ow
is either less than 1 or greater than 4.
However, this recommendation does not account for the effect of
K
ow
flux (e.g. the product of K
p
from water and the water solubility) may
be a more suitable indicator for dermal absorption potential.
10.1.7 Applicability domain for QSPeR
The applicability domain for QSAR is defined as the physico-
chemical, structural, or biological space, knowledge or information
on which the training set of the model has been developed, and for
which it is applicable to make predictions for new compounds
(Jaworska et al., 2003). At present, no formal methods exist to
define such an applicability domain. However, it is accepted practice
that a QSAR should not be used to make predictions outside of its
applicability domain (Cronin & Schultz, 2001). If a global QSPeR
can be based on log K
ow
and molecular weight, then an applicability
domain may be defined relatively easily and may be shown graph-
ically on a two-dimensional plot of log K
ow
and molecular weight,
which will probably be elliptical in shape, due to the paucity of low
molecular weight molecules that are hydrophobic. The CEFIC
Working Group recommended that the applicability domain should
be defined for any QSPeR developed and that all predictions should
be made only for chemicals within the applicability domain (Jones et
al., 2004).
10.1.8 Maximum fluxes
Maximum flux QSPeRs are of particular value, as they apply to
all vehicles that do not affect the skin adversely. A prediction of
on water solubility, and, as discussed in chapter 4, maximum
100
fluxes less than the maximum flux can be made on the basis of
concentrations being expressed in terms of fractional solubilities for
a given vehicle (Roberts et al., 2002). Maximum flux QSARs are
generally based on the premise that transport through the stratum
corneum can be related to the solubility of solutes in the stratum
corneum, the diffusivity of solutes through the stratum corneum, and
the presence of any interfacial resistances (Kasting et al., 1987;
Magnusson et al., 2004a). While the fuller expression of the QSAR
with molecular weight and log K
ow
is intrinsically non-linear, the
resulting regression is only marginally better than a linear relation-
ship between log of the maximum flux (mol/cm
2
per hour) and
molecular weight (P < 0.001, n = 278, r
2
= 0.688) (Magnusson et al.,
2004a):
Log J
max
= 4.52 0.0141 MW [Equation 10]
10.1.9 Rules as an alternative to QSPeRs
QSARs can also be analysed using principal components
analysis and related techniques. Such an approach has been used to
broadly model skin permeability and cytotoxicity and to discrim-
inate between corrosive and non-corrosive chemicals (Barratt,
1995a,b). Skin penetrants have also been classified as good,
bad, or intermediate, based on rules defined by the solutes
physicochemical properties (Magnusson et al., 2004b). Such rules
are more commonly used to predict the likely skin toxicity of
compounds (e.g. Gerner et al., 2004).
10.2 Mathematical modelling
Mechanistically based models relate dermal absorption to
physicochemical parameters, such as diffusion coefficients and
partition coefficients, that depend on the diffusing chemical and the
vehicle. These parameters can be derived from experiments or
estimated using QSAR equations developed from experimental
measurements. In the simplest models, skin is treated as a single
pseudo-homogeneous barrier, represented as either a membrane or a
stirred compartment. More complex models include additional skin
layers, such as the viable epidermis and/or dermis.
101
Membrane-based mathematical models have been developed for
calculating the rate and extent of dermal absorption for a variety of
exposure situations (Roberts & Anissimov, 2005). Cleek & Bunge
(1993) described one- and two-membrane models for the skin that
represent the stratum corneum alone and in combination with the
viable epidermis. In these models, it was assumed that the chemical
concentration in the vehicle remained constant during the exposure.
Bunge and colleagues (Cleek & Bunge, 1993; Bunge & Cleek,
1995; Bunge et al., 1995; Bunge & McDougal, 1999) also proposed
strategies for estimating the physicochemical parameters that these
models require when the vehicle is water. These models were
included in documents by the USEPA (1992, 2004b). Reddy et al.
(1998) extended these models to include the loss of chemical in the
skin at the end of exposure by desquamation of the stratum corneum.
In this model, they included the barrier effect of the viable epidermis
and again assumed that the chemical concentration did not change
during the exposure. This desquamation is likely to affect the
stratum corneum reservoir effect for only the most lipophilic solutes
(Roberts et al., 2004).
The penetration of a volatile compound or mixture from the
skin surface after topical application involves both diffusion in the
stratum corneum and evaporation from the vehicle. Kastings group
(Kasting & Saiyasombati, 2001; Saiyasombati & Kasting, 2003;
Kasting & Miller, 2006) have described this process and developed
appropriate models. Stempfer & Bunge (2004) have also presented a
one-membrane model of the stratum corneum, allowing for evapora-
tion of absorbed chemical. They showed that for volatile com-
pounds, a significant fraction of the chemical in the stratum corneum
at the end of the exposure will probably evaporate. For semi-volatile
compounds, the amount that would evaporate will depend on both
the vapour pressure (which affects evaporation rate) and the absorp-
tion rate through the skin.
In the Skinperm model for prediction of the permeation of
substances (Skinperm 3.1 model; Wilschut et al., 1995; ten Berge,
2005), the skin was considered to consist of the following com-
partments: protein layer of the stratum corneum, lipid layer of the
stratum corneum, and aqueous layer below the stratum corneum.
The overall permeation coefficients were expressed mathematically
as a complex of permeation coefficients, representing the
102
permeation through these layers. The regression coefficients were
derived from the experimental results and were based on the
database established by Wilschut and co-workers. The values of the
regression coefficients in the model were: b1 = 1.736; b2 = 0.7219;
b3 = 0.059 93; b4 = 0.000 297 6; and b5 = 4.209. For the
permeation coefficient, the 95% confidence limits were provided.
These 95% confidence limits were based on the original variances
and covariances of the derived regression equation of the QSAR
predicting the permeation coefficient, dependent on the
octanol/water partition coefficient and on the molecular weight. The
model predicts, among other parameters, the cumulative amount
absorbed into the skin (ABS
skin
) and K
p
. In order to calculate the
time-weighted average skin flux (J
skin, average, model
), the value of
ABS
skin
is divided by the exposure concentration, C
exp
, the exposure
duration, t
exp, der
, and the exposed skin area, A
skin
. An example of the
use of this model with xylene is given in Kezic et al. (2004).
The recent modelling efforts by Krse & Kezic (2004) allow for
variation of concentration in an aqueous vehicle and include barrier
contributions from both the stratum corneum and viable epidermis.
Like Bunge and colleagues, Krse & Kezic (2004) have assumed
local equilibrium at the waterstratum corneum and stratum
corneumviable epidermis interfaces, described by partition coeffi-
cients and diffusion coefficients in each layer. The model is based
on Ficks law of diffusion within each layer (and based on a
diffusion coefficient for the chemical in each layer). The model has
more parameters (to be fitted), but it describes the time course of
permeation more thoroughly, and it is in principle more appropriate
for predicting the consequences of non-steady-state doses. It has
been fitted to data describing the time course of permeation of
chemical into the skin in vitro.
The rate-limiting step of permeation is often diffusion through
the stratum corneum. However, for some compounds (highly
lipophilic compounds) that have a much higher solubility in the skin
than in water (e.g. by a factor of 1000), the diffusion within the
aqueous solution towards the skin surface may become a rate-
limiting factor (Kasting & Robinson, 1993).
Mathematical modelling plays a key role in linking the permea-
bility coefficient obtained from tests under idealized, infinite dose
103
conditions (i.e. steady-state conditions) to those that will occur
under the finite dose conditions more typical of occupational expo-
sure (i.e. non-steady-state conditions).
Mathematical modelling has also been used to estimate dermal
absorption of contaminants from, for example, water and soil.
USEPA (1992) presents a two-compartment diffusion model for
estimating dermal absorption of contaminants from water. USEPA
(2004b) presents a modification to this model that accounts for
losses of lipophilic compounds, which are absorbed in the stratum
corneum and lost via desquamation.
Mathematical models have also been described for predicting
dermal absorption from chemicals. USEPA (2004b) presents the
traditional per cent absorbed approach for estimating dermal absorp-
tion from soils. More mechanistic models are described in Bunge &
Parks (1998).
10.3 Mathematical pharmacokinetic models of
percutaneous penetration
Recent reviews on mathematical pharmacokinetic models of
percutaneous penetration are given by Roberts et al. (1999),
McCarley & Bunge (1998a,b, 2001), Reddy et al. (1998), and
Roberts & Anissimov (2005). The rate constants in compartmental
pharmacokinetic models can be related to the physicochemical
parameters, such as diffusion coefficients and partition coefficients
of the stratum corneum and additional skin layers. However, espe-
cially in a one-compartmental representation of the skin, these
relationships require simplifying assumptions, which are sometimes
not fully stated. McCarley & Bunge (1998a,b, 2000, 2001) and
Reddy et al. (1998) have derived several different pharmacokinetic
rate constants based on different assumptions applied in compart-
mental models. They have also showed that calculations made with
these different rate constants can sometimes produce significantly
different results that are not consistent with membrane models of
skin, which the compartmental pharmacokinetic models are meant to
simulate. Reddy et al. (1998) have presented model calculations that
include concentration variation in the vehicle and blood when the
stratum corneum is the rate-limiting barrier. Additional calculations
104
made by Roberts et al. (1999) have validated and extended this
analysis.
In such models, the algebraic equations that accurately represent
the partition processes and diffusion migration of a molecule
through the different layers of the stratum corneum are written down
and solved within certain chosen simplifying assumptions (Fitzpat-
rick et al., 2004). Such a set of equations has been solved ana-
lytically for both steady-state and non-steady-state transport through
a two-membrane composite representing the lipophilic stratum cor-
neum and the hydrophilic viable epidermis layers (Cleek & Bunge,
1993). Recently, Wang et al. (2006) have described a two-dimen-
sional microscopic model of the stratum corneum, recognizing vary-
ing hydration and permeability of corneocytes. A key outcome of the
model is a mechanistic description of the dependency of the stratum
corneum permeability coefficient on the octanol/water partition
coefficient and other parameters.
Estimates of dermal absorption are used in exposure assessment
to calculate the internal dose of persons contacting, for example,
pesticides and are a critical part of risk assessments. An exponential
saturation model with a lag time was validated against a classic
dermal absorption study of 12 pesticides administered to human
volunteers. The model gave dermal absorption estimates consistent
with reported values in the literature (Thongsinthusak et al., 1999).
In occupational exposure to toxic chemicals, dermal absorption
may result from multiple short-term exposures as well as from long-
term exposures. Corish et al. (2004) describe the application of two
diffusion-type numerical models of percutaneous penetration to non-
steady-state time course data generated by participants in the
EDETOX project. The first is a new numerical model developed by
J. Krse (Krse & Kezic, 2004) and implemented using the
Anissimov & Roberts (1999, 2000, 2001) (see also Roberts et al.,
1999; Roberts & Anissimov, 2005). Both models allow finite and
infinite doses to be modelled.
Berkeley Madonna package (http://www.berkeleymadonna.com).
The second is an implementation using Mathematica (http://
www.wolfram.com) of an existing pair of models developed by
105
11. USE OF DERMAL PENETRATION STUDIES IN
RISK ASSESSMENT
Risk assessment is a process by which the extent of exposure is
compared against the hazard (intrinsic toxicity) of the chemical to
determine whether it is likely to result in harm to the exposed
individual(s). Exposure to a chemical can be by oral, inhalational, or
dermal routes; however, in an occupational and many environmental
and consumer settings, the latter two are the major routes.
To determine the hazard or intrinsic toxicity of a chemical, a
comprehensive array of toxicity tests is performed, from which the
critical effect and a no-observed-adverse-effect level (NOAEL)
are derived. An uncertainty factor (sometimes called a safety factor),
which is chosen in recognition of intra- and interspecies variability
(maximum 10-fold for each) and the adequacy of the toxicological
database (IPCS, 1994), is applied to the NOAEL, to give a guidance
value. Alternatively, a margin of safety of exposure can be cal-
culated for a specific scenario by comparing the NOAEL with the
actual exposure conditions.
Since most laboratory animal testing is by oral administration,
but the predominant route of exposure for pesticides, for example, is
by skin absorption, the extent of dermal absorption needs to be
determined to perform an occupational risk assessment (route-to-
route extrapolation).
Although the scope of this document is focused on dermal
absorption assuming normal healthy skin, it should be recognized
that both the intrinsic local toxicity of the chemical and the skin
condition can influence the degree to which the chemical is absorbed
Skin irritation, sensitization, and phototoxicity are part of the
formal risk assessment process for local skin effects. These effects
most commonly occur after acute or short-term exposure. Personal
protective equipment (e.g. gloves/coveralls) is recommended when
appropriate.
(see chapters 3 and 12).
106
Assessment of dermal absorption is an important aspect of the
overall risk assessment of chemicals coming in contact with the skin.
One reason is that inhalation exposures to chemicals have decreased
as a result of improved control technologies and reduction of occu-
pational exposure limits (OELs), thereby increasing the contribution
of dermal exposure. In certain scenarios, dermal exposure may be
greater than respiratory exposure, and intoxications due to skin
exposure have been documented. The skin is not an almost imper-
meable barrier to chemicals, as was originally thought. Now there
are many databases describing skin permeation of chemicals (see
the risk assessment of pesticides, biocides, cosmetics, pharmaceu-
ticals, and industrial chemicals. The potential exposure can occur
during the manufacturing process, transport, and the end use of
ronment.
For pesticides, biocides, cosmetics, and industrial chemicals,
the internal exposure is calculated on the basis of relative absorption
(percentage of dose applied) under realistic exposure conditions
(e.g. area dose and formulation). Although conventional risk assess-
ments are usually not performed for pharmaceuticals because of the
benefit derived through deliberate dosing, the exposure can be cal-
culated using plasma AUC, excretion data, and pharmacological
effect data. The peak exposure is defined by the maximum plasma
concentration, whereas total exposure is defined by the AUC. Other
methods of calculation of internal dose include steady-state flux
estimations and biologically based modelling (McDougal &
Boeniger, 2002). At present, these methods are not widely applied in
risk assessment, with the exception of dermal exposure to large
amounts of chemicals e.g. incidents at work and contaminated
(swimming) water (Walker et al., 1996; Moody, 1998).
