r/EPA
Urrtsri States
Environments! Protection
Agency
Office of Pesticides and
Toxic Substances
Washington, D.C. 204(10
Pesticidss and Toxic Substances
EPA-560/11-82-002
January 1982
Selected Issues in Testing for Dermal Toxicity, Including Irritation,
Sensitization, Phototoxicity, and Systemic Toxicity
EPA 560/11-82-00:
-------
This report was prepared as an account of work spon-
sored by an agency of the United States Government.
Neither the United States Government nor any agency
thereof, nor any of its employees, contractors, subcontrac-
tors, or their employees, makes any warranty, expressed or
implied, nor assumes any legal liability or responsibility for
the accuracy or completeness of the information contained
herein nor for any third party's use or the results of such use
of any information, apparatus, product, or process dis-
closed in this report, nor represents that its use by such third
party would not infringe privately owned rights.
This report has been reviewed by the Office of Pesti-
cides and Toxic Substances, U .S. Environmental Protection
Agency, and approved for publication. Approval does not
signify that the contents necessarily reflect the views and
policies of the United States Government or the U.S. Envi-
ronmental Protection Agency, nor does mention of trade
names or commercial products constitute endorsement or
recommendation of use.
-------
EPA 560/11-82-002
January 1982
DERMATOTOXICITY
Selected Issues in Testing for Dermal Toxicity,.
Including Irritation, Sensitization, Phototoxicity,
and Systemic Toxicity
S. Chaube
K. J. Falahee
C. S. Rose
H. E. Seifried
T. J. Taylor
J. A. Winstead
Contract No. 68-01-6176
Project Officer - Charles Auer
Technical Advisors - Norbert P. Page, Daljit Sawhney
Assessment Division
Office of Pesticides and Toxic Substances
Washington D.C., 20460
OFFICE OF PESTICIDES AND TOXIC SUBSTANCES
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON D.C. 20460
-------
TABLE OF CONTENTS
PREFACE vii
EXECUTIVE SUMMARY ix
UNRESOLVED ISSUES AND RESEARCH RECOMMENDATIONS xiii
DERMAL IRRITATION
1.0 SUMMARY 1
2.0 INTRODUCTION 2
2.1 Objective 2
2.2 Definitions 2
2.3 Historical . r 2
2.4 pH of Test Agents 7
3.0 SPECIES SELECTION 9
3.1 Animal Tests 9
3.2 Interspecies Comparison 9
4.0 TESTING PROCEDURES 18
4.1 Patch Test Unit 18
4.1.1 Immersion 27
4.1.2 Occlusion 27
4.1.3 Conclusions on Patch Test Unit 28
4.2 Abrasion . . . 29
4.3 Application of Dry Test Substances 33
4.4 Application Site 35
4.5 Exposure and Observation Period 35
4.6 Enhancements to Visual Scoring 37
5.0 REPRODUCIBILITY AND SCORING 38
5.1 Reproducibility 38
5.2 Other Scoring Methods 39
5.3 Recommendations 40
6.0 CONCLUSIONS 41
7.0 REFERENCES 42
-------
TABLE OF CONTENTS (Continued)
TABLES
1-1 Evaluation of Skin Reactions 4
1-2 Primary Irritant Response Categories in the Rabbit ... 4
1-3 Comparison of Methods Used for Dermal Irritation .... 5
1-4 Summary of Primary Irritation Scores 10
1-5 Relative Irritancy, Man vs Seven Other Species 13
1-6 Ranking of Skin Irritation Results With Surfactants On
Human and Animal Skin 14
1-7 Irritation Response of Human, Guinea Pig, and Rabbit
Skin to Chemicals/Household Products 15
1-8 Assessment of Irritation Mouse Ear 16
1-9 Summary of Testing Procedures Used in Dermal Studies . . 19
1-10 Four-Hour Patch Test Results 30
1-11 Effect of Abrasion on Skin Irritation in Humans .... 34
FIGURES
1 Patch Test Unit 25
2 Al Test 25
3 Finn Chamber 26
4 KI Chamber 26
DERMAL SENSITIZATION
1.0 SUMMARY 47
2.0 INTRODUCTION 49
2.1 Objectives 49
2.2 Scope 49
2.3 Definitions 50
2.4 The Sensitization Test 50
2.5 Methods for Sensitization 50
2.6 Experimental Animal 51
3.0 PREDICTIVE TEST METHODS FOR SKIN SENSITIZATION USING
GUINEA PIGS 52
3.1 Draize Test 52
3.2 Freund's Complete Adjuvant Test . 56
3.3 Open Epicutaneous Test 57
3.4 Closed Patch Test 58
3.5 Maximization Test 59
3.6 Optimization Test . 61
3.7 Split-Adjuvant Test 62
3.8 Footpad Test 64
4.0 COMPARATIVE RESULTS OF SENSITIZATION TESTS IN THE GUINEA PIG 66
111
-------
TABLE OF CONTENTS (Continued)
5.0 HUMAN SKIN SENSITIZATION TEST PROCEDURES 74
5.1 Schwartz and Schwartz-Peck Test 74
5.2 Shelanski-Shelanski Test 74
5.3 Modified Draize Test 74
5.4 Maximization Test 77
6.0 APPLICATION OF PREDICTIVE TESTS TO KNOWN CONTACT ALLERGENS . 79
6.1 Human Tests 79
6.2 Guinea Pig Tests 79
7.0 USE OF HUMAN TESTS 84
8.0 CONCLUSIONS 87
9.0 RECOMMENDATIONS 88
10.0 REFERENCES 89
TABLES
2-1 Predictive Tests for Guinea Pig Skin Sensitization ... 53
2-2 Comparative Results of Sensitization Test in Guinea
Pig 68
2-3 Percent of Animals Reacting per Group in the
Optimization, Maximization and Epidermal Test 70
2-4 Comparison of Six Protocols for Development
of Delayed Hypersensitivity to Seven Compounds 71
2-5 Allergenicity of 32 Incriminated Compounds for
Humans and Determinations of Their Allergenicity in the
Guinea Pig by Four Test Methods 73
2-6 Predictive Patch Tests for Human Skin Sensitization . . 75
2-7 Predictive Patch Test in Humans 80
2-8 Reproducibility of Maximization Test Grades in Humans . 81
2-9 Grades of Allergenic Potency by the Maximization
Tests in Humans and Guinea Pigs 82
2-10 Sensitization Rate in Guinea Pig Closed Patch Test .