11.1 Decision-making process for setting dermal
absorption values
There are various ways to determine the actual skin absorption
of a substance, and the methodology for this has been described in
previous chapters (in vitro experimentation, in vivo animal studies,
and studies with human volunteers). In addition, order-of-magnitude
estimates of dermal absorption can be made by considering data on
products. In addition, exposure to chemicals can occur in the envi-
chapter 9). Determination of dermal absorption is a key element of
Use of Dermal Penetration Studies in Risk Assessment
107
physicochemical properties of the substance, such as molecular
weight and log K
ow
ment may vary depending on the chemical or product area, and
guidance has been developed by various regulatory agencies. This
section provides an overview of the current practices.
11.1.1 Default values
In the absence of any experimental dermal absorption data, one
should assume the worst-case scenario, in which 100% of the
chemical at a relevant dose will be absorbed. If this initial, very
conservative, risk assessment indicates that the exposure level is
acceptable/tolerable, a quantitive estimate of dermal absorption is
not required. Otherwise, an assessment of dermal absorption is the
next step.
In Europe, regulatory authorities involved in risk assessment are
using a rule based on molecular weight and log K
ow
to distinguish
between chemicals with high and low potential for dermal absorp-
tion (EC, 2003, 2004):
10% dermal absorption for those chemicals with a molecular
weight > 500 and log K
ow
smaller than 1 or higher than 4;
otherwise assume 100% dermal absorption.
The reason for assuming 10% as the lower limit was that the data
presented in the literature indicated the occurrence of dermal
absorption for tested compounds even beyond the extremes of log
K
ow
and/or molecular weight values (EC, 2003, 2004). However, by
expert judgement, a deviation from these 10% or 100% values can
be chosen on a case-by-case basis, taking into account all the data
available (e.g. water solubility, ionogenic state, molecular volume,
oral absorption, and dermal area dose in exposure situations in
practice). Appropriate values for oral absorption can be obtained by
absorption, distribution, metabolism, and excretion (ADME) studies,
using low dose levels.
[Comment of the IPCS Task Group: The validity of using the
physicochemical properties to obtain the default criteria is unclear.
A preferable method may be to use dermal absorption rates based on
the QSAR between maximum flux and molecular weight or rules for
(chapter 10). The use of these data in risk assess-
108
recognized that this rate will usually be an overestimate of dermal
absorption, as maximum flux does not take into account lag time and
relevant dosing conditions.]
In Europe, results from acute toxicity studies (dermal and oral)
are not considered suitable for estimation of skin absorption (EC,
2004). Acute doses, especially those using gavage administration,
result in relatively fast oral absorption, resulting in a peak concen-
tration in the body, whereas absorption after dermal exposure is
generally more gradual. Differences in toxicity between oral and
dermal exposure could be the result of different first-pass effects
in the liver and the skin. The North American Free Trade Agreement
Technical Working Group on Pesticides agreed that in the absence
of an acceptable in vivo dermal absorption study, dermal absorption
and absorption via the oral route would be considered equivalent
(i.e. up to 100%) (Hakkert et al., 2005). The California Department
of Pesticide Regulation uses a default of 50% for regulatory pur-
poses based on an upper-bound estimate of dermal absorption of 40
pesticides, which were obtained from studies in rats (Donahue,
1996).
For the occupational risk assessment of pesticides in Australia,
a default dermal absorption value of 100% is usually used in the
absence of any chemical-specific experimental data. However, this
value may be reduced if there are suitable dermal absorption data
from a related chemical (i.e. same class). Other factors include a
consideration of the molecular size or a comparison of oral and
dermal repeated-dose toxicity of the substance (APVMA, 1997).
11.1.2 Measured values
The most reliable data for determining absorption through
human skin are obtained from in vivo human volunteer studies
performed under occupationally relevant test conditions (Ross et al.,
2005). These could be both controlled volunteer studies and bio-
monitoring studies. In addition, some cosmetic ingredients have
been studied under controlled in-use conditions (e.g. Nohynek et al.,
2006). For technical and ethical reasons, the conduct of these studies
is limited and closely regulated.
good, bad, and intermediate permeants (see chapters 4 and 10). It is
109
Recently, OECD test guidelines for in vivo (OECD Test
Guideline 427) and in vitro (OECD Test Guideline 428) dermal
absorption studies have been adopted (OECD, 2004b,c) to encour-
age a harmonized approach in their conduct. In addition, criteria on
the performance of in vitro studies have been developed in the
cosmetics area (SCCNFP, 2003b). In all guidelines, it is emphasized
that it is essential that studies reflect the anticipated exposure situa-
tion (e.g. dose, formulation, exposure time). It should be realized
that the data on external exposure are often measured or estimated
averages over the total exposed area. Since skin absorption is
dependent on the applied dose, improved accuracy of exposure data
will lead to more realistic skin absorption data.
In animal studies, absorption is determined on the basis of the
excretion profiles and the amount in the carcass of the laboratory
dose is critical for the calculation of the dermal absorption in in vivo
studies (Chu et al., 1996; EC, 2004). The decision about the quantity
that remains bound in the skin can be based on the excretion curve
a decline of radioactivity in the excreta at the end of the experi-
ment indicates that the dose at the application site may not become
completely systemically available (Thongsinthusak et al., 1999; see
In vitro dermal absorption studies on pesticides have been used
for risk assessment purposes for more than 20 years within the EU
under Directive 91/414. Other regulatory authorities are increasingly
accepting in vitro data as part of the risk assessment process.
Despite the fact that in vitro studies have been shown to be in gener-
there is still controversy over how in vitro data are used in risk
assessment. An important issue of concern is the presence of (lipo-
philic) test substances remaining in the skin at the end of the experi-
ment. Provided that skin levels are included as absorbed, results
from in vitro methods are considered adequate for risk assessment
(in the absence of in vivo data). However, the fate of the compound
remaining in the skin is important and may need be to be determined
in additional measurements (see section 6.4 and chapter 12).
In vitro dermal absorption studies are preferably carried out
with human skin preparations; pig skin is considered a suitable
animal at the end of the experiment (see chapter 7). The skin-bound
chapters 7 and 12).
ally good agreement with in vivo dermal absorption (chapter 8),
110
alternative. In vitro studies with rat skin can be used, but this leads
to a more conservative estimate, as absorption by human skin is
usually lower than that by rat skin (EC, 2004). If appropriate dermal
absorption data are available from rats in vivo and for rat and human
skin in vitro, the in vivo human absorption value can be calculated
according to the following equation (EC, 2004; van Ravenzwaay &
Leibold, 2004):
In vivo human absorption =
in vivo rat absorption in vitro human absorption
in vitro rat absorption
11.1.3 Values from mathematical skin permeation models (e.g.
QSARs/QSPeRs)
As the present QSARs are based on absorption data from
chemicals in aqueous solutions at infinite dose, it is important that
methods are developed to apply the data to realistic exposure
scenarios (low dose, formulated substances, repeated exposure,
mixtures with potential cumulative or synergistic effects). The use of
QSARs may prove to be a useful screening tool and may prove
valuable for a group of closely related pure substances (EC, 2003;
Jones et al., 2004) for predicting dermal absorption potential (see
11.2 Use of relative absorption values versus flux (and
their derived permeability coefficients)
Dermal absorption of chemicals is most often expressed as a
percentage of the dose that is in contact with the skin. Until now,
fluxes (and the derived permeability coefficient values) have not
been incorporated in occupational risk assessment in the United
States, but are used to estimate the degree of an acute exposure from
a large amount/volume of a chemical (incidental splash, contam-
inated swimming water) (Walker et al., 1996). However, the USEPA
is promulgating a final rule under the Toxic Substances Control Act
(TSCA) that requires manufacturers, importers, and processors of 34
chemicals to conduct in vitro dermal absorption rate studies. These
chemicals are of interest to the Occupational Safety and Health
Administration of the United States Department of Labour, and the
chapter 10).
111
data will be used to evaluate the need for skin designations for these
chemicals (USEPA, 2004a).
Methods for conducting dermal risk assessments for the water
and soil pathways at Superfund sites are given in USEPA (2004b).
The document gives K
p
values for over 200 organic chemical con-
taminants in water, calculated using a mathematical model based on
absorption data from Flynns (1990) data set on 90 hydrophilic
cients for inorganic compounds, USEPA (2004b) presents a table
compiled from specific chemical experimental data as modified
from USEPA (1992) and Hostynek et al. (1998). The USEPA
(2004b) guidance document presents recommended default exposure
values for all variables for the dermalwater and dermalsoil path-
ways for Superfund sites. The modelling of dermal absorption from
soils and powders in vivo has recently been reviewed (Bunge et al.,
2005).
An approach for deciding whether to consider dermal absorp-
tion in a risk assessment is by comparing it with other pathways of
exposure. For example, for a contaminated drinking-water scenario,
the dermal pathway (i.e. bathing) is evaluated only if the screening
analysis suggests that it will contribute at least 10% of the dose from
drinking the water (USEPA, 2004b).
11.3 Other topics related to risk assessment
Harmonization of skin notation and dermal absorption in sus-
topics.
chemicals (see chapter 10). For guidance on permeability coeffi-
ceptible populations are discussed in chapter 12 on controversial
112
12. CONTROVERSIAL TOPICS IN THE ASSESSMENT
OF DERMAL ABSORPTION
Although there has been a movement towards harmonization of
methodology (OECD, 2004a,b,c), there are still many topics in
dermal absorption assessment that are controversial. This chapter
touches on some of these issues and refers the reader, where
necessary, to the relevant sections in the present document or to
other sources of information.
12.1 QSARs/QSPeRs
The OECD guidelines are not strict protocols. However, the
acceptance and use of these guidelines should lead to a more reliable
database. Can reliable QSPeRs be constructed from such an
improved database, or are stricter protocols necessary (e.g. that
suggested by a CEFIC Working Group; Jones et al., 2004) (see
The acceptance of QSARs/QSPeRs themselves is still a matter
of controversy. On the one hand, dermal absorption data are lacking
for thousands of chemicals; on the other hand, variability of data is
controversial. Further, guidance is needed on how to use QSAR data
in risk assessment. At present, most QSARs give estimates of K
p
values (or relative absorption over 24 h), which need to be
translated into a parameter that can be applied in risk assessment.
12.2 Reduction of intralaboratory/interlaboratory
variation
There is still a debate going on about how in vitro data could or
should be used in risk assessment. An evaluation of available data
on in vitro dermal absorption was performed under the auspices of
the OECD (2000). Because the available studies comparing the in
vitro and in vivo test results contained many variables (different
species, thickness and types of skin, exposure duration, vehicles,
etc.), an evaluation/consensus was found to be difficult. Some of the
variables in in vitro determinations and in in vivo experiments were
chapter 10 and Appendix 2)?
Controversial Topics in the Assessment of Dermal Absorption
113
duct carefully controlled in vitro and in vivo dermal absorption
studies. Some of these topics (e.g. skin thickness, vehicle effects,
appropriate receptor fluids, tape stripping) have also been addressed
in a recent European project (i.e. EDETOX; see section A2.3 in
Appendix 2).
In general, studies have shown that properly conducted in vitro
measurements can be used to predict in vivo absorption (chapter 8).
The acceptance of in vitro measurements in lieu of in vivo studies by
conditions using accepted protocols.
12.3 Consequences of reservoir effect for risk
assessment
Another controversial issue is the presence of test substance in
the various skin layers i.e. absorbed into the skin but not passed
into the receptor fluid (see sections 3.5 and 6.4). In particular, very
lipophilic compounds are difficult to investigate in vitro due to their
low solubility in most receptor fluids. If the amount retained in the
skin is also counted as being absorbed, a more acceptable but con-
servative estimate can be made. Water-soluble substances can be
tested more accurately in vitro because they more readily diffuse
into the receptor fluid. If skin levels are included in the overall per
cent absorption figure, results from in vitro methods seem to ade-
quately reflect those from in vivo experiments and support their use
as a replacement of in vivo testing (EC, 2003).
Different approaches are taken by different bodies. ECETOC
(1993) base their measurements of percutaneous absorption on
receptor fluids only. In the cosmetic guidelines issued by the Euro-
pean Cosmetic Toiletry and Perfumery Association (COLIPA)
(Diembeck et al., 1999) and the SCCNFP (2003b), the material
remaining in the epidermis and dermis, in addition to that in the
receptor fluid, is considered as being systemically available, but not
the test substance remaining in the stratum corneum at the end of the
study. In OECD Test Guideline 428 (OECD, 2004c), skin absorp-
tion may sometimes be expressed using receptor fluid data alone.
However, when the test substance remains in the skin at the end of
discussed in chapter 6 and chapter 7, respectively. Studies on inter-
and intralaboratory variation (chapter 8) reinforce the need to con-
regulatory authorities (chapter 11) necessitates controlled study
114
the study (e.g. lipophilic test substances), it may need to be included
in the total amount absorbed. The OECD (2004a) guidance docu-
ment notes that skin fractionation (e.g. by tape stripping) may be
performed to further define the localization of the test substances
within the skin as required by the objectives of the study. Alter-
natively, distribution within the skin can be determined by taking
vertical sections and using autoradiography or other analytical tech-
niques to visualize the test substance.
A recent publication has discussed this topic and concludes that
when the movement of chemicals from a skin reservoir to the
receptor fluid is shown to occur, it is appropriate to add skin levels
to receptor fluid values to obtain a more realistic estimate of dermal
absorption (Yourick et al., 2004).
12.4 Relevance of percutaneous measurements to data
required by risk assessors: finite and infinite
exposures
Ideally, estimates of dermal absorption required by risk assess-
ors should be as close as possible to real exposure conditions. To
achieve this, experiments should be conducted under finite dose
conditions, using vehicles, concentrations of chemicals, and periods
of exposure that reflect in-use conditions (Benford et al., 1999; EC,
2004). In practice, most in vitro dermal absorption studies are
carried out under infinite exposure conditions. At present, there are
attempts to use modelling to link permeability coefficients measured
under infinite exposure (steady-state) conditions to conditions more
typical of occupational exposure (non-steady-state conditions) (see
Krse & Kezic, 2004).
12.5 Single- versus multiple-exposure regimes
It is suggested that experiments using multiple-dosing regimes
would be more comparable with occupational exposure scenarios.