and the Human Repeated Insult Patch Test 83
2-11 Evaluation of Some Common Sensitizers by the
Diagnostic and Predictive Tests 85
IV
-------
TABLE OF CONTENTS (Continued)
Page
PHOTOTOXICITY
1.0 SUMMARY 93
2.0 PHDTOSENSITIVITY 94
3.0 PHOTOTOXICITY TESTING 97
3.1 Animal Models 97
3.2 Human Testing . . . 97
4.0 PHDTOALLERGY TESTING 101
4.1 Animal Models 101
4.2 Human Testing 101
5.0 CONCLUSIONS 103
6.0 REFERENCES 104
TABLES
3-1 Comparison of Phototoxic and Photoimmunologic
Reaction 95
3-2 Phototoxic and Photoallergic Chemicals 96
3-3 Phototoxicity Testing in Animals 98
3-4 Phototoxicity Testing in Humans 100
3-5 Photoallergy Testing in Guinea Pig 102
SYSTEMIC DERMAL TOXICITY
1.0 SUMMARY 105
2.0 INTRODUCTION 106
3.0 FACTORS INFLUENCING ABSORPTION AND TOXICITY 107
3.1 Properties of Test Agents 107
3.2 Species Differences 107
3.2.1 Recommendations 109
3.3 Regional Variation in Absorption 110
3.4 ^n Vitro vs In Vivo Absorption 110
3.5 Use of AbrasTon Ill
3.6 Dermal LD50 Vs Oral LD50 112
4.0 ACUTE DERMAL TOXICITY TESTING 113
4.1 Duration of Exposure 113
5.0 LONGER TERM DERMAL TOXICITY TESTING 117
6.0 EXAMINATION 124
6.1 Irritation Assessment ..... 124
-------
TABLE OF CONTENTS (Continued)
Page
6.2 Histopathology and Clinical Measurements 124
6.2.1 Clinical Chemistry 124
6.2.2 Hematology 126
6.2.3 Recommendations 126
7.0 PUBLIC COMMENTS 128
8.0 CONCLUSIONS 129
9.0 REFERENCES 131
APPENDIX
Comparison of Oral and Dermal Lethality 134
TABLES
4-1 Acute Dermal LD50 of Test Compounds in the
Rabbit and Guinea Pig 109
4-2 Effect of Anatomic Region on Absorption of
Pesticides in Humans 110
4-3 Absorption in Human Skin Ill
4-4 Relationship Between Toxicity and Safety of Pesticides . 112
4-5 Comparison of Guidelines for Acute Dermal Toxicity . . . 114
4-6 Comparison of Guidelines for Subchronic Dermal Toxicity 119
FIGURE
4-1 Absorption Variation by Species 108
-------
PREFACE
The Environmental Protection Agency (EPA) is formulating a uniform set
of toxicity testing guidelines based on those recommended by the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA), the Toxic Substances
Control Act (TSCA), the Interagency Regulatory Liaison Group (IRLG), and the
Organization for Economic Cooperation and Development (OECD). In support of
this activity, EPA's Office of Pesticides and Toxic Substances has requested
Tracor Jitco to critically review, summarize, and evaluate the available
information on dermatotoxicity testing. Tracor Jitco has examined dermal
irritation, sensitization, phototoxicity, and systemic toxicity.. These test-
ing procedures were reviewed and assessed in terms of scientific rationality,
effective use of animal resources, reliability in predicting effects in humans,
practicality, and cost-effectiveness.
To evaluate the various protocols used in dermatotoxicity studies for
approximately the last 20 years, literature searches were made using computer-
ized data bases and published indexes. Relevant articles published earlier
were manually retrieved by "tree searching" of references cited in the
original article. Data bases searched included: Toxline and Medline, BIOSIS,
SCISEARCH, CHEMICAL ABSTRACTS, EXCERPTA Medica, NIOSHTIC, NTIS and RTECS.
Information was also obtained from laboratories engaged in dermatotoxicity
testing and from recognized experts.
After receiving this information a critical review was performed by
Tracor Jitco toxicologists. The next step in the process was to evaluate the
literature and public comments relative to the specific EPA guidelines. For
example, there is disagreement on the best choice of species for particular
studies. More than one species is justifiable in many cases. Other topics
requiring additional refinement included: the use of abraded skin, the patch
test methods of scoring, degree of occlusion, clinical chemistry measurements
and histopathological studies. When possible an attempt was made to compare
and contrast the accepted guidelines with other available information to arrive
at an independent recommendation or conclusion.
The data obtained from the literature survey are organized into four
major sections in this report: the first section deals with Dermal Irritation,
the second with Dermal Sensitization, the third with Dermal Phototoxicity and"
the fourth with Systemic Dermal Toxicity.
Each section is organized as a separate unit complete with information
on the objective and scope of the survey, a description of the pertinent find-
ings, tables and figures, conclusions and/or recommendations and a summary.
In a Federal Register notice of January 27, 1981, the EPA stated that
its toxicity testing requirements would be satisfied by following the OECD
guidelines. These guidelines include provisions for acute dermal toxicity,
repeated dose dermal toxicity (21/28 day study), subchronic dermal toxicity
(90 day study), dermal irritation/corrosivity and skin sensitization. The
guidelines will be periodically modified and revised when necessary. The
information in the following reports is to be used in support of revision
VI1
-------
petitions by the EPA, and as a reference source for established investigators
and those just entering the field.