However, most data from dermal absorption studies are from single-
exposure regimes. Data on the effects of repeated exposure are
scarce and conflicting. Some data show that repeated exposures may
alter dermal absorption (e.g. Roberts & Horlock, 1978; Wester &
Maibach, 1996). However, others show no differences. For example,
also chapter 10; section A2.3.4 in Appendix 2; Jones et al., 2004;
115
the effects of single and multiple dosing (for 1421 days) on the
dermal absorption of six test compounds (methylprednisolone
aceponate, azone, malathion, estradiol, hydrocortisone, and testos-
terone) have been compared. No significant changes in absorption
were observed following multiple dosing (Wester et al., 1983, 1994;
Bucks et al., 1985; Tauber & Matthes, 1992). Pendlington et al.
(2004) found that in a repeated-dose experiment with caffeine, the
amount recovered from each skin compartment mirrored the number
of doses applied.
12.6 Barrier integrity test for skin barrier function of
human skin in skin penetration tests
OECD Test Guideline 428 (OECD, 2004c) recommends the use
of a barrier integrity test when performing skin penetration studies
for regulatory submission. There is much debate at the moment as to
whether barrier integrity tests actually correlate to epidermal proper-
ties (Gordon Research Conference, 2005). If skin samples do not
exhibit a permeability coefficient K
p
for tritiated water above 2.5
10
3
cm/h, they are rejected as being damaged. In a recent study
(Roper et al., 2004) on K
p
values from 1110 human skin samples,
230 (21%) were rejected because they did not fulfil the barrier
integrity criteria. However, it is likely that many of these rejected
samples were atypical rather than damaged, resulting in an under-
estimation of absorption in such an individual. Further, this is an
unnecessary wastage of valuable human skin samples, leading to a
truncation of the actual population frequency distribution of K
p
at
high K
p
(see also section 6.2).
12.7 Dermal absorption in susceptible populations
Risk assessment of dermal exposure is mostly related to healthy,
adult individuals. However, skin contact with chemicals occurs in
everyday life, and all parts of a population are potentially exposed.
Concern for children as a potentially susceptible population for
exposure to toxic environmental agents has increased dramatically in
the last few years (Daston et al., 2004). As full-term neonates have a
well developed stratum corneum, it is generally believed that the age
of the child has little bearing on dermal permeability (USEPA,
1992). However, the stratum corneum of premature infants is less
116
effective than that of full-term infants or adults (Barker et al., 1987;
USEPA, 1992), and dermal absorption has to be considered in
scenarios where these neonates are dermally exposed to contam-
inants present in bath water or to chemicals in hygienic or diaper
rash products. Furthermore, risks from dermal exposure to environ-
mental chemicals may differ between children and adults for a
variety of reasons, including differing behavioural patterns, anatom-
ical and physiological differences, and developmental differences of
vital organs such as the brain, which may result in different end
organ effects (Mancini, 2005).
Enhanced dermal absorption has also been reported in skin
affected by disease. Certain genetic defects in lipid metabolism or in
the protein components of the stratum corneum produce scaly or
ichthyotic skin with abnormal barrier lipid structure and function
(Madison, 2003). The inflammatory skin diseases psoriasis and
atopic dermatitis also show decreased barrier function. The psoriatic
skin allows more penetration of not only low molecular weight
substances but also large proteins (Schaefer et al., 1977; Gould et
al., 2003). In atopic dermatitis epidermis, a significant decrease in
the ceramide content correlates with an increased rate of water loss
at the skin surface a sign of an impaired barrier (Humbert, 2003).
This is consistent with the findings of Jakasa et al. (2005), who
found increased stratum corneum penetration of polyethylene gly-
cols of a wide range of molecular weights through the clinically
normal skin of atopic dermatitis patients. Abnormal barrier functions
of clinically normal skin in atopic dermatitis were also reported by
Hata et al. (2002), who found increased dermal absorption of hydro-
philic dyes in the skin of atopic dermatitis patients. As a conse-
quence, the impaired barrier functions of clinically normal skin in
atopic dermatitis may predispose inflammatory processes evoked by
irritants and allergens. This is a point of concern, since the preva-
lence of atopic dermatitis in industrialized countries is dramatically
increasing.
Besides already damaged skin in disease, the skin barrier can be
compromised in a wide variety of ways: physical damage (e.g.
burned, shaved skin), chemical damage (e.g. detergents, solvents),
occluded skin (e.g. wearing of gloves), increased hydration (e.g.
excessive hand washing), and even psychological stress (USEPA,
1992; Choi et al., 2005). It has been shown that even slight damage
117
to the skin achieved by sodium lauryl sulfate may lead to increased
percutaneous penetration of chemicals covering a wide range of
solubilities (Nielsen, 2005). Higher dermal absorption was also
reported in skin damaged by acetone (Benfeldt et al., 1999; Tsai et
al., 2001) and by tape stripping (Benfeldt et al., 1999; Tsai et al.,
2003).
In conclusion, there is a line of evidence suggesting that the
compromised skin barrier is not uncommon. This condition may
make the skin more susceptible to irritants, sensitizers, and disease.
In addition, a compromised barrier makes the skin more permeable
and facilitates dermal uptake. Therefore, when evaluating the health
risk associated with skin exposure, susceptible subgroups in the
population should be taken into account. Furthermore, evaluation of
potentially increased penetration due to chemical or physical dam-
age should be included in risk assessment.
12.8 Skin notation
The skin notation was introduced almost 50 years ago as a
qualitative indicator of hazard related to dermal absorption at work.
It is designated if:
skin exposure to a defined area, duration, etc. increases the sys-
temic dose by a given percentage, compared with inhalation
exposure at the OEL; and/or
realistic dermal exposure at the workplace has been shown to
cause adverse effects (Johanson, 2003).
A major complication is that the skin notation is often used as
an instrument for risk management (Nielsen & Grandjean, 2004).
Workplace exposure needs to be assessed in quantitative terms, and
a qualitative hazard indicator, such as the skin notation, is not very
useful for risk assessment or risk management. It has been suggested
that skin notation should relate to the potential for toxicity following
relevant dermal exposure (Sartorelli, 2002; Nielsen & Grandjean,
2004).
In many countries, compounds considered a skin hazard are
identified by skin notation on the list of OELs. In general, the
notation has the purpose of drawing attention to the fact that
118
cutaneous exposure to these compounds can significantly contribute
to total systemic exposure. Irritating and corrosive compounds do
not have a skin notation.
12.8.1 Skin notation criteria in different countries
Vague criteria and lack of good data have led to different
classifications for the same chemical by different authorities and
organizations. The criteria used to assign such skin notations vary
considerably between countries and institutions and are generally
qualitative (Johanson, 2005). The different criteria are shown in
Table 9.
Table 9. Criteria for assigning a skin notation in different countries
a
Country Criteria used to assign a skin notation by that country
Denmark When known that the substance can be absorbed
via skin
Norway Substances that can be taken up via skin
Finland Absorbed amounts and health risks cannot be
evaluated only from air concentrations
Sweden Substances easily taken up by the body via skin
Germany (MAK) When dermal exposure increases the body burden
EU (SCOEL) Substantial contribution to total body burden via
dermal exposure
USA (ACGIH) Potential significant contribution to overall exposure
by the cutaneous route, including mucous
membranes and the eyes, either by contact with
vapours or, of probable greater significance, by direct
skin contact with the substance
The Netherlands
(DECOS)
More than 10% contribution to total exposure
European industry
(ECETOC)
More than 10% contribution to total exposure
ACGIH, American Conference of Governmental Industrial Hygienists;
DECOS, Dutch Expert Committee on Occupational Standards; ECETOC,
European Centre for Ecotoxicology & Toxicology of Chemicals; MAK,
German Senate Commission on the Investigation of Health Hazards of
Chemical Compounds in the Work Area; SCOEL, Scientific Committee for
Occupational Exposure Limits.
a
From Johanson (2003).
119
A review of the use of the skin notation employed by many of
the worlds health and safety authorities identified inconsistencies
(Fiserova-Bergerova et al., 1990). For example, the United Kingdom
Health and Safety Executive currently assigns a skin notation to over
120 chemicals, while the American Conference of Governmental
Industrial Hygienists (ACGIH) applies the skin notation to over 160
substances (Semple, 2004). The ACGIH notations have been
criticized in the past for inconsistencies in documentation. Varying
criteria for assignment are due to a lack of information on dermal
absorption rates of chemicals (Semple, 2004). The German criteria
for designation of a skin notation (discussed by Drexler, 1998) are
that the maximal allowable concentration (MAK) for the substance
is not sufficient to protect dermally exposed persons from adverse
effects on their health. The German definition has therefore intro-
duced adverse health effects as part of the criterion, but does not
include quantitative terms (Nielsen & Grandjean, 2004).
12.8.2 Quantitative approaches
Although the concept of comparing dermal uptake with uptake
following inhalation is central to most skin notation definitions, the
exact terminology has yet to be decided. One approach for a
quantitative term is to consider the dermal LD
50
. Chemicals with
dermal LD
50
values below 2000 mg/kg body weight are normally
regarded as a health risk to humans (Nielsen & Grandjean, 2004).
The present use of skin notation does not reflect this proposal, as
only a few countries have included all such chemicals on their lists
of chemicals with a skin notation. An inherent problem with LD
50
values is that they refer only to acute toxicity, with death as the
single registered outcome. Thus, an acute LD
50
value will not cover
non-lethal or chronic toxicity, accumulation, or the effects of
repeated dosing (Nielsen & Grandjean, 2004). Fiserova-Bergerova
et al. (1990) recommended two reference values as criteria for skin
notation: 1) the dermal absorption potential, which relates to dermal
absorption raising the dose of non-volatile or biological levels of
volatile chemicals 30% above those observed during inhalation
exposure to the threshold limit value / time-weighted average (TLV-
TWA) only, and 2) the dermal toxicity potential, which triples
biological levels as compared with levels observed during inhalation
exposure to the TLV-TWA only. The Netherlands uses the criterion
that dermal exposure of the hands and forearms for 1 h must lead to
120
uptake exceeding 10% of that received by inhalation for 8 h at the
OEL for a skin notation (Semple, 2004).
12.8.3 New approaches
A comparison of dermal absorption rates between OEL listed
chemicals with skin notation showed variation by several orders of
magnitude, and the contribution of dermal absorption ranged from
negligible to a major fraction of the absorbed dose (Johanson,
2003). The current qualitative skin notation does not reflect this.
A dermal uptake index (D in Equation 11, pD in Equation 12)
that relates the contributions of dermal absorption rate (P
skin
) to the
respiratory uptake rate via inhalation at the OEL (P
resp
) has been
proposed (Johanson, 2005):
D = (P
skin
/P
resp
) k [Equation 11]
pD = log
10
D + 8 (with 8 including the constant k
in Equation 11) [Equation 12]
Dermal occupational exposure limits (DOELs) have also been
proposed (Bos et al., 1998), in which a limit is related to the total
dose deposited on the skin during a work shift (Semple, 2004). The
uncertainties that may restrict the use of DOELs in quantitative risk
assessment include:
difficulty in evaluating the extent of contaminated skin;
regional variations in skin permeability;
lack of percutaneous penetration data; and
influence of workers behaviour on skin contamination (Sartor-
elli, 2002).
The National Institute for Occupational Safety and Health in the
United States is currently revising its criteria for skin notation (S.
Soderholm, personal communication, 2005). There is an obvious
need for harmonization of the skin notation both within the EU and
with the United States and a strong argument for a quantitative
component.
121
12.9 Dermal absorption of nanoparticles
Nanoscale materials have at least one dimension less than
100 nm, a scale 1000 times smaller than most microscale materials
tested to date. This significant reduction in size has resulted in
topical products containing nanoscale materials and has generated
concern about the potential nanoparticle skin exposure. Cosmetic
products containing non-ghosting sunscreens and nano-liposome-
based skin care products, certain window sprays, paints, varnishes,
and coatings are examples of nanoscale products (SCENIHR, 2005).
It is, however, unclear exactly how well nanoparticles will penetrate
the skin and what their toxicological impact will be (Tsuji et al.,
2006). Kim et al. (2004) have shown that, when nanoparticles are
administered in the dermis, they localize to regional lymph nodes,
potentially via skin macrophages and Langerhans cells, raising
potential concern for immunomodulation if nanoparticles penetrate
the skin. Tsuji et al. (2006) suggest that potential sources of toxicity
are skin or organ cytotoxicity, long-term toxicity subsequent to
accumulation in skin and other organs, metabolism to toxic particles,
and toxicity of photoactivated nanoparticles.
Titanium dioxide and zinc oxide nanoparticles have been
included in sunscreens, as they scatter UV A but not visible light and
therefore enable a transparent sunscreen. Several studies of micro-
scale and nanoscale materials have identified titanium in the epider-
mis and dermis following application of titanium dioxidecontaining
sunscreens, whereas other studies found metal oxide or fluorescent
polystyrene nanoparticles only in the hair follicles and skin furrows
and no material below the surface of the stratum corneum (Tan et
al., 1996; Lademann et al., 1999, 2001; Alvarez-Roman et al.,
2004). Lansdown & Taylor (1997) suggested that microfine titanium
dioxide penetrates the skin when applied in castor oil, but their
studies used rabbits. Bennat & Muller-Goymann (2000) suggested
that titanium dioxide penetrates human skin when applied as an oil
in water emulsion, with penetration being greater in hairy skin.
Microparticles have been identified in the lymph nodes and
dermis of individuals who walked barefoot in African rift valleys
and developed elephantiasis of the feet and legs, suggesting that
external force may be necessary for particle penetration to occur
(Corachan et al., 1988; Blundell et al., 1989). This association of
122
external pressure with particle translocation was extended by in vitro
studies demonstrating translocation of fluorescent particles, 1 m or
smaller, into the epidermis of flexed human skin, as at the wrist
(Tinkle et al., 2003).
The importance of the surface characteristics of nanoscale mat-
erials was reinforced by recent studies of transcutaneous vaccine
delivery (Kohli & Alpar, 2004). Whereas negatively charged fluor-
escent particles of 50 and 500 nm permeated pig skin, neutral and
positively charged particles did not. Surprisingly, negatively charged
100- and 200-nm fluorescent particles also did not penetrate. Addi-
tional research found that the total concentration of charge was
critical. Zeta potential and crystallinity are critical parameters for
successful penetration of nanoscale lipid vesicles and lipid solid par-
ticles into the skin (Souto et al., 2004).