This report has been reviewed by individuals within the EPA, but has
not been externally peer reviewed. The authors would like to thank James
Murphy, Cheryl Peterson, and Mark Townsend of the EPA for their technical
review and helpful suggestions.
Vlll
-------
EXECUTIVE SUMMARY
A major objective of toxicity testing guidelines is to ensure the gath-
ering of data which can be used to reliably predict effects in humans prior to
exposure of the general population to potentially hazardous substances. Guide-
line tests should be cost-effective, specific, and designed so as to minimize
the numbers of animals used and the trauma to each animal.
This report examines the four categories of Dermatotoxicity testing:
Dermal Irritation, Dermal Sensitization, and Systemic Toxicity, all of which
presently, have testing guidelines, and Phototoxicity, for which guidelines are
currently being developed.
In conducting this assessment, a number of areas and issues were con-
sidered in detail. These include species selection, testing procedures/
protocols, sensitivity and enhancement of reaction to test agents, predicta-
bility of human response from animal data, and test result evaluation.
Dermal Irritation
Although dermal irritation studies historically, have been widely con-
ducted on rabbits, the findings of this report suggest that the guinea pig
should also be considered as an acceptable animal model, as the sensitivity of
this species is comparable to that of the rabbit for many compounds.
Other species of animals have also been used in dermal irritation stud-
ies. These include the minature swine, rat, mouse, piglet, beagle, baboon,
hairless rat, hairless hamster, and hairless mouse. In addition to reliably
detecting irritancy, species selection criteria should include practical con-
siderations. For example, the monkey, is rarely used because of cost and non-
availability, even though its response mimics that of the human.
Factors affecting the results of dermal irritation studies include:
1) the patch test unit, 2) the degree of occlusion, 3) use of abrasion,
4) application of the test substance, 5) the application site, 6) the duration
of exposure and observation and 7) the techniques used in evaluating the test
results.
The simple gauze patch is the most commonly used technique. The use of
an occlusive chamber that ensures reproducibility in the amount of substance
applied to a given area of skin is a recent development that deserves further
evaluation. In the standard patch test, present guidelines allow for varying
the degree of occlusion, the application site, and the method of moistening a
solid substance. Nevertheless, a need may exist to control these variables.
On the other hand, the use of abrasion and the length of exposure and the
observation period are factors that generally are rigidly defined. Greater
flexibility in these criteria may, however, permit the development of more
information on a compound. While increased flexibility will allow the
investigator to enhance the sensitivity of the test to suit conditions of use
and population exposure, constancy in the length of the exposure and observa-
tion periods contributes to comparability of test results with different
compounds.
IX
-------
Variability in the results of dermal irritation studies can result from
the methods used for evaluation or scoring. The development of a reference
set of photographs or slides depicting the various grades of irritation could
facilitate the evaluation by ensuring consistent grading, terminology usage,
and scoring. A possible alternative scoring method that should be considered
is the use of a quanta1 response, i.e., the IT50 (number of days before notice-
able irritation occurs in 50% of test animals) or the ID50 (the concentration
that produces noticeable irritation in 50Z of the test animals).
The data evaluated in this review suggest that the use of a tier-like
strategy may be an effective means of obtaining information on the dermal
irritation potential of test substances. As a preliminary step, test materials
may be measured for pH. According to the OECD guidelines, substances with a
pli of 2 or less or 11.5 or greater do not need to be tested for dermal irrita-
tion based on the assumption that they will be irritating. Where substances
are not screened out by pH determinations, the hairless mouse could be used as
a test species for the prescreening of irritancy. Further characterization of
irritancy could be obtained by testing in the rabbit or guinea pig. Depending
on anticipated use and exposure hazards of the test materials, human subjects
could then be used to test compounds of low or marginal irritancy.
Dermal Sensitization
Dermal sensitization can be defined as the delayed immune response to
an antigenic substance applied to the skin. The guinea pig is the species
most widely usii because it is generally effective in determining the sensiti-
zation potential of a chemical. In certain cases, however, this test species
has not demonstrated the subtle effects observed in humans. The OECD lists
eight guinea pig methods which it considers acceptable. These can be divided
into three groups depending on whether the route of administration is intra-
dermal, epicutaneous or a combination of the two. These tests involve an /
initial exposure to the test material (induction) followed by a rest period of
approximately 2 weeks and a second exposure (challenge). A positive challenge
reaction indicates sensitivity, assuming that the control group shows no
response. The Maximization and Closed Patch tests are currently the most
widely used, although no one method has been sufficiently validated to support
its selection as the method of choice. The Maximization test provides more
reliable detection of weak sensitizers than the earlier Draize method. Based
on a small amount of published data, the Optimization and Open Epicutaneous
tests also appear effective in providing information on sensitization responses
to test agents.
In testing regimens requiring exact prediction of response in a large
human population (i.e., allergy testing), the use of human subjects is a common
practice. In all instances, prescreening of substances in guinea pigs is
necessary prior to testing in humans. The Maximization test in humans most
accurately predicts the response to the general population. Use of this test
to validate guinea pig test methods has demonstrated good quantitative agree-
ment between the response of the guinea pig and man to a variety of test
chemicals.
-------
A satisfactory skin sensitization program for chemicals, pesticides,
drugs, paints and coatings, toiletries and cosmetics may involve both the
guinea pig and man. Positive (strong sensitization) results in the guinea pig
preclude the testing on humans; a weak or negative response may indicate the
need for further characterization and carefully controlled testing in small
groups of human subjects. For the latter case, the Maximization test should
be used.
Phototoxicity
Ultraviolet light can alter a chemical that normally produces no toxic
responses into one causing direct toxicity or allergenicity. With the excep-
tion of fragrance ingredients, only a small number of chemicals have been
extensively studied for phototoxic potential. Light activation of the test
substance can elicit either a toxic or sensitivity response. The guinea pig,
hairless mouse and minature swine have been used to study phototoxicity, but
present studies do not permit evaluation of the most appropriate species for
testing.
In methods that have been used for phototoxicity studies, the test
agent is either applied topically to the skin of the test animal or delivered
by intraperitoneal injection. After a specified time interval, the test site
is irradiated with ultraviolet light and reactions are scored. The OECD is
currently developing guidelines in this area.
Systemic Toxicity
Dermal toxicity testing is done to determine whether a substance can be
absorbed in quantities sufficient to produce systemic effects, as well as the
nature of such effects. Some of the factors that influence the degree of irri-
tation produced by an agent will also influence its systemic toxicity. These
include characteristics of test agents such as pH and lipid/water solubility
and specific test procedures, such as abrasion of the skin and the methods used
to apply test substances. A dermal study alone will rarely be sufficient to
completely characterize the toxic effects of an agent, as it provides informa-
tion on the effects produced by only one route of exposure. The OECD guide-
lines suggest testing the rat, rabbit or guinea pig. With the IRLG guidelines,
preference is given to the rabbit. The data evaluated indicate that the rat
is a more appropriate species to study systemic effects after dermal exposure.
This is primarily because much of the available toxicity data resulting from
tests by other routes of exposure have been obtained from the rat. If LD50
values from different routes are compared in the rat, the relative rate of
percutaneous absorption of a series of compounds can be estimated.
A comparison of LD50 values for rabbits and rats shows that, in more
than 75% of the documented cases, the LDSO's varied by less than a factor of
four, with neither species clearly showing greater sensitivity. It has also
been shown that the LD50 values were similar whether a rabbit was used for 24
hours or a rat was used for 4 hours.
XI
-------
Dermal toxicity studies of longer duration are limited in their practi-
cality and cost-effectiveness. Animal restraint, patch attachment and
laboratory personnel are necessary for long periods. In addition, systemic
effects can be adequately determined by administration of the agent by a more
cost-effective route. Likewise, extensive clinical chemistry measurements and
histopathology studies should be selectively performed, depending on the
intended use of the substance. Inclusion of these additional measurements can
be appropriate, however, in tests carried out by other routes.
When dermal toxicity studies are performed, particular attention should
be paid to the size of the patch, the area of skin in relation to the size of
the animal, the degree of occlusion, and the concentration and amount of test
substances to allow development of consistent data.
XI1
-------
UNRESOLVED ISSUES AND RESEARCH RECOMMENDATIONS
Dermal Irritation
o Further evaluation of guinea pig, rabbit and hairless mouse
o Validation of occlusive chambers in animals
o Validation of IT50- and ID50 in animals
o Preparation of an illustrated guide for the grading of dermal irritation
o Further studies to determine the optimum exposure period and degree of
occlusion for industrial chemicals
Sensitization
o Extensive cross-validation of the eight guinea pig test methodologies with
a variety of industrial and other chemicals
Phototoxicity
o Further examination to determine the appropriate animal model for
phototoxicity testing
Systemic Toxicity
o Determination of the appropriateness of the rabbit, guinea pig and rat for
long-term studies
o Establishment of minimum and optimum exposure periods in acute testing
Kill
-------
DERMAL IRRITATION
1.0 SUMMARY
A major goal of testing substances for dermal irritation is reliability
in predicting the human response. All guidelines, with the exception of the
National Academy of Sciences (1977), recommend the use of the albino rabbit as
the test species. The rabbit is primarily used because it is readily avail-
able, easy to handle, and as it has been preferred historically, use of the
rabbit allows comparison of results with a large number of test substances in
the same species.
Humans come into contact with a myriad of chemicals such as solvents,
industrial compounds, pesticides, detergents, household chemicals, and cos-
metic products. The chemical industry is interested primarily in the effects
of short-term exposure from spills and more prolonged exposure from normal
usage. Tests attempting to predict the expected effects in humans should make
allowances for possible delays in decontaminating clothing and skin and for
the protection of individuals who show a much greater sensitivity than the
normal population. The cosmetic and toiletries industries are most interested
in preventing even mild irritation that might occur in a small fraction of
exposed consumers. These industries use the direct approach through paid
volunteer test subjects, avoiding the uncertainty of interspecies extrapolation
and providing adequate prediction of human irritation responses. Results of
several studies support the use of the guinea pig because of practical
considerations and its ability to predict the human response. The hairless
mouse has also been studied to a limited extent. In actuality, the choice of
test species should be based on practical considerations, cost-effectiveness,
and on the ability to detect irritation effects from expected exposures.
The Federal Hazardous Substances Act, in 16 CFR 1500.4, stipulates that
human data should be taken into account in evaluating a substance. Studies in
humans are generally performed to characterize possible responses to
commercial products and substances that are intended for use on human skin or
present other special exposure hazards. Most human studies employ patch
testing procedures with modifications to detect irritation effects under
conditions of anticipated product use and exposure.
-------
2.0 INTRODUCTION
2.1 Objective
The objective of this survey is to investigate the factors affecting
the predictability of dermal irritation studies and the reproducibility of
their results, in particular with reference to the OECD (1981) guidelines.
The main purpose for measuring dermal irritation is to differentiate between
substances that cause moderate or strong irritation or corrosion of the skin
and those compounds which do not. Three major areas of concern are addressed
in the following pages: selection of species for testing, testing procedures,
and evaluation of results. In some cases modifications of the current
guidelines have been suggested when appropriate.
2.2 Definitions
Irritation is the local inflammatory response of the skin to direct
injury by a single, repeated, or prolonged contact with a chemical without the
involvement of an inmunological mechanism. Erythema and edema are the macro-
scopic manifestations of irritation. Corrosion (the correct pathological term
is erosion) is the direct chemical action on normal living skin which results
in its disintegration and irreversible alteration at the site of contact.
Corrosive chemicals cause ulceration, necrosis, fissuring, and with time, the
formation of scar tissue.