Nanoscale materials also display novel chemical and physical
properties, and the interaction of these materials with biological
systems is uncertain. Several studies demonstrate that the epidermis
can respond physiologically to particulate exposure. An in vivo
study demonstrated the development of murine immune hypersen-
sitivity following cutaneous application of beryllium oxide particu-
late, and an in vitro study confirmed single wall carbon nanotube
induction of oxidative stress in a keratinocyte cell line (Shvedova et
al., 2003).
Solid lipid nanoparticles have been used since the early 1990s
in cosmetic products as an alternative system to emulsions, lipo-
somes, and polymeric nanoparticles. Muller et al. (2002) reviewed
this area. To date, emphasis appears to have been placed on mea-
suring the skin absorption of drugs incorporated in the solid lipid
nanoparticles rather the penetration of solid lipid nanoparticles per
se (Santos et al., 2002; Borgia et al., 2005). Solid lipid nanoparticles
can lead to macrophage cytotoxicity at high concentrations but, in
general, appear to be safe, toxicologically acceptable carrier systems
(Scholer at al., 2000).
In conclusion, nanoscale material science is an emerging field in
which the benefits and adverse consequences of exposure and der-
mal absorption are not understood. If hairy animals are used, this
may lead to an overestimation of dermal absorption after nano-
123
particle application, as most nanoparticle absorption appears to be
via the follicles. Objective research will be critical over the next 5
10 years to promote responsible development that maximizes posi-
tive biological interaction and minimal adverse health effects. In the
interim, public and corporate concern for the environmental and
human health and safety of nanomaterials and products is high.
Assessment of exposure risk and dermal absorption requires careful
evaluation of the composition of the nanomaterials, their physical
and chemical properties, the potential for exposure, the magnitude of
exposure, the conditions of exposure, and, if absorption proves to be
significant, the route of exposure. Established risk assessment meth-
odologies can be used to build a framework to evaluate the current
nanomaterials; however, this framework should be dynamic and
revised as new safety data become available.
124
13. CONCLUSIONS AND RECOMMENDATIONS
1. Human skin in vitro and in vivo should be universally
recognized as the gold standard in dermal absorption risk
assessment.
2. In vitro and in vivo human experimental studies using
standardized, well controlled methods are needed to appro-
priately assess human dermal absorption of chemicals,
including formulated products, that come into contact with
the skin.
3. Further efforts are needed to improve and harmonize meth-
odology in order to minimize variability in in vitro dermal
absorption measurements. Interlaboratory comparative
studies should include appropriate internal and external
quality controls, i.e.:
use of a validated analytical technique;
use of a standard membrane and test solution as an
additional control;
cross-check using pure reference chemicals;
confirmation of barrier integrity;
consistent and appropriate skin preparation; and
other recommendations of the OECD test guidelines
(OECD, 2004a,c).
4. There should be more research aimed at addressing the lack
of repeated-dose dermal absorption experiments that
simulate relevant exposure conditions.
5. In risk assessment, maximum flux, either measured or
estimated from molecular weight, should be used in prefer-
ence to K
p
or percentages of the chemical absorbed. Adjust-
ment should be made to account for non-saturating
exposure.
6. Collection of more data on highly lipophilic chemicals
under in-use conditions is needed to facilitate understand-
Conclusions and Recommendations
125
ing of the effect of various vehicles, receptor fluids, and
mixtures on results obtained in dermal absorption studies.
7. There is a need for more in vivo and in vitro data on the
dermal absorption of chemicals in damaged skin (e.g.
mechanical damage, UV damage, skin irritants, sensitizers,
and solvents) and in diseased skin to assist the risk assess-
ment process.
8. Greater efforts should be made to correlate in vitro and in
vivo data and to develop reliable prediction models.
9. The regression of the absorption time course will always
give a more accurate estimate of dermal absorption param-
eters than the usual techniques and may be more valuable
in finite dose estimation, in comparing different dose
exposure scenarios, and in extrapolating to different occu-
pational exposure situations.
10. Further efforts to evaluate QSARs for risk assessment
purposes and to prepare guidance on their use are encour-
aged. However, the inherent limitations of QSARs to mimic
a highly complex biological process must be recognized,
and approaches being developed in the pharmaceuticals
area to model drugskin component interactions hold
promise for the future.
11. Databases containing measured and well defined skin
absorption data are a key first step in development of
QSARs and for research to improve understanding of the
dermal absorption process for chemicals. Such databases
have been developed by industry, researchers, and govern-
ment institutions and should be supported, maintained, and
updated. Means of sharing data should be explored, recog-
nizing that in some instances the data are proprietary.
12. Noting that different sectors (pesticides, industrial chem-
icals, cosmetics, pharmaceuticals, etc.) have long-
established but different usages for some key terms (e.g.
permeation, penetration, absorption, resorption, systemic
absorption), it is important that terms are clearly defined in
126
scientific articles to aid interpretation. Similarly, care needs
to be taken in making comparisons between studies.
13. It needs to be recognized that emerging technologies such
as nanoparticles may have unanticipated toxicological
consequences following contact with the skin.
14. Development and standardization of non-invasive and
semi-invasive human in vivo methods, such as stratum
corneum stripping, Fourier transform infrared, Raman,
multiphoton, and confocal spectroscopy, and microdialysis,
should be encouraged.
127
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163
APPENDIX 1: GUIDELINES AND PROTOCOLS
A1.1 Historical perspectives
Although methodology for and assessment of percutaneous
absorption have been advancing for several decades, it is only
comparatively recently that interest in this area has expanded from
the drugs and cosmetics branches to the field of chemical risk
assessment. In the last decade, a number of documents have been
prepared, reviewed internationally, and published for various asso-
ciations or regulatory bodies. The following is intended only as a
short guide to the available publications, which have, in part, been
stepping stones in the preparation of the OECD test guidelines
(OECD, 2004a,b,c). Details are to be found in the original docu-
ments.
A1.2 ECETOC (European Centre for Ecotoxicology &
Toxicology of Chemicals)
ECETOC (1993) was prepared and reviewed by experts in the
field of percutaneous absorption on behalf of ECETOC. With
emphasis on industrial chemicals rather than on drugs or cosmetics,
this publication describes methods for studying percutaneous
absorption, discusses the relevance of results when forming inter-
species comparisons, and reviews the assessment of dermal expo-
sure.
A1.3 ECVAM (European Centre for the Validation of
Alternative Methods )
Howes et al. (1996) is the Report and Recommendations of the
ECVAM Workshop, which was held in 1994. The participants
comprised scientists from both academia and industry. The aim of
the workshop was to review the status of in vivo and in vitro
methods for studying dermal absorption at that time, with particular
emphasis on recommending and optimizing in vitro methods to
reduce the number of animals needed for testing.
The group noted that the regulatory requirements for submis-
sion of relevant data on dermal penetration varied worldwide, from
164
the very demanding and precise in vivo (rat) protocols of the
USEPA for dermal absorption of pesticides (e.g. USEPA, 1998) to
absence of guidelines. In Europe, the requirement for percutaneous
absorption data was usually fulfilled as part of the ADME studies
(pharmacokinetics/toxicokinetics) undertaken for new chemicals,
drugs, and pesticides, as well as experimental data on the delivery of
drugs. There was/is increasing pressure on industry to reduce the
number of animals used in safety testing.
The ECVAM Report discusses mechanisms and modelling of
skin penetration, in vitro and in vivo methods for measuring per-
cutaneous absorption, and skin metabolism.
A1.4 COLIPA (European Cosmetic Toiletry and Perfumery
Association)
Owing to a potential ban on the use of animals in the testing of
cosmetic products and their ingredients (6th amendment to the
European Cosmetics Directive 93/35/EEC), a Task Force of
COLIPA met in 1995 to develop test guidelines for in vitro assess-
ment of dermal absorption and percutaneous penetration of cosmetic
ingredients (Diembeck et al., 1999). This publication was the basis
for further activities by the cosmetics industry (see also Steiling et
al., 2001).
A1.5 SCCNFP (Scientific Committee on Cosmetic
Products and Non-Food Products Intended for
Consumers)
SCCNFP (2003a) contains notes of guidance for testing of
cosmetic ingredients for their safety evaluation. Only one subchapter
(3-4.4) is related specifically to dermal/percutaneous absorption.
The document points out the wide variety of terms used in
definitions. For cosmetics, the SCCNFP makes a clear distinction
between dermal absorption and dermal adsorption, the latter being
defined as the amount of topically applied test substance present in
or sticking to the stratum corneum. It is considered not to be
systemically available and is excluded from the risk assessment for
cosmetic products (see Diembeck et al., 1999; also Steiling et al.,
2001).
Appendix 1
165
Dermal/percutaneous absorption is defined as the amount of
dermally applied substance remaining in the residual skin (excluding
the stratum corneum) plus the amount of dermally applied substance
that has transpassed the skin and is detected in the receptor fluid.
The sum is considered to be systemically available (= dermal
bioavailability) (see Diembeck et al., 1999).
Test formulations and concentrations tested should be an
adequate representation of the final cosmetic products. In cases
where dermal absorption studies are not available, a default value
for dermal absorption of 100% is applied in the calculation of the
margin of safety.
Details of SCCNFP basic criteria for in vitro testing of cosmetic
ingredients are given in SCCNFP/0750/03 (SCCNFP, 2003b). The
SCCNFP has also published guidance in particular on in vitro
dermal absorption of cosmetic ingredients and its assessment
(SCCNFP, 2003b).
A1.6 USEPA (United States Environmental Protection
Agency)
A1.6.1 USEPA (1998)
USEPA (1998) is intended to meet testing requirements of the
United States Federal Insecticide, Fungicide, and Rodenticide Act
and the TSCA. The guideline has been designed and validated using
the laboratory rat, and this is the required species for testing. Other
animal species were considered but were rejected. It is recom-
mended that the same strain of rat used for metabolism and toxicol-
ogy studies be used for dermal absorption testing. For risk assess-
ment, the absorption rates determined in the rat can be used as a
modest overestimate of human dermal absorption or to perform a
kinetic evaluation.
A1.6.2 USEPA (2004a)
In their Final Test Rule (USEPA, 2004a; update of USEPA,
1999) under section 4(a) of the TSCA, manufacturers, importers,
and processors of 34 (formerly 47) chemical substances of interest
to the Occupational Safety and Health Administration (OSHA) will
166
be required to conduct in vitro dermal absorption rate testing. These
dermal absorption rate data are to be used to support OSHAs
development of skin designations for the chemical substances. For
measuring the permeability constant (K
p
), the test standard specifies
the use of static or flow-through diffusion cells and non-viable
human cadaver skin. It also requires the use of radiolabelled test
substances unless the analytical methods used have an equivalent
sensitivity. For compounds that may damage the skin with prolonged
contact, a short-term absorption rate measurement is more appro-
priate. The six parameters (choice of membrane, preparation of
membrane, diffusion cell design, temperature, testing hydrophobic
chemicals, and vehicle) are similar for the determination of either of
the two percutaneous absorption rate values. In contrast, the
remaining two parameters (dose and study duration) are different for
the two percutaneous absorption rate values.
A1.6.3 USEPA (1992)
Dermal Exposure Assessment Principles and Applications is an
interim document and details procedures for estimating permeability
coefficients of toxic chemicals and for evaluating the dermal
absorbed dose. This is updated in USEPA (2004b).
A1.6.4 USEPA (2004b)
This document is the Supplemental Guidance (Part E) to the
Risk Assessment Guidance for Superfund, Volume 1: Human Health
Evaluation Manual. USEPA (2004b) incorporates and updates the
USEPA (1992) document and contains methods for conducting
dermal risk assessments for the water and soil pathways at Super-
fund sites. The USEPA has a web site where update information can
The permeability factor K
p
has been identified as one of the
major parameters contributing to uncertainty in the assessment of
dermal exposures. Since, according to this document, the variability
between predicted and measured K
p
values is no greater than the
variability in interlaboratory replicated measurements, the USEPA
(2004b) document recommends the use of a mathematical model to
predict K
p
values (given in Appendices A and B of that document)
based on equations estimated via an empirical correlation as a
function of K
ow
and molecular weight (Potts & Guy, 1992), obtained
be found (http://www.epa.gov/oswer/riskassessment).
Appendix 1
167
from an experimental database (the Flynn [1990] data set) using
absorption data of about 90 chemicals from water through human
skin in vitro.
Based on the Flynn (1990) data set, Equation 3.8 (of the
USEPA 2004b document) was derived and can be used to predict
the K
p
of chemicals with K
ow
and molecular weight (MW) within the
Effective Prediction Domain determined via a statistical analysis:
Log K
p
= 2.80 + 0.66 log K
ow
0.0056 MW (r
2
= 0.66)
However, there are some chemicals that fall outside the
Effective Prediction Domain for determining K
p
, in particular
those with a high molecular weight and high K
ow
values (lipophilic
compounds accumulating in the stratum corneum). It is suggested in
this document that a fraction absorbed term be applied to account
for the loss of chemical due to desquamation of the outer skin layer
and a corresponding reduction in the absorbed dermal dose. For
halogenated chemicals, the predicted K
p
could be underestimated
due to the lower ratio of molar volume related to molecular weight
compared with those included in the Flynn (1990) data set.
The USEPA (2004b) document gives a table (Exhibit B-2) of
predicted K
p
values for 209 organic chemical contaminants in water.
The USEPA (2004b) guidance document presents recom-
mended default exposure values for all variables for the dermal
water and dermalsoil pathways.
For guidance on permeability coefficients for inorganic com-
pounds, USEPA presents a table compiled from specific chemical
experimental data as modified from USEPA (1992) and Hostynek et
al. (1998).
A1.7 EC (European Commission)
A1.7.1 EC (2003)
In Appendix IVB of the EC Technical Guidance Document on
Risk Assessment (EC, 2003), the contribution that dermal exposure
may make to systemic body uptake and its estimation are discussed.
168
Although guidelines give a general description of the experimental
design, it is important for risk assessment that the anticipated expo-
sure conditions be taken into account (Benford et al., 1999).