2.3 Historical
One of the earliest descriptions of the use of the patch test for dermal
testing was provided by Fabre in 1898. After handling a caterpillar and suf-
fering a reaction, Fabre tested an extract of the insect's hair and described
his procedure as follows:
"When the etheral infusion is reduced by evaporation to a few drops, I
soak a slip of blotting-paper folded in four, so as to form a square measuring
something over an inch...Lastly, the square of paper,...is applied to the
under surface of the forearm. A thin water-proof sheeting covers it, to
prevent it from drying too rapidly; and a bandage holds it in place."
The bandage was kept in place for ten hours during which time nothing
happened. Fabre then developed an acute eczematous reaction.
"I experience an increasing itch and a burning sensation acute enough
to keep me awake for the greater part of the night. Next day, after 24 hours
of contact, the poultice is removed. A red mark, slightly swollen and very
clearly outlined, occupies the square which the poisoned paper covered. The
skin feels sore, as though it had been cauterized, and looks as rough as sha-
green. From each of its tiny pustules trickles a drop of serous fluid, which
hardens into a substance similar in color to gum arabic. This oozing con-
tinues for a couple of days or more. Then the inflamation abates; the pain,
hitherto very trying, quiets down, the skin dries and comes off in little
flakes. All is over, except the red mark, which remains for a long time, so
tenacious in its effects is the extract of Processionary (the caterpillar
-------
genus). Three weeks after the experiment, the little square on the forearm
subjected to the poison is still discolored."
Many of the practices used by Fabre, including the inch square patch on
the inside of the forearm and the occlusive covering held in place for several
hours foreshadowed the general approach to dermal irritation testing developed
in the years that followed.
The original Draize test called for a patch test on previously clipped
albino rabbits using, both intact and abraded skin sites. Abrasions were de-
fined as minor incisions through the stratum corneum that did not reach the
dermis and induce bleeding (Draize, 1955). The test chemical was introduced
under gauze patches applied to the backs of albino rabbits, which were clipped
free of hair. Applications were made on three rabbits with intact skin and
three with abraded skin. The patches were secured by adhesive tape, and the
entire trunk of the animal was wrapped in rubberized cloth for 24 hours. The
animals were immobilized during the exposure period, the patches were removed
after 24 hours, and the resulting reaction evaluated visually for erythema and
edema according to the scoring system shown in Table 1-1. The reaction was
also scored after 72 hours. A Primary Irritation Index (PII) was obtained by
averaging the 24- and 72-hour reaction scores. The PII was then used to rate
the chemical as a non-irritant, a moderate irritant or a severe irritant
(Table 1-2). The Draize method is summarized in Table 1-3.
In 1959, the procedure was finalized with the addition of the step of
wetting solids to be tested with an appropriate solvent prior to testing. The
procedure was published in the Food, Drug, and Cosmetic Law Journal. The
Draize procedure was adopted by the Food and Drug Administration as the
official test for primary skin irritation and has since been legislated under
provisions of the Federal Hazardous Substances Act (FHSA) (29 CFR 13009 191.11)
(Table 1-3). The Consumer Product Safety Act (CPSA) (15 U.S.C. 2051; 1972)
vested authority for the FHSA to the Consumer Product Safety Commission (CPSC)
for determining hazard labelling. Presently, the method of testing primary
irritant substance can be found at 16 CFR 1500.41. Since evaluation of
irritation threshold is necessary for labelling a product, FHSA states that a
test agent is considered to be a primary irritant if it scores 5 or higher in
the test (see Table 1-2). Further adaptations of the Draize procedure have
deemphasized this cut off for primary irritation versus non-irritation due to
reported variability both in absolute scoring of the standard FHSA protocol as
well as rank ordering of irritation of compounds (Weil and Scala, 1971).
In 1972, the FDA reported a revision of the test procedures that re-
duced the exposure period to 4 hours and required the detailed evaluation of
the corrosive effects due to a concern "that the skin irritation test does not
realistically reflect the skin contact that could reasonably be expected from
exposure,... (as)...immediate steps would probably be taken to flush or remove
a substance when irritation is perceived". This proposal, excluding the re-
quirements for testing substances on abraded skin, has been adopted by the
Department of Transportation for classifying corrosive substances (49 CFR
173.1200 Appendix A).
-------
Table 1-1. Evaluation of Skin Reactions: Single Application
A. Erythema and eschar formation
No erythema
Very slight erythema (barely perceptible)
Well defined erythema
Moderate to severe erythema
Severe erythema (beet redness to slight
eschar formation-injuries in depth)
Total possible erythema (or score)
B. Edema formation
No edema
Very slight edema (barely perceptible)
Slight edema (edges of area well defined by
definite raising)
Moderate edema (area raised approximately 1 mm)
Severe edema (raised more than 1 mm and extending
beyond area of exposure
Total possible score
Score
0
1
2
3
4
4
0
1
2
3
)
4
Total possible score for primary irritation
(Primary Irritation Index, PII)
a(Draize et al. 1944)
Table 1-2. Primary Irritant Response Categories in the Rabbit
(Draize et al., 1944)
Response Category Mean Score (Primary Irritation Index - PII)
Negligible 0 - 0.4
Slight 0.5 - 1.9
Moderate 2 - 4.9
Strong (Primary Irritant) 5 - 8.0
-------
Table 1-3. Comparison of Methods Used for Denial Irritation
SPECIES
SEX
AGE
WEIGHT
NUMBER
NUMBER OF
PATCHES PER
COMPOUND
DOSE
PATCH SIZE
PATCH MATERIAL
PATCH TAPE
AND COVER
O.E.C.D.
ALBINO RABBIT
N.S.C
ADULT
N.S.
3 minimum
N.S.
0.5 ml undiluted
liquid or
0.5 gm solid or
seni-solid
6cm2
Gauze Patch
Nonirritating tape
loosely held by
I.R.L.G.
SAMEb
N.S.
SAME
2-3 Kg
6 minimum
3 for limit test
plus 3 additional if
results equivocal
1
SAME
SAME but prenoisten
sample and gauze with
water or solvent
SAME
SAME
Nonirritating tape
loosely held using
DRAIZB
SAME
N.S.