The duration and frequency of exposure as well as the level of
exposure may vary tremendously exposure may be incidental or
continuous. Studies addressing more than one relevant exposure per
unit area are recommended, as well as the use of various exposure
times and vehicles. For risk assessment, the percentage of absorption
is the most useful parameter. Ideally, 1) the exposure duration of the
study should be as long as or longer than the anticipated exposure
duration, and 2) the concentrations tested should include the lowest
concentration anticipated.
The Technical Guidance Document presents the problems
encountered when making risk assessments 1) when studies are not
available (default values) and 2) from in vitro and in vivo data.
the present report.
A1.7.2 EC (2004)
This guidance document on dermal absorption has been pre-
pared for the EC Directorate E1 Plant Health (EC, 2004). There-
fore, the emphasis is on guidance for notifiers and EU Member
States on the setting of dermal absorption values to be used in risk
assessment for users of plant protection products reviewed for
inclusion in Annex I of Directive 91/414/EEC. Inclusion of active
substances in this annex is possible only if the products containing
them can be used with acceptable risk to humans (i.e. operators,
workers, bystanders). The dermal route is the main exposure route
for most pesticides for operators applying them as well as for
workers and bystanders.
The EC (2004) document contains an overview of dermal
absorption and the methodologies used in measuring dermal absorp-
tion. It discusses the decision-making processes for setting absorp-
tion percentages and includes a proposal for a tiered approach to
risk assessment for operator exposure, using default dermal absorp-
tion percentage or dermal absorption percentage determined experi-
Some of this discussion is given in chapter 11 on risk assessment in
mentally (see also chapter 11 in the present report).
Appendix 1
169
In the absence of experimental data, the occupational exposure
is based on models for example, the European Predictive Opera-
each calculating external dermal and inhalation exposure. For
risk assessment, these external exposure data are compared with
toxicity data (acceptable operator exposure level or AOEL, defined
as an internal value and expressed in mg/kg body weight per day;
EC, 2001). In order to compare the external exposure with the inter-
nal AOEL, external exposure data have to be converted into internal
levels. The studies should be performed in accordance with OECD
Test Guidelines 427 (OECD, 2004b) and 428 (OECD, 2004c) and
the associated Guidance Document No. 28 (OECD, 2004a).
A1.8 OECD (Organisation for Economic Co-operation and
Development)
A1.8.1 OECD (2004a)
This guidance document was written to guide scientists unfamil-
iar with the procedures of skin absorption studies and to support
technical aspects of the OECD skin absorption test guidelines
(OECD, 2004b,c).
A1.8.2 OECD (2004b)
This guideline describes details of the in vivo studies commonly
performed on rats but also on hairless species as well as other
species having skin absorption rates more similar to those of
humans.
A1.8.3 OECD (2004c)
This guideline describes in vitro methods used for studying
dermal absorption. Skin from human or laboratory animal sources
can be used. It is recognized that the use of human skin is subject to
national and international ethical considerations and conditions.
Both static and flow-through diffusion cells are acceptable.
Normally, donor chambers are left unoccluded during exposure to a
finite dose of a test preparation. However, for some studies, infinite
applications are necessary when donor chambers may be occluded
tor Exposure Model (EURO POEM; http://www.pesticides.gov.uk)
(see also chapter 6).
170
APPENDIX 2: PAST AND PRESENT INITIATIVES ON
EXCHANGE OF INFORMATION AND
HARMONIZATION OF METHODOLOGY ON DERMAL
ABSORPTION
In the last few years, there have been several initiatives to accel-
erate progress in international harmonization of methodology and
protocols of dermal absorption, culminating in the publication of the
OECD test guidelines in 2004 (OECD, 2004a,b,c; see Appendix 1).
There have been initiatives from the regulatory side in the
United States (USEPA, 1999, 2004a,b) and in Europe. In Europe,
there have been two large projects: the Dermal Exposure Network
(19971999) and, leading on from it, the EDETOX project. From
the industrial side, CEFIC has supported two projects associated
with QSARs linking 1) physicochemical properties to permeation
data and 2) measured with calculated values, so that in the future it
may be possible to predict the data for a large number of chemicals
rather than undertake expensive testing of chemicals. Here, an
overview is given of these projects, the results of which are also
cited in other parts of the document.
In addition, it should be emphasized that this interest and
progress in dermal absorption harmonization have been a result of
the momentum from the biannual meetings of the Prediction of
Percutaneous Penetration (PPP) Conferences (later the Perspectives
in Percutaneous Penetration Conferences; section A2.5) and Gordon
Research Conferences (section A2.6) and, more recently, the Inter-
national Conference on Occupational and Environmental Exposures
of Skin to Chemicals (section A2.7).
A2.1 OECD Test Guidelines
The most important recent international initiative was the effort
to harmonize the protocols for in vitro and in vivo testing (see also
ment became apparent in 1997 when national experts from a number
of member countries could not agree on the possibilities and
chapters 6 and 7 and Appendix 1). The need for a guidance docu-
Appendix 2
171
limitations of the in vitro test. The draft outlines were presented in
2000, and the final guidelines were published in 2004 (OECD,
2004a,b,c). The guidance document (OECD, 2004a) provides addi-
tional technical background to both the in vivo and in vitro methods
for skin absorption, as described in Test Guidelines 427 (OECD,
2004b) and 428 (OECD, 2004c), respectively.
A2.2 Dermal Exposure Network (19971999)
At an international meeting of experts from European countries
and the United States in June 1994, it was decided to form a
European network on dermal exposure to encourage development of
projects in the area and pool research capabilities, to harmonize
techniques, expertise, and knowledge, and to guide the EC in devel-
oping a comprehensive and harmonized risk assessment strategy in
the workplace. Under the Environment and Health Programme of
the EC, the Dermal Exposure Network was established, with mem-
bers comprising skin experts from many countries in Europe.
Meetings of the Dermal Exposure Network of the EC started in
1997, and five subgroups were organized: Risk Assessment, Biolog-
ical Monitoring, Percutaneous Penetration, Skin and Surface Con-
tamination, and Contribution of Different Sources (Sartorelli et al.,
2000).
The work of the Percutaneous Penetration Subgroup of the
Dermal Exposure Network focused on the standardization and
validation of in vitro experiments necessary to obtain internationally
accepted penetration rates for regulatory purposes. The key items
discussed were:
the use of percutaneous penetration data in risk assessment;
in in vitro studies, the factors influencing
- the choice of cell characteristics,
- the choice of donor phase,
- receptor fluids;
the presentation of in vitro percutaneous penetration results;
existing guidelines on in vitro percutaneous penetration studies;
prediction of plasma levels from penetration data;
the influence of cutaneous metabolism on skin absorption;
172
criteria for the selection of reference compounds for in vitro
percutaneous penetration;
the use of microdialysis for the determination of percutaneous
penetration of hazardous substances in vivo; and
correlation between in vitro and in vivo experiments.
The members of the Percutaneous Penetration Subgroup ana-
lysed the guidelines on in vitro percutaneous penetration studies
presented by various organizations and suggested a standardization
of in vitro models for percutaneous penetration taking into account
their individual experiences, literature data, and guidelines already
in existence. The subgroup also presented a number of short papers
of up-to-date information on the key issues to focus the existing
knowledge and knowledge gaps in the field of percutaneous pen-
etration. The publication (Sartorelli et al., 2000) is an outcome of
work of this subgroup during the Dermal Exposure Network Project
(19971999).
From this network, two research projects developed that were
funded by the EU. These were RISKOFDERM dermal exposure
monitoring and EDETOX. Within RISKOFDERM: Risk Assess-
ment of Occupational Dermal Exposure to Chemicals, 15 European
institutes/organizations from 10 European member states worked
together with the following major aims (van Hemmen et al., 2003;
van Hemmen, 2004):
to develop a validated/benchmarked predictive model for esti-
mating dermal exposure for use in generic risk assessment for
single chemicals; and
to develop a practical dermal exposure risk assessment and
management toolkit for use by small and medium-sized enter-
prises, and others, in actual workplace situations.
A predictive model for dermal exposure assessment was
produced based on the conceptual model for dermal exposure of
Schneider et al. (1999) (Marquart et al., 2003). In a further part of
the project, a toolkit for hazard, exposure, and risk assessment of
dermal exposure based on label information and material safety data
sheets was developed (Oppl et al., 2003). During this project, a
series of quantitative dermal exposure measurements were carried
out (references published in Annals of Occupational Hygiene,
Appendix 2
173
Volume 48, Number 3, 2004). The next step would be to implement
the results in European regulations on notification of chemicals in
current frameworks and for workplace risk assessments of chemical
compounds.
A2.3 EDETOX (Evaluations and Predictions of Dermal
Absorption of Toxic Chemicals)
EDETOX was a 3-year multipartner EU project (20012003/4)
funded under the Framework V Programme to generate new data on
dermal absorption of chemicals (EDETOX, undated). The consor-
tium comprised 12 participants from seven EU member states. The
project aimed to define robustness and standardize in vitro systems
for predicting percutaneous penetration and to compare these with
relevant in vivo studies, mainly in humans. It also aimed to use the
standardized in vitro methods to generate occupationally relevant
dermal absorption data acceptable for risk assessment and data to
evaluate QSARs and predictive models of skin penetration of health-
related chemicals. A database of evaluated literature and data
generated during the project has been established and used in the
QSAR and predictive models (Williams, 2004a,b). The results of the
EDETOX project were presented in a research day at the Perspec-
tives in Percutaneous Penetration conference in 2004 (see section
A2.5). The full report of the EDETOX project is completed, and a
2004). An updated list of publications is available on the web site.
A2.3.1 Work package 1: Robustness of in vitro methodology
A2.3.1.1 Intra- and interlaboratory variation in in vitro dermal absorption
methodology
A major part of the EDETOX project was a study into the intra-
and interlaboratory variation in dermal absorption data generated by
in vitro methods The dermal absorptions of benzoic acid, caffeine,
and testosterone were determined following a detailed protocol
using human skin (nine laboratories) and rat skin (one laboratory)
(EDETOX, 2004; van de Sandt, 2004). There was some variability
between the results due, to a large extent, to interindividual varia-
bility in absorption between samples of human skin. The results are
summary is available at http://www.ncl.ac.uk/edetox (EDETOX,
discussed in chapter 8. The observations from this study stimulated
174
further investigations of particular aspects of optimization of experi-
mental conditions.
A2.3.1.2 Effects of experimental conditions on dermal absorption
The influence of skin thickness on percutaneous penetration of
caffeine, testosterone, butoxyethanol, and propoxur through human
skin in vitro was investigated (Wilkinson et al., 2004). Skin thick-
ness influenced the absorption rate of lipophilic molecules to a
greater extent than that of less lipophilic molecules. Physicochem-
ical properties must be taken into account when considering skin
preparation.
In vivo (rodent) and in vitro (human and rodent) absorption data
generated for five pesticides of varying lipophilicity in a range of
formulations were considered to assess the contribution of the
stratum corneum reservoir to absorbed chemical for risk estimations.
Inclusion of the material remaining in the stratum corneum appeared
to overpredict the level of absorption in vivo when using the recom-
mended in vivo/in vitro calculation (OConnor & Cage, 2004;
A2.3.2 Work package 2: In vivo studies in volunteers
A2.3.2.1 Human in vivo studies of dermal absorption: their relation to in vitro
prediction
Human in vivo studies have served as the gold standard to
evaluate in vitro results. For a range of chemicals of different
physicochemical properties (2-butoxyethanol [aqueous solutions of
different composition], trichloroethene, m-xylene vapour, and caf-
feine), the percutaneous absorption was determined in parallel in
human volunteers and in vitro with human skin using the same dose,
vehicle, and application time (EDETOX, 2004; Kezic, 2004). The
percutaneous absorption of 2-butoxyethanol from aqueous solution
was much higher than that of neat 2-butoxyethanol (Jakasa et al.,
2004b), and this was predicted from the parallel in vitro studies. In
volunteer studies, dermal absorption was determined using two
different methods: microdialysis and biomonitoring. The comparison
of 2-butoxyethanol data between the laboratories performing the in
vivo experiments in volunteers showed good agreement; the
apparent steady-state flux differed by a factor of 2. The correlation
OConnor et al., 2004) (see chapters 6 and 12).
Appendix 2
175
benzo[a]pyrene, and di(2-ethylhexyl)phthalate at a range of concen-
trations were conducted in the rat (Kezic, 2004).
Parallel studies in which dermal microdialysis and urinary
metabolite excretion were determined following application of 50%
butoxyethanol in water were also conducted. Microdialysis studies
of dermal absorption of pyrene in volunteers were compared with in
vitro studies. The microdialysis technique has been modified for in
vitro tests e.g. studying the absorption of three low molecular
weight (radiolabelled) test compounds (toluene, 50% aqueous
butoxyethanol, and propoxur) using glass diffusion cells (Maas et
al., 2004). In a further experiment, an infinite dose of non-radio-
labelled pyrene in ethanol was investigated using in vitro micro-
dialysis (Wellner & Korinth, 2004).
A2.3.3 Work package 3: In vitro measurement of dermal absorption
In the EDETOX project, finite dose data were generated for 60
chemicals at occupationally relevant exposures using the previously
standardized guidelines for in vitro studies. Chemicals chosen were
from classes for which dermal absorption may pose a risk and risk
assessment will be required. New data included effect of sweat on
absorption of metal, effect of water on butoxyethanol dermal absorp-
tion, effect of vehicle on caffeine and pesticide absorption, dermal
absorption data for an extended series of glycol ethers, natural oils,
and pesticides, and absorption and metabolism of aromatic amines
(EDETOX, 2004).
A2.3.3.1 In vitro dermal absorption of metals
Studies into the in vitro percutaneous absorption of metal pow-
ders (nickel, cobalt, and chromium) (Venier et al., 2004) showed
that metallic ions can easily permeate the skin. Using the Franz cell,
it was possible to measure a flux of ions through the skin for cobalt
and nickel but not for chromium (Larese Filon et al., 2004). A
similar study was described by Sartorelli et al. (2004). Synthetic
sweat promoted the absorption of metals through the skin.
between in vivo and in vitro data was good (see chapters 6, 7, and
8). Parallel in vivo/in vitro studies with butoxyethanol, pyrene,
176
A2.3.3.2 Dermal absorption of pesticides
In an in vitro percutaneous penetration study of five pesticides
covering a range of solubilities and molecular weights, it was shown
that molecular weight as well as solubility affect dermal penetration.
After short-term occupational exposures, an exposure assessment
based on penetrated pesticide at the end of the work shift may under-
estimate the exposure. This suggests that data used by the regulatory
agencies should include maximum flux (or K
p
), lag time, as well as
an estimation of the potential importance of the skin reservoir
(Nielsen et al., 2004a,b). Further studies on pesticides are in the
process of publication.
A2.3.3.3 Dermal absorption of aromatic amines
Absorption and local metabolism and adduct formation of
aromatic amines such as trimethylamine (Kenyon et al., 2004a) and
4,4'-methylenedianiline (Kenyon et al., 2004b) were studied.
A2.3.4 Work package 4: Modelling and prediction of absorption
A2.3.4.1 EDETOX database
A database has been compiled giving details of in vitro and in
vivo studies on percutaneous penetration of chemicals. This data-
in vivo and in vitro percutaneous absorption and distribution data
from all available sources together with the physicochemical data for
each chemical for use in occupational risk assessment. The database
currently contains 4800 studies for 320 chemicals. Data have been
assessed against specific inclusion criteria. Less than half of the
studies satisfied the criteria for required details (Soyei & Williams,
2004).
A2.3.4.2 Modelling of dermal absorption
Because of the complex nature of the skin membrane, the
modelling of dermal absorption was confined to 1) QSARs that
relate the dermal absorption of a molecule to some of its physico-
chemical properties and 2) the modelling of the macroscopic
www.ncl.ac.uk/edetox). The purpose of the database was to compile
base is freely available on the EDETOX web site (http://
Appendix 2
177
behaviour of the absorption process using diffusion equations or
compartmental models.
1) QSARs for dermal absorption
Existing QSARs for percutaneous penetration were evaluated
(Fitzpatrick et al., 2004; Golden et al., 2004). Infinite dose (sat-
urated aqueous) data were generated for 21 chemicals for com-
parison with the data set gathered by Flynn (1990) or by Cronin et
al. (1999) as the basis of predictive QSAR models used today. It was
identified that there was a need for further investigations of the
effect of vehicle, formulation, multiple doses, and mixtures on
absorption.
2) Further modelling of dermal absorption data
Another approach has been to model the absorption process
using diffusion equations or compartmental models that represent
the vehicle, the sink, and the various layers of the skin (Corish,
2004). A comparative analysis of non-steady-state data using two
diffusion models has been presented (Corish et al., 2004; Krse &
Kezic, 2004).
A mechanistically based mathematical model was developed
during the project, which was used to interpret infinite and finite
absorption data for triclosan, malathion, testosterone, parathion, and
caffeine (Corish et al., 2004; J. Krse, unpublished data, 2006). This
model enables the time courses of various variables to be simulated,
and modelling of a variety of exposure regimes, such as single and
multiple dosing from different application forms, can be handled.
The development of these models and their testing through applica-
tion to data measured in the EDETOX project have resulted in
considerable progress towards the development of a more reliable
predictive tool for the estimation of the extent of dermal penetration
of a chemical based on its physicochemical properties.
178
A2.3.5 Work package 5: Dissemination of results and discussion of
skin notation
The EDETOX project showed that outcomes from both
predictive modelling and the design of in vitro protocols influence
generation of data suitable for improving existing QSAR models and
building novel models for dermal absorption. EDETOX recom-
mended a refinement of the in vitro protocol and generation of
further data to improve QSARs. This has also been highlighted by
CEFIC (see section A2.4).
EDETOX found that there was a need for further investigations
of the effect of vehicle, formulation, multiple doses, and mixtures on
absorption and suggested that data should be generated for QSARs
that take into account the effect of vehicles.
Further work was recommended in the area of an EU-wide skin
notation system that might be semiquantitative.
Further research is required to identify and define factors con-
tributing to interindividual variability in dermal absorption in order
to be able to predict how permeable an individuals skin might be.
The importance of local metabolism and generation of toxic
metabolites producing local skin damage such as irritation and
sensitization following penetration was emphasized and should be
further investigated.
EDETOX recommended that future research should concentrate
on development of finite dose models to mathematically define the
profile of dermal penetration through skin both in vitro and in vivo
at exposure relevant to low-level, short-term doses. These should be
extended to investigate the effects of multiple exposures, exposure
to mixtures, and vehicle and formulation effects on the dermal
absorption profile of chemicals. It is important to compare finite and
infinite doses and effects of vehicles on the absorption profile in
detail to provide a database of information with which to validate
this model.
A2.3.5.1 Recommendations from EDETOX (htp://www.ncl.ac.uk/edetox)
Appendix 2
179
EDETOX recommended that the use of the direct modelling
approach should be pursued to assess its use for risk assessment in
areas such as the new EU REACH regulations on use of chemicals.
The EDETOX project concentrated on the use of human skin in
in vitro systems, as results with this can be directly extrapolated to
risk assessment for humans. However, with reduced availability of
human skin for investigation due to ethical issues, further investiga-
tions of alternatives such as pig or rodent skin should be initiated.
A2.3.5.2 Skin notation
Preliminary discussions were held on skin notation concerning
a) the strengths and weaknesses of the present approach to the use of
skin notations in different EU and non-EU countries and b) possible
ways of refining the skin notation in order to make it usable for
prevention in occupational settings (Nielsen & Grandjean, 2004;
Nielsen et al., 2004c).
A2.4 European Chemical Industry Council (CEFIC)
initiatives
A2.4.1 Determination of the optimal physicochemical parameters to
use in a QSAR approach to predict skin permeation rate
Approaches funded by CEFIC and previously published (Patel
& Cronin, 2001; Moss et al., 2002; Patel et al., 2002) were
of this document.
A2.4.2 CEFIC workshop on methods to determine dermal permeation
for human risk assessment (Utrecht, 1315 June 2004) (taken
from Jones et al., 2004)
A2.4.2.1 Introduction
The proposed new European chemicals strategy involving the
Registration, Evaluation, Authorisation and Restriction of Chemicals
(REACH) is likely to involve 10 000 industrial chemicals and many
more mixtures. Data on the potential for dermal uptake are available
for only a small number of these chemicals, and these were often
considered at the workshop and have been discussed in chapter 10
180
obtained not using a standardized protocol. Obtaining human in vivo
or in vitro data on all REACH chemicals is not feasible. The
European Chemical Industry (through CEFIC, the European
Chemical Industry Council) recognized the need to establish a
systematic tiered approach to predict dermal permeation of chemi-
cals for risk assessment.
With this in mind, a meeting sponsored by the CEFIC Long-
range Research Initiative (LRI) was convened to reach a consensus
on methods to determine dermal absorption in vitro, and it was
intended that data produced from a limited number of these studies
would be used to develop a QSAR linking physicochemical proper-
ties to permeation data so that, ideally, it would be possible to
predict the data for a large number of chemicals rather than under-
take expensive testing.
Approximately 20 experts in skin permeation, risk assessment,
and QSAR were invited from academia, contract testing labora-
tories, industry, regulatory agencies, and the EC. At the meeting,
presentations and discussions spanned the assessment of dermal
exposure, permeation measurements in the laboratory, and the
application of permeation data to risk assessment and brought
together the views from two preceding conferences: the April 2004
PPP Conference and a 2004 QSAR meeting (11th International
Workshop on Quantitative StructureActivity Relationships in the
Human Health and Environmental Sciences, 913 May, Liverpool,
Before the meeting, there had been concerns that the QSAR
predictions that had previously been made might be unreliable, as
they had been based on in vitro tests conducted using a variety of
methods over more than a decade. However, discussions at the meet-
ing led to the conclusion that the existing databases for K
p
, despite
some inherent variability due to methodological differences, were
acceptable for derivation of the existing QSARs. Further, it was not
expected that further data would produce any significant revision of
the QSARs. However, it was recognized that the existing database
was primarily from chemicals from specialized sectors or selected
on the basis of physicochemical properties, and the meeting
recommended that generation of in vitro data and K
p
for a range of
relevant industrial chemicals would be valuable in reassuring all
England; http://www.cefic-lri.org).
Appendix 2
181
stakeholders of the validity and relevance of QSARs within the
broad application area of REACH. Chemicals should be selected as
being of greatest relevance to high volume production chemical
manufacture and should provide a good coverage of the range of
physicochemical properties needed to produce data that will support
the development of QSAR models.
Further to this was the discussion of the relevance of infinite
dose studies to realistic risk assessment scenarios, which usually
correspond to finite dose conditions. Data used for QSARs have up
to now been those from infinite in vitro assays, which are most
suitable for establishing a stable maximum flux and calculating a
p
, and which can be related by QSARs to
physicochemical properties.
Therefore, a link must be established between finite and infinite
dose experiments, thus linking the QSAR-derived information with
the inputs required for risk assessment. This link relies on
mathematical modelling, which requires that a sound theoretical
basis is used in the interpretation of data from each experiment and
should improve the reliability of parameters calculated from experi-
mental data. The model also enables extrapolation to predict absorp-
tion under different dosing conditions.
In a further contribution, data for a range of pesticides were
presented, which showed how predictions of absorption (based on
QSAR-predicted K
p
) compare with systemic absorption in vivo
measured in rats (Zendzian, 2000; Reddy & Bunge, 2002). Such
data could be used to predict a safety factor such that the safety
factor multiplied by the predicted systemic absorption would
encompass the majority of the set of measured values for the in vivo
systemic absorption.
A2.4.2.2 Proposed finite and infinite dose protocols (Appendices 2 and 3 of
the CEFIC protocol)
These protocols have been suggested as the most effective and
timely way of obtaining data robust enough to construct a QSAR
model that could be acceptable to all interest groups.
182
A2.4.2.3 Chemicals to be selected for testing
The following recommendations were made to CEFIC by the
meeting members for the selection of chemicals for developing the
acceptance of QSARs as a valid and reliable method of predicting
the permeability for industrial chemicals:
The chemicals should span the physicochemical space of the
parameters log K
ow
from 3 to 7 and molecular weight from 30
to 1000 (an elliptical space on a two-dimensional plot of
chemicals by these two parameters).
The chemicals should be chosen from chemicals that are pro-
duced in high volume in industry.
The chemicals should be chosen in conjunction with industry.
If possible, the chemicals should be radiolabelled versions of
the production chemicals (by selecting chemicals for which
radioactive versions are available).
If some non-radiolabelled chemicals are used (to obtain the
range of physicochemical characteristics), then the chosen
chemicals need to be suitable for sensitive chemical analysis.
A2.4.2.4 Strategy recommendations
The CEFIC Workshop recommended a two-stage strategy:
Stage 1 Build on an already extensive data set for aqueous
soluble chemicals and the QSARs that have been made for these
chemicals.
1) To promote the recognition of the likely reliability of
QSAR predictions of dermal permeation for industrial
chemicals. The CEFIC Workshop suggested a project mea-
suring the permeation of a selection of about 50 chemicals
using the proposed infinite dose protocol.
2) To establish the validity of using the mathematical models
to extrapolate from infinite dose to finite dose. The model
should be used to extrapolate from infinite dose to predict
Appendix 2
183
the results of a finite dose experiment before the results of
the finite dose experiment are available.
It is important, especially for QSAR, that variability be min-
imized. Recent studies concerning robustness and variability (van de
Sandt, 2004; Chilcott et al., 2005) have highlighted the problems
that still exist in the field of in vitro dermal absorption measure-
ments.
The CEFIC Workshop recommended that the first stage would
use a wide range of chemicals with the standard aqueous solution
protocol. The data from this project would be directly comparable
with those of the existing data set already used for the development
of QSARs and could be extrapolated using mathematical models for
comparisons with finite dose data.
Stage 2 of the CEFIC Workshop recommendations involves
more detailed testing of those chemicals in subset 1 together
with more lipophilic chemicals that cannot be tested in the
aqueous solution protocol. Further, there would be investi-
gations into the effects of various donor fluids, receptor fluids,
and mixtures.
The aim is to facilitate the development of models bridging the
results from Stage 1 (standard aqueous solution test conditions) to
Stage 2 (more complex and more realistic exposure conditions for
an extended range of chemicals). A further objective would be a
standardized protocol for chemicals in solution in other liquids.
A2.4.2.5 Recommendations of CEFIC Workshop for use of their proposed
new data in risk assessments
1) Assume either 100% absorption or 10% default assumption for
high molecular weight and log K
ow
<1 or >4; and then, if
necessary,
2) Use saturated water concentration and K
p
to calculate an
estimate of maximum flux, allowing for any effects from the
vehicle; then, if necessary,
3) Use the more complete mathematical model with diffusion
coefficients and partition coefficients to obtain a best estimate
184
of the flux and dose for the likely occupational exposure
concentration (i.e. finite dose).
Future developments in determining dermal permeation should
be conducted in close liaison with developments in dermal exposure
assessment (Kromhout et al., 2004; Semple, 2004).
A2.5 Perspectives in Percutaneous Penetration (PPP)
Conferences
The purpose of the Perspectives in Percutaneous Penetration
(formerly Prediction of Percutaneous Penetration) or PPP Confer-
ences is to provide a forum for the presentation and discussion of all
the latest research, development, and technology concerning the
penetration of exogenous compounds through the skin. The meetings
are of relevance and interest to those involved in dermatological
research, the development of formulations, risk assessment, and
regulatory affairs relating to the dermal effects of molecules within
the agrochemical, cosmetic, and pharmaceutical fields within indus-
trial, academic, and governmental domains. The PPP Conferences
The first Prediction of Percutaneous Penetration Conference
took place in Manchester, United Kingdom, in 1989, the second
conference in Southampton, United Kingdom, in 1991, and the third
and fourth in La Grande Motte, France, in 1993 and 1995.
Subsequently, the conference was renamed Perspectives in Percuta-
neous Penetration, and conferences have taken place in 1997, 1998,
2000, 2002, and 2004. Proceedings of the first five conferences were
published in full (Scott et al., 1990, 1991b; Brain et al., 1993, 1996,
1998b), but the later conference publications (Vol. 6b [1998], Vol.
7b [2000], Vol. 8b [2002], Vol. 9a [2004]) were abstracts only. The
10th conference is to be held in April 2006 in La Grande Motte.