N.S.
N.S.
6
4
SAME undiluted
SAME but dissolv solids
in appropriate solvent
1 in2
SAME - 2 layers thick
Adhesive tape
- entire trunk
N. A. 8. (1977)*
ALBINO GUINEA PIG
PREFERRED, RABBIT
N.S.
YOUNG ADULT
N.S.
6
N.S.
SAME
SAME but moisten with
solvent (SOX slurry)
1 in2
GAUZE or other inert
emi-absorbant
material i.e. 2- or
12-ply gauze, non-
woven cotton fabric
or cellulose pad*
porous tape
with rubberized
D.O.T.
ALBINO RABBIT
N.S.
N.S.
N.S.
6 minimum
N.S.
SAME
SAME as OECD
1 in2
GAUZE, 2-ply
SAME as Draize
seraiocclusive
dressing
Occlusive dressing
may be used
Access to patch
should be prevented
impermeable material
(rubberized cloth or .
plastic fiber)
Not recommended unless
human exposure warrants
Immobilized animals in
stocks or Newman Harness
wrapped with impervious
material (rubberized
cloth)
N.S.
N.S.
cloth or stockinette'
N.S.
Do not occlude
Collars or restraint N.S.
-------
Table 1-3. Comparison of Methods Used for Derouil Irritation
(Continued)
EXPOSURE
WASHING
ABRASION
EXAMINATION
O.E.C.D. I.B.L.G.
4 hours 4 hours
Allowed N.S.
Not required Not required
1/2 to 1, 24, 48, and SAME
72 hrs. after patch
DBAIZB
24 hours
N.S.
Abrasion
skin for
+ nomal
each animal
Read after 24 hr.
exposure and 48 hrs.
N.A.S.(1977)
4 hour*
M.S.
Not reconmended
SAME except no
reading at 48 hrs.
D.O.T.
4 hours
Allowed
Not required
After 4 hours
and at 48 hours
SCORING
CORROSION
MISCELLANEOUS
removal
Draize Scoring
Note if corrosive
Further observation
if needed,
Record previous
lesions/toxic effects
Describe degree and
nature of irritation
SAME, report
average of all values at
all times of observation
Note if corrosive
Possibly use histo-
pathology
later
SAME
Add erythema and edema
values for abraded and
intact skin at both tines
and divide by four
Not considered
SAME
Add erythema and
edema scores at each
time, use highest
ean score
Not considered
Corrosion test seen
at 7 days
Evaluate corrosion
only
Note if corrosive
'from National Academy of Sciences (1977)
"Same as OECD
cNot Specified
-------
The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA - 40 CFR
162) requires that pesticides be labeled with toxicity categories (from I to
IV). The degree of dermal irritation at 72 hours is one of the criteria for
assignment to a toxicity category. In June 1975 and August 1978, the EPA pub-
lished proposed FIFRA test guidelines for measurement of dermal irritation.
TSCA guidelines were proposed in May and June of 1979 (40 FR 26802, 43 FR
37336, 44 FR 27334 and 44 FR 44054). Due to differences in wording and
approach between FIFRA and TSCA test guidelines, the more broadly based
Interagency Regulatory Liaison Group (IRLG) and later the Organization for
Economic Cooperation and. Development (OECD) guidelines were adopted. These
two methodologies, along with those of the FHSA (CPSC), DOT and the National
Academy of Sciences (1977), are compared in Table 1-3. Differences in the
guidelines are discussed in the following sections. The OECD guidelines have
taken into account and addressed many of the scientific issues which have
arisen from public comment on the FIFRA and TSCA guidelines. Corrosivity is
to be noted as part of the OECD reporting procedure and is specifically men-
tioned in the section on Minimum Premarket Data of the TSCA guidelines but
scoring of corrosion per se is not required. Adoption of this procedure will
allow OECD guidelines to fulfill DOT Regulations.
Friedman, in his General Referee Report on Toxicological Testing to the
AOAC commented: "Toxicological testing procedures have, until recently,
not been considered suitable subjects for standardization. The determination
of whether a given material has the capability of evoking a biological response
is usually a problem of such complexity that it does not lend itself to stan-
dardization. There are stages, however, in the development of any problem. In
the beginning there is a need for a maximum of innovation and creativity in
devising the approaches and techniques that might be useful. In the case of a
complex problem, this stage where standardization is contra-indicated may last
a long time...The danger of standardization lies in the possibility of fixing
systematic errors into a procedure and achieving a desired degree of reproduci-
bility at the price of accuracy and innovation," (Goldberg, 1975). Some
testing procedures to be discussed are still in the developmental stage;
others are well established. They are analyzed here in light of new
scientific discoveries and thinking over
the last two or three decades.
2.4 pH of Test Agents
The design of a skin irritation test should take into account the
physical and chemical properties of the compound. According to the OECD
guidelines, substances with a pH of 2 or less or 11.5 or greater do not need
to be tested for dermal irritation based on the assumption that they will be
highly irritating to the skin.
Guillot and coworkers (1981) studied the effect of pH on the primary
cutaneous irritation responses produced by a variety of substances. Measure-
ments of pH were performed on the test agents; the pH of solid materials was
measured from a saturated solution of the pulverized substance in distilled
water. Substances were tested on the clipped skin surface of the scarified
right flank and on the intact left flank of albino rabbits. Liquid test
agents were applied to gauze pads and occluded to the application site. Solid
substances were pulverized, applied to the site, covered with a gauze pad that
-------
was moistened with water, and occluded to the skin. Of highly acidic sub-
stances, dimethylsulfate, a liquid with a pH of 1.0, was severely irritating,
while two solid substances, aluminum nitrate, pH 0.8; and oxalic acid, pH 1.0,
were non-irritating and moderately irritating, respectively. Four solid sub-
stances with pH values between 2.1 and 2.7 were all non-irritating. Of highly
alkaline substances, one solid with a pH of 10.7 (scouring powder) was non-
irritating, while four liquids showed the following pH values and corresponding
results: pH 10.1, non-irritating; pH 10.8, slightly irritating; pH 10.5 and
10.7, moderately irritating. The above results show that solid acidic sub-
stances in solution with a pH of 2 or less cannot be assumed to be severely
irritating. The apparent discrepancy between the results of highly acidic
liquids and solids may be explained by the different properties (e.g., wetting
properties) of these substances as tested. In addition, further studies or
additional data would be useful to more fully validate the OECD test exclusion
for highly alkaline substances. The OECD exemption is a reasonable one, but
it does not prohibit further testing if indicated by a need to know more about
either a specific chemical or the physicochemical properties that enhance or
retard irritancy.
-------
3.0 SPECIES SELECTION
3.1 Animal Tests
The use of the rabbit as the preferred test species is evidenced by the
large amount of dermal irritation information on this species in the Registry
of Toxic Effects of Chemical Substances (RTECS). Eighty-five percent of over
2,000 RTECS entries report test results with the rabbit, 7.5% with the human,
4Z with the mouse and 3Z with the guinea pig.
The majority of the human data comes from the Research Institute for
Fragrance Materials Monographs on essential oils and other aromatics published
in Food and Cosmetic Toxicology. This is also the primary source of mouse
skin irritation data.
The choice of proper species for measuring dermal irritation and pre-
dicting the effects of chemicals on humans has been actively discussed since
its requirement in the Federal Hazardous Substances Labeling Act in 1961. The
OECD and IRLG guidelines both specify the use of the albino rabbit. The
Department of Transportation also requires the use of rabbits in the identifi-
cation of corrosive substances (49 CFR 173.1200 Appendix A). As a result,
rabbits have been used to generate a vast majority of the available data in
the open literature. Despite the wide use of rabbits, the National Academy of
Sciences (1977), suggests that the guinea pig and not the rabbit be used as
the preferred test animal based on its response being more like that of human
skin over a wice range of materials and the more economical requirements for
space and caging.
3.2 Interspecies Comparison
Roudabush et al. (1965) compared the acute effects of 14 chemicals on
the skin of rabbits and guinea pigs using procedures described in 21 CFR 191.11
(Federal Hazardous Substances Act). Their results indicated that the responses
of these two species are essentially equivalent when scores for intact and
abraded skin were averaged. In the guinea pig, scores were higher compared to
rabbit on intact skin and lower on abraded skin (Table 1-4). Household ammonia
and trisodium phosphate acted as primary irritants only in the guinea pig*
Accordingly, and in consideration of cost and space requirements, the authors
concluded that there is sound justification for using the guinea pig as an
alternative species to the rabbit for skin irritation studies.
The rabbit is not a good model for moderately and minimally irritating
materials (NAS, 1977) as these substances often show stronger response in the
rabbit than in humans. It does however, correctly identify compounds which
are highly irritating to humans, thus avoiding unnecessary risk in exposing
the volunteer test force to these compounds.
In attempting to more accurately predict human response, other animals
have been tested with mixed results. These include the guinea pig, miniature
swine, rat, piglet, beagle, baboon, hairless rat, hairless hamster and hairless
mouse. The guinea pig and the hairless mouse have been used more often than
any of the others.
-------
Table 1-4. Summary of Primary Irritation Scores in Rabbit and Guinea Piga
Compound
Acetic acid,
4Z aqueous
Ammonia,
household
Borax
Boric acid
2-Butanone
Carbon
tetrachloride
Gasoline
Kerosene
Phenylhydrazine
Sodium
carbonate
Sodium
sulfite
Sucrose
Trisodium
phosphate
Water,
distilled
Range
Animal
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Rabbit
Guinea pig
Intact
Skin
1.5
1.9
3.2
5.6
1.6
1.5
0.8
2.7
0.8
1.6
2.8
4.1
2.0
3.9
1.9
3.6
1.8
3.6
2.1
2.1
1.6
2.4
0.4
1.5
3.4
6.3
1.1
1.3
0.4-3.4
1.3-6.3
Abraded
Skin
3.6
1.9
5.0
5.0
2.3
1.4
2.5
1.4
2.7
2.4
4.0
2.2
2.9
2.8
3.2
2.6
3.0
2.6
5.0
2.6
3.1
1.7
2.5
0.9
5.1
5.1
1.8
1.0
1.8-5.1
0.9-5.1
Average.^
2.6
1.9
4.1
5.3
2.0
1.4
1.7
2.1
1.8
2.0
3.4
3.1
2.4
3.3
2.5
3.1
2.4
3.1
3.5
2.3
2.4
2.0
1.4
1.2
4.2
5.7
1.4
1.1
1.4-4.2
1.1-5.7
10
-------
Table 1-4. Summary of Primary Irritation Scores in Rabbit and Guinea Piga
(Continued)
Compound
Average
Correlation
coefficient to
Animal
Rabbit
Guinea pig
Rabbit
Guinea pig
Intact
Skin
1.79
2.87
0.943
0.803
Abraded
Skin
3.34
2.40
0.962
0.861
Average^
2.56
2.69
0.847
rabbit, intact
plus abraded
aAdapted from Roudabush et al (1965)
bThe calculations of this primary irritation score is described in 21CFR
191.11. In the other columns for intact or abraded skin, the sum of the
values at 24 and 72 hours was divided by 2 rather than 4.
11
-------
Davies et al. (1972) found considerable interspecies variation in dermal
irritation, as summarized in Table 1-5. The major interest of these authors
was the screening of cosmetic ingredients, where false positives are acceptable
but false negatives are not. Therefore, they felt that a test species should
not be chosen based on its resemblance to man but based on which is the most
sensitive, namely the rabbit and guinea pig. Testing in the dog and miniature
swine give false negative results for cream shampoo, propylene glycol,
aluminum chlorohydrate, and sodium lauryl sulfate.