A2.6 Gordon Research Conferences
The Gordon Research Conferences provide an international
forum for the presentation and discussion of frontier research in the
biological, chemical, and physical sciences and their related
technologies. The Gordon Research Conferences on the Barrier
Function of Mammalian Skin were established in 1989 to provide
are biannual (http://www.pppconference.org).
Appendix 2
185
an interdisciplinary scientific forum focusing on the development,
structure, and function of the barrier of mammalian skin. The
conferences take place every 2 years (e.g. 1999, 2001, 2003, 2005).
The next conference is scheduled for August 2007 in Rhode Island,
USA. The programme of lectures and speakers for the last 10 years
can be found through the Gordon Research Conferences home page
(http://www.grc.uri.edu).
A2.7 International Conference on Occupational and
Environmental Exposures of Skin to Chemicals
The first meeting (September 2002) in Washington, DC, USA,
was organized by the National Institute for Occupational Safety and
Health and was convened to bring together scientists with many
areas of expertise (dermatologists, occupational hygienists, labora-
tory researchers, policy-makers, and occupational physicians) to
focus on the science, knowledge gaps, and policy opportunities
related to occupational and environmental exposures of the skin to
chemicals (Chen & Sartorelli, 2005).
A second meeting was held in June 2005 in Stockholm, Sweden
and a workshop were held for each of six themes: 1) Irritants and
Wet Work, 2) Quantitative Risk Assessment, 3) Exposure Reduc-
tion, 4) Process-Based, Qualitative Risk Assessment, 5) Systemic
Toxics, and 6) Allergens.
(http://www.cdc.gov/niosh/topics/skin/OEESC2). A plenary session
186
RESUME
On dsigne par lexpression gnrale de rsorption ou
dabsorption dermique, cutane ou percutane le transport de
substances chimiques de la surface externe de la peau vers lintrieur
du revtement cutan et la circulation gnrale. Le prsent document
de la srie des Critres dhygine de lenvironnement donne une vue
densemble de labsorption percutane et de son application
lvaluation du risque que reprsentent les substances chimiques. Il
expose et analyse en outre un certain nombre de points intressants
concernant labsorption percutane. Celle-ci peut se produire
loccasion dune exposition des substances chimiques, des cos-
mtiques ou des produits pharmaceutiques lors de lexercice dune
activit professionnelle, dans lenvironnement ou encore lorsquun
consommateur expose sa peau des produits de ce genre.
Membrane vivante, la peau est un organe complexe. Elle assure
des fonctions de protection, de rgulation de la temprature centrale
et des pertes en eau, de dfense et de rparation. La peau est
constitue dune rgion externe, lpiderme, et dune rgion interne,
le derme. Lpiderme comporte diverses couches cellulaires dont la
plus extrieure, appele stratum corneum ou couche corne,
reprsente la principale barrire la pntration des substances
chimiques trangres. Lorsquil est fonctionnel, lpiderme est
capable de mtaboliser les substances chimiques qui traversent la
couche corne. Le derme, qui sert de substrat physiologique lpi-
derme avasculaire, est parcouru par les vaisseaux sanguins, les nerfs
sensitifs et les vaisseaux lymphatiques cutans. La peau comporte
galement un certain nombre dannexes telles que les follicules
pileux ou encore les glandes sudoripares et les glandes sbaces qui
prennent naissance dans le derme sous-papillaire.
La mesure de la permabilit cutane donne des rsultats
extrmement variables. Les diffrences peuvent tre considrables
dune espce lautre. On sait peu de choses au sujet des variations
en fonction de lge, sinon que la structure du revtement cutan
volue avec celui-ci; en revanche, il ne semble pas que la perm-
abilit cutane varie avec le sexe ou lappartenance ethnique. La
rsorption percutane dpend galement de la localisation anatom-
ique, de ltat du tgument et de son degr dhydratation.
Rsum
187
Au nombre des facteurs qui jouent un rle dans labsorption
percutane figurent : 1) les proprits physico-chimiques de la
substance tudie; 2) les proprits physico-chimiques et autres du
vhicule dans lequel cette substance est dissoute; 3) les interactions
entre la substance tudie et le revtement cutan; 4) les proprits
de la peau et le mtabolisme cutan; 5) certains facteurs lis au
systme dpreuve dans lequel seffectue la mesure par exemple,
dose et volume de la substance tudie, occlusion ou non occlusion
du territoire cutan tudi, systme dpreuve in vitro ou in vivo ou
encore dure dexposition.
Des quations et des modles thoriques ont t tablis pour
rendre compte du transport dune substance chimique qui diffuse
travers la peau. En rgle gnrale, le flux en rgime stationnaire (J
ss
)
et le coefficient de permabilit (K
p
) sont les principaux paramtres
que lon dtermine dans les tests in vitro au cours desquels on
sarrange pour que la concentration de la substance pntrante
assure le maintien dune valeur constante (infinie) de la dose. On
admet dsormais que la dtermination du flux maximum, du temps
ncessaire pour que le flux atteigne sa valeur maximale, du temps de
raction, des teneurs rsiduelles retenues dans la couche corne et
du bilan massique dans des conditions dapplication relles sont
de premire importance pour lestimation de lexposition.
La mesure du mtabolisme dune substance chimique en contact
avec la peau peut tre importante pour lvaluation de la scurit
comme de lefficacit. Certaines substances peuvent tre mtabo-
lises de manire apprciable lors de la rsorption intradermique,
processus qui peut conduire la formation de mtabolites actifs ou
inactifs. Il est donc important de mesurer cette mtabolisation pour
une bonne valuation de la scurit. On a montr que des produits
chimiques toxiques comme le benzo[a]pyrne subissaient une
activation cutane, dautres substances tant susceptibles dtre
hydrolyses ou conjugues dans le derme avec pour consquence
une moindre disponibilit dans lorganisme. En gnral, il est
possible de conserver sa viabilit un chantillon de peau plac
dans une cellule de diffusion in vitro condition dutiliser un tgu-
ment frais et un tampon physiologique. Il est recommand de
vrifier la viabilit de lchantillon en mesurant lactivit des
enzymes de mtabolisation appropries.
188
Les paramtres qui caractrisent la permabilit de la couche
corne restent, pour la plupart, inchangs lorsquun chantillon est
prlev sur le corps. Il sensuit quil existe une bonne corrlation
entre les mesures effectues in vitro et in vivo sur les mmes
substances chimiques (du moins dans le cas de substances hydro-
philes). Les tests in vitro peuvent tre valablement substitues aux
tests in vivo et prsentent un certain nombre davantages par rapport
lexprimentation sur des animaux entiers ou sur des sujets
humains volontaires. Les mthodes in vitro mesurent la diffusion des
substances chimiques dans et travers la peau jusqu un comparti-
ment liquidien et on peut utiliser des chantillons non viables pour
mesurer uniquement la diffusion ou des chantillons frachement
prlevs et mtaboliquement actifs pour dterminer simultanment
la diffusion et la mtabolisation intradermique. La ligne directrice
exprimentale 428 de lOrganisation pour la coopration et le
dveloppement conomiques (OCDE) prconise lharmonisation des
mthodes de mesure. Outre ceux qui sont indiqus plus haut,
dautres facteurs exprimentaux influent sur labsorption percutane
in vitro, savoir lpaisseur de lchantillon de peau, les variations
de temprature lintrieur du systme dpreuve et la composition
du liquide rcepteur. On peut utiliser des cellules de diffusion
statiques ou coulement continu. Il existe aussi dautres tech-
niques, qui ncessitent des dispositifs plus labors, telles que le
prlvement par ruban adhsif ou lutilisation dune peau artificielle
ou reconstitue.
Les mthodes in vivo permettent de dterminer la rsorption
percutane de la substance tudie ainsi que son passage dans
lorganisme. Ces tudes in vivo ont, par rapport aux tests in vitro, le
principal avantage dutiliser un systme dpreuve physiologique-
ment et mtaboliquement intact. Les tudes de pntration
percutane effectues in vivo utilisent des animaux de laboratoire,
des rongeurs en gnral, mais peuvent galement tre pratiques sur
des sujets humains volontaires. Cette exprimentation sur volon-
taires humains est largement pratique pour ltude des produits
pharmaceutiques et, dans une moindre mesure, dautres produits
chimiques. Sur le plan mthodologique, le nec plus ultra est lexpri-
mentation humaine. Toutefois, toute tude in vivo soulve des
questions dordre thique. Le principal inconvnient de lexpri-
mentation sur animaux de laboratoire tient au fait quils ont une
Rsum
189
permabilit cutane et une absorption systmique diffrentes de
celles de lHomme.
Des tudes sur volontaires humains montrent que lexposition
professionnelle des liquides (solvants, par exemple) peut entraner
une trs forte absorption percutane. Labsorption percutane par
exposition des vapeurs peut contribuer de manire importante
labsorption totale de certains produits volatils, comme les thers de
glycol.
Les rsultats dtudes in vitro sur labsorption percutane sont
de plus en plus souvent communiqus aux autorits responsables en
vue de lhomologation de produits chimiques industriels, de cos-
mtiques ou encore de produits phytiatriques ou phytopharmaceu-
tiques. De nombreuses tudes comparant labsorption percutane in
vitro et in vivo chez lHomme et lanimal ont t publies.
Lorsquelles sont correctement effectues en suivant les lignes
directrices de lOCDE pour ce genre dexprimentation, les tudes
in vitro permettent une bonne prvision de labsorption in vivo.
On a accumul depuis plusieurs dcennies une somme impor-
tante de donnes relatives la pntration percutane de nombreux
produits chimiques, pesticides, cosmtiques et produits pharma-
ceutiques. Ces donnes rsultent dtudes sur volontaires humains,
dexprimentations in vivo sur modles animaux, dtudes in vitro
utilisant des chantillons de peau prlevs par exemple sur des sujets
humains, des rongeurs, des porcs ou des cobayes et, plus rcemment,
dtudes in vitro sur peau synthtique.
Au cours des cinquante dernires annes, on a tent, maintes
reprises, de faire des prvisions au sujet de la vitesse et de lampleur
de labsorption percutane de manire rduire les tudes in vitro
ou in vivo ncessaires. Cest l un besoin encore plus important au
vu de la multiplication des problmes dthique poss par lexpri-
mentation humaine et animale comme dailleurs des considrations
dordre conomique et chronologique imposes par la rglemen-
tation. Les relations quantitatives structure-permabilit (RQSP)
sont des relations de nature statistique entre le flux en rgime
stationnaire dun compos donn et divers descripteurs physico-
chimiques ou proprits structurales de la molcule. Les relations
quantitatives structure-activit (RQSA) sont galement prises en
190
compte dans les considrations relatives la scurit et lefficacit,
notamment en ce qui concerne lirritation, la sensibilisation cutane,
le mtabolisme, les effets chimiques et la clairance. Les RQSA inter-
viennent donc plusieurs stades de lvaluation de la scurit
chimique.
On fait appel des modles mathmatiques pour simuler la
dynamique du partage, de la diffusion, du mtabolisme dautres
processus qui interviennent dans labsorption percutane, ce qui peut
permettre de prdire dans quelle proportion et quelle vitesse une
substance chimique donne va pntrer travers le revtement
cutan. Ces modles mathmatiques sont dune importance primor-
diale pour tablir la relation qui lie le coefficient de permabilit et
les donnes de flux fournies par lexprimentation en rgime station-
naire (cest--dire dose infinie) aux estimations de labsorption en
prsence de doses finies qui sont plus reprsentatives de lexposition
professionnelle (cest--dire en rgime non stationnaire).
Lorsquon procde lvaluation du risque, lestimation initiale
de labsorption percutane est habituellement obtenue en oprant en
plusieurs stades, la marge de scurit maximale tant dfinie par le
cas le plus critique et des estimations plus labores permettant une
meilleure approximation de la marge relle. Dans un premier temps,
on suppose donc que labsorption est de 100 % en labsence de toute
donne. On procde ensuite une deuxime valuation plus raliste
de labsorption percutane en prenant en compte les proprits
physico-chimiques de la substance tudie et de son vhicule. La
troisime stade comporte la prise en considration de toute donne
exprimentale sur labsorption percutane tire de tests in vitro ou in
vivo. Si, lissue de ces trois tapes, le calcul montre que le risque
est inacceptable, le meilleur moyen den affiner lvaluation consiste
sappuyer sur les valeurs effectives de lexposition.
Au cours des dernires annes, il y a eu, pour une part sous
linfluence de la rglementation, un certain nombre dinitiatives en
vue de faire progresser plus rapidement lharmonisation au niveau
international des mthodes et des protocoles exprimentaux. Ces
initiatives ont abouti en 2004 la publication par lOCDE de lignes
directrices exprimentales applicables aux tudes sur labsorption
percutane. Cette coopration internationale se traduit par un certain
nombre de projets tels quune tude internationale de validation
Rsum
191
laquelle participent 18 laboratoires, le projet europen EDETOX
(European Evaluations and Predictions of Dermal Absorption of
Toxic Chemicals), ainsi que divers autres projets subventionns par
lindustrie ou encore des confrences telles que la confrence
intitule Perspectives in Percutaneous Penetration (prcdemment
intitule Prediction of Percutaneous Penetration ou PPP) ou les
Gordon Research Conferences. Les donnes existantes au sujet du
flux transcutan et du coefficient dabsorption ont t recueillies
dans des bases de donnes et analyses. On a galement progress
dans llaboration de RQSA permettant de relier la pntration
transcutane aux proprits physico-chimiques des substances chim-
iques. On parvient ainsi prdire de mieux en mieux la pntration
transcutane dun grand nombre de produits chimiques. Ces
dveloppements peuvent vraisemblablement contribuer rduire
lexprimentation humaine et animale des produits chimiques qui est
coteuse et exigeante sur le plan thique.
Malgr les succs obtenus jusquici dans lharmonisation inter-
disciplinaire au niveau international, des amliorations peuvent
encore tre apportes sur un certain nombre de points qui sont
encore en discussion. Il sagit notamment du degr de variation
intralaboratoire et interlaboratoires des rsultats dtudes in vivo et
in vitro, de lacceptation des RQSP, de leffet de rservoir des
substances chimiques dans la couche corne et de son interprtation
pour lvaluation du risque, de lintrt des mesures de labsorption
percutane eu gard aux donnes demandes par ceux qui sont
chargs de lvaluation du risque et enfin de lutilisation du test
dintgrit de la barrire cutane pour valuer la fonction de
protection de la peau. Parmi les autres points envisager figurent la
valeur de labsorption percutane dans les populations sensibles, la
ncessit dharmoniser le systme de notation cutane et labsorp-
tion percutane des nanoparticules.
Le Groupe de travail a formul des recommandations sur les
points suivants : intrt dutiliser des chantillons de peau humaine
plutt que de peau animale; conception des tudes et harmonisation
de la mthodologie; corrlation entre les donnes in vivo et in vitro
et laboration de modles prdictifs fiables; incitation soutenir,
grer et actualiser les bases de donnes; poursuite de lvaluation
des RQSA aux fins de lvaluation du risque et prparation dun
guide en vue de leur utilisation.
192
RESUMEN
Absorcin cutnea (percutnea, a travs de la piel) es un
trmino general que describe el transporte de sustancias qumicas
desde la superficie externa de la piel a su interior y a la circulacin
sistmica. El presente documento de la serie de Criterios de Salud
Ambiental contiene un panorama general de la absorcin cutnea y
su aplicacin a la evaluacin del riesgo de las sustancias qumicas.
Adems, se exponen y examinan temas actuales de inters en esta
esfera. La absorcin cutnea se puede producir a partir de la
exposicin de la piel a productos qumicos, cosmticos y farma-
cuticos en el trabajo, en el medio ambiente o en el consumo.
La piel es un rgano complejo y una membrana viva. Tiene
funciones de proteccin, de regulacin de la temperatura corporal y
la prdida de agua y de defensa y reparacin. Se compone de una
regin externa, la epidermis, y una regin interna, la dermis. La
epidermis est formada por varias capas de clulas, la ms externa
de las cuales, el estrato crneo o capa crnea, funciona como barrera
principal para la entrada de sustancias qumicas extraas. La
epidermis viable puede metabolizar las sustancias qumicas que
pasan a travs del sustrato crneo. La dermis proporciona apoyo
fisiolgico a la epidermis no vascular y en ella se localizan los vasos
sanguneos, los nervios sensoriales y los vasos linfticos cutneos.
La piel contiene tambin otros apndices, como los folculos
pilosos, las glndulas sudorparas y las glndulas sebceas, cuya
formacin tiene lugar en la dermis subpapilar.
En la medicin de la permeabilidad cutnea hay una variabili-
dad considerable. Puede haber diferencias importantes en la permea-
bilidad entre especies. Es poco lo que se conoce acerca de la
variacin debida a la edad, aunque la estructura de la piel cambia
con el tiempo; sin embargo, la condicin sexual o tnica no parece
ser fuente de variacin de la permeabilidad. La absorcin percutnea
depende asimismo del lugar anatmico, del estado de la piel y de su
hidratacin.
Los factores que influyen en la absorcin percutnea a travs de
la piel son los siguientes: 1) propiedades fisicoqumicas del
Resumen
193
compuesto de prueba; 2) propiedades fisicoqumicas y de otro tipo
del vehculo en el que est disuelto dicho compuesto; 3) inter-
acciones entre este compuesto o el vehculo y la piel; 4) propiedades
y metabolismo de la piel; y 5) factores inherentes al sistema de
prueba utilizado para la medicin, por ejemplo la dosis y el volumen
de la sustancia de prueba, la oclusin o no de la zona de prueba, la
utilizacin de sistemas de prueba in vitro o in vivo y la duracin de
la exposicin.
Se han elaborado ecuaciones y modelos tericos para describir
el transporte de una sustancia qumica por difusin a travs de la
piel. Los principales parmetros evaluados en los experimentos in
vitro son normalmente el flujo estacionario (J
ss
) y el coeficiente de
permeabilidad (K
p
), en los cuales la concentracin de la sustancia
penetrante en la solucin donante se mantiene en condiciones de
dosificacin (infinita) constante. Ahora se reconoce que la esti-
macin del flujo mximo, el tiempo necesario para alcanzar el flujo
mximo, el tiempo de demora, las cantidades residuales (reservorio)
retenidas en el estrato crneo y el balance de masa en condiciones
de aplicacin reales son de importancia capital para las estimaci-
ones de la exposicin.
La medicin del metabolismo de una sustancia qumica en
contacto con la piel puede ser importante en las evaluaciones tanto
de la eficacia como de la inocuidad. Algunas sustancias qumicas se
pueden metabolizar de manera significativa durante la absorcin
cutnea, lo que podra dar lugar a la formacin de metabolitos
inactivos o activos. Por consiguiente, la medicin de este metabo-
lismo puede ser importante en una evaluacin apropiada de la
inocuidad. Se ha demostrado que sustancias qumicas txicas como
el benzo[]pireno se activan en la piel, mientras que otras pueden
sufrir reacciones de hidrlisis y/o conjugacin, con la consiguiente
disminucin de la disponibilidad de esas sustancias por el organ-
ismo. En general se puede mantener la viabilidad de la piel en una
celda de difusin in vitro utilizando piel fresca y un tampn
fisiolgico. Se recomienda verificar esta viabilidad mediante la
medicin de la actividad de las enzimas metablicas correspon-
dientes.
La mayor parte de las propiedades de permeabilidad del estrato
crneo se mantienen inalteradas despus de su extraccin del
194
organismo. As pues, existe una buena correlacin entre las
mediciones obtenidas en experimentos de difusin cutnea tanto in
vivo como in vitro utilizando las mismas sustancias qumicas (por lo
menos para los compuestos hidroflicos). Los experimentos in vitro
son un sistema sustitutivo adecuado de los estudios in vivo y ofrecen
varias ventajas sobre los experimentos con animales enteros o
voluntarios humanos. Con los mtodos in vivo se mide la difusin de
las sustancias qumicas hacia el interior de la piel y a travs de ella
hasta alcanzar un reservorio fluido y se puede utilizar piel no viable
para medir slo la difusin o piel fresca metablicamente activa para
medir al mismo tiempo la difusin y el metabolismo cutneo. La
Directriz 428 de Prueba de la Organizacin de Cooperacin y
Desarrollo Econmicos (OCDE) alienta la armonizacin de la
metodologa. Otros factores experimentales que afectan a la
absorcin cutnea in vitro, adems de los ya mencionados, son el
grosor de la muestra de piel, las variaciones de temperatura en el
sistema de prueba y la composicin del fluido receptor. Se pueden
utilizar celdas de difusin in vitro estticas o de flujo continuo.
Otras tcnicas que requieren un ulterior perfeccionamiento son la
extraccin con tiras adhesivas (tape stripping) y la utilizacin de
piel artificial o reconstituida.
Los mtodos in vivo permiten determinar el grado de absorcin
cutnea, as como sistmica, de la sustancia de prueba. La ventaja
principal de realizar un estudio in vivo en lugar de in vitro es que se
utiliza un sistema intacto desde el punto de vista fisiolgico y
metablico. Se realizan estudios de penetracin cutnea in vivo en
animales de laboratorio, normalmente roedores, y en voluntarios
humanos. Estos ltimos se han utilizado ampliamente para productos
farmacuticos de aplicacin humana y en menor medida para otras
sustancias qumicas. El nivel mximo de garanta se obtiene
mediante estudios in vivo con personas. La realizacin de cualquier
estudio in vivo conlleva cuestiones ticas. La principal desventaja de
utilizar animales de laboratorio es que tienen una permeabilidad
cutnea y una disposicin sistmica diferentes en comparacin con
las personas.
Los resultados de los estudios realizados con voluntarios
humanos han puesto de manifiesto que la exposicin ocupacional a
lquidos (como disolventes) puede dar lugar a una absorcin cutnea
considerable. La absorcin cutnea a partir de vapores puede
Resumen
195
contribuir de manera importante a la total de algunas sustancias
voltiles, como los teres de glicol.
Cada vez se presentan ms estudios de absorcin cutnea in
vitro con fines de registro de sustancias qumicas industriales,
cosmticos y productos de proteccin de los cultivos. Se han
publicado numerosos estudios en los que se comparan los resultados
obtenidos in vitro e in vivo con animales de laboratorio y con
personas. Los estudios in vitro realizados de manera adecuada
siguiendo las directrices de prueba de la OCDE han puesto de
manifiesto que los mtodos in vitro pueden proporcionar una buena
prediccin de la absorcin cutnea in vivo.
Durante decenios se ha obtenido informacin abundante sobre
la penetracin percutnea de un gran nmero de sustancias qumicas,
plaguicidas, cosmticos y productos farmacuticos. Los estudios han
incluido experimentos con voluntarios humanos, estudios in vivo
con modelos animales, estudios in vitro con piel extrada por
ejemplo de personas, roedores, cerdos y cobayas, y ms reciente-
mente estudios in vitro con piel sinttica.
Durante los 50 ltimos aos se han realizado numerosos
intentos de predecir la velocidad y el grado de la absorcin cutnea,
para de esta manera reducir la necesidad de realizar pruebas in vitro
e in vivo. Esta necesidad es incluso mayor en respuesta a las
crecientes dificultades ticas con respecto a la realizacin de experi-
mentos con personas y animales de laboratorio, as como a las
consideraciones econmicas y temporales impuestas por la legis-
lacin, sobre todo con respecto a la evaluacin del riesgo de las
sustancias qumicas industriales. La relacin cuantitativa entre la
estructura y la permeabilidad es una relacin derivada estadstica-
mente entre el flujo estacionario de un compuesto y diversos
descriptores fisicoqumicos y/o propiedades estructurales de la
molcula. En las consideraciones relativas a la eficacia y la inocui-
dad tambin se reconoce la relacin cuantitativa entre la estructura y
la actividad en relacin con la irritacin, la sensibilizacin cutnea,
el metabolismo, los efectos qumicos y la eliminacin. Por
consiguiente, este tipo de relacin interviene en varios niveles de la
inocuidad qumica.
196
Se han utilizado modelos matemticos para simular la dinmica
de la distribucin, difusin, metabolismo y otros procesos que
intervienen en la absorcin cutnea y pueden conducir a la predic-
cin del grado y la velocidad de la permeacin qumica a travs de
la piel. La creacin de modelos matemticos desempea una funcin
esencial en la vinculacin del coeficiente de permeabilidad y los
datos relativos al flujo obtenidos en pruebas realizadas en
condiciones estacionarias (es decir, dosis infinita) con la estimacin
de la absorcin para las aplicaciones de dosis finitas que son ms
habituales en la exposicin ocupacional (es decir, condiciones no
estacionarias).
En la evaluacin del riesgo, la estimacin inicial de la absorcin
cutnea se suele obtener mediante la utilizacin de un mtodo
estratificado, en el que el mayor margen de inocuidad se define por
el caso peor y las estimaciones ms ajustadas definen mejor el
margen real. Por consiguiente, como primer paso se supone que la
absorcin es del 100% cuando no se dispone de datos. En segundo
lugar se proporciona un estimacin ms realista del grado de
absorcin cutnea mediante un anlisis de las propiedades fisico-
qumicas de la sustancia qumica y del vehculo. En tercer lugar se
realiza un estudio de todos los datos experimentales de absorcin
cutnea in vitro e in vivo. Si al final de estas etapas se calcula un
riesgo inaceptable, la mejor manera de ajustar la evaluacin del
riesgo es mediante los datos de exposicin real.
En los ltimos aos, debido en parte a presiones normativas, ha
habido varias iniciativas para acelerar los progresos en la armoniza-
cin internacional de la metodologa y los protocolos, que cul-
minaron con la publicacin en 2004 de las directrices de prueba de
la OCDE para los estudios de absorcin cutnea. Esta colaboracin
internacional incluye proyectos tales como un estudio de validacin
internacional con la participacin de 18 laboratorios, el proyecto de
evaluaciones europeas y predicciones de la absorcin cutnea de
sustancias qumicas txicas (EDETOX) y proyectos patrocinados
por la industria, as como conferencias, por ejemplo sobre las
Perspectivas en la penetracin percutnea (anteriormente, Predic-
cin de la penetracin percutnea, o PPP) y las Gordon Research
Conferences. Los datos disponibles sobre los flujos cutneos y los
coeficientes de permeabilidad se han recogido en bases de datos y se
han analizado. Se ha avanzado en el perfeccionamiento ulterior de
Resumen
197
las relaciones cuantitativas entre la estructura y la actividad que
vinculan los datos de permeacin con las propiedades fisicoqumicas
de las sustancias qumicas. Por consiguiente, cada vez son mayores
las posibilidades de predecir de manera ms fidedigna los datos de
penetracin para un gran nmero de sustancias qumicas. Un posible
resultado es la reduccin de las pruebas que son costosas y exigentes
desde el punto de vista tico de las sustancias qumicas utilizando
animales de laboratorio y personas.
A pesar del xito alcanzado hasta ahora en la armonizacin
internacional interdisciplinaria, hay algunos aspectos susceptibles de
mejora y que siguen siendo objeto de debate. Son el grado de
variacin en los estudios in vitro e in vivo dentro de un laboratorio y
entre laboratorios; la aceptacin de la relacin cuantitativa entre la
estructura y la permeabilidad; el efecto de reservorio de las
sustancias qumicas en el estrato crneo y su interpretacin en la
evaluacin del riesgo; la pertinencia de las mediciones de la
absorcin cutnea para los datos que necesitan los evaluadores del
riesgo; y la utilizacin de la prueba de integridad de la barrera para
la funcin protectora de la piel. Otros temas que se pueden examinar
son la absorcin cutnea en poblaciones susceptibles, la necesidad
de armonizar la anotacin cutnea y la absorcin cutnea de nano-
partculas.
El Grupo de Trabajo formula recomendaciones sobre los bene-
ficios de la utilizacin de la piel humana en comparacin con la piel
de animales de laboratorio; la formulacin del estudio y la armoni-
zacin de la metodologa; la correlacin de los datos obtenidos in
vitro e in vivo y la creacin de modelos de prediccin fidedignos; el
fomento del apoyo, el mantenimiento y la actualizacin de las bases
de datos; y la promocin de la evaluacin de la relacin cuantitativa
entre la estructura y la permeabilidad con fines de evaluacin del
riesgo y la preparacin de orientaciones sobre su utilizacin.

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not usually important in humans (see chapter 3).

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