Brown (1971) compared the irritant effects of surfactants (labeled Tl
through T9) on humans, rabbits, guinea pigs and hairless mice without occlu-
sion. When the gross and histological irritancy is graphed on an arbitrary
scale of 1 (least irritating) to 5 (most irritating), the rabbit and the guinea
pig give equivalent responses which are reasonably predictive of human response
giving only false negatives for T-9. The hairless mouse was less accurate,
giving a false negative score for T-9 and a higher reading for T-l. The
results are depicted in Table 1-6.
Marzulli and Maibach (1975) compared the results of a 16-day cumulative
irritation test in rabbits (uncovered test site) with those from a 21-day test
in man (covered test site) using antimicrobials, sunscreens, acids and alkalis,
detergents, anti-perspirants, vitamin E preparations, cosmetics, and an anti-
psoriatic. Results for 60 test materials showed a significant correlation
(r = 0.30; P less than 0.02) between the scores obtained for rabbits and man.
In most cases, the cumulative scores were higher for rabbits, indicating a
greater sensitivity. However, oxalic acid and sulisobenzone gave higher scores
in man. The authors concluded that repeated application serves as a valuable
modification to the Draize test and that the greater responsiveness of the
rabbit offers a useful margin of safety for prediction of irritation in man.
Griffith and Buehler (1977) compared skin response in humans with
rabbits and guinea pigs using a patch test. Twenty-four chemicals and house-
hold products were tested and scored. Highly lipophilic compounds (soaps and
detergents) showed the greatest tendency toward exaggerated response in
rabbits. Both species gave 16 "correct" results w'.ien compared with man, but
tended to err on the high side with "weak" irritants. The results for the
guinea pig (intact skin) were more often on the low side for slight or moder-
ate human skin irritants, as shown in Table 1-7. It should be noted that for
industrial chemicals (Table 1-4) the guinea pig scored higher in general,
however, ammonia tested by each group gave opposite results.
Tests for primary irritation using the albino mouse ear skin (Uttley
and Van Abbe, 1973) (Table 1-8) and the back of the hairless mouse (Brown,
1971; Kastner, 1977; and Homberger et al., 1962) have been suggested as pre-
liminary screening procedures before initiating patch testing in man. The
sensitivity observed in 24-hour patch tests using the hairless mouse has more
closely resembled human response to over 60 compounds than that observed for
either the rabbit or guinea pig (Kastner, 1977). The compounds tested included
fatty acids, alcohols, amides, detergents, esters, and cosmetic bases. Hair-
less mice have also proven to be useful in the testing of marketed sunscreening
preparations (Gloxhuber, 1976).
12
-------
Table 1-5. Relative Irritancy, Man vs. Seven Other Species8
Species
Relative irritancy
Similar to Man
More than Man
Less than Man
Mouse
Rabbit
Mini pig
Piglet
Dog
Baboon
Lanolin
Guinea pig Lanolin
Lanolin
Lanolin
Thioglycolate paste
Sodium lauryl sulphate
(5Z)
Para-phenylenediamine
Propylene glycol
Lanolin
Aluminium chlorhydrate
Sodium lauryl sulphate
(5%)
Para-phenylenediamine
Lanolin
Thioglycolate paste
Sodium lauryl sulphate
Aluminium chlorhydrate
Para-phenylenediamine
Thioglycolate paste
Aluminum chlorhydrate
Sodium lauryl sulphate
(1% and 5%)
Para-phenylenediamine
Thioglycolate paste
Propylene glycol
Aluminium chlorhydrate
Sodium lauryl sulphate
(1Z)
Lanolin
Thioglycolate paste
Cream shampoo
Propylene glycol
(12 and 5Z)
Cream shampoo
Thioglycolate paste
Propylene glycol
Cream shampoo
Sodium lauryl sulphate
(5%)
Para-phenylenediamine
Cream shampoo
Propylene glycol
Aluminum chlorhydrate
Sodium lauryl sulphate
(1Z)
Cream shampoo
Cream shampoo
Aluminium chlorhydrate
Sodium lauryl sulphate
(1Z and 5Z)
Para-phenylenediamine
Propylene glycol
Sodium lauryl sulphate
(1Z)
Propylene glycol
Aluminium chlorhydrate
Cream shampoo
Para-phenylenediamine
Sodium lauryl sulphate
aAdapted from Davies, et al. (1972)
13
-------
Table 1-6. Blanking of Skin Irritation Results with Surfactants
on Human and Animal Skina
Irritancy
Rating
Least
Irritant 1
2
3
4
Most 5
Irritant
Not Tested
Uncovered Exposures
Human Arm
Immersion
. Tests Rabbits Guinea Pig
T3 T6b Tl T2 T3 Tl T2 T3
T4 T5 T6 T4 T5 T6
T9 T9
Tl
T5 T7 T7
T9 T8 T8
T8
T2 T4 T7
"Hairless"
Mice
T2 T3 T6
T9
Tl T5 T7
T8 T4
aAdapted from Brown (1971), (Gross and/or Histological Examination)
bSurfactants are labelled Tl through T9
14
-------
Table 1-7. Irritation Response of Human, Guinea Pig, and Rabbit Skin
to Chemicals/Household Products3
Material
Isopropyl alcohol, 100%
Table salt, 50%
Sodium tri poly phosphate 50%
Sodium carbonate, 50%
Sulfuric acid, 10%
Sodium lauryl sulfate, 50%
Acetic acid, 10%
Cg - CIQ fatty acid, 100%
Sodium metasilicates, 50%
Potassium hydroxide, 10%
Detergent granules:
Low carbonate, 50%
High carbonate, 50%
Enzyme, 50%
Phosphate, 50%
Coconut oil soap, 50%
Antiperspirant, 100%
Liquid detergent, 100%
Lemon juice, 100%
Liquid cleaner, 100%
Liquid shampoo, 100%
Pine oil cleaner, 100%
Household ammonia, 100%
Metasilicate-carbonate
detergent, 50%
Hypochlorite bleach, 100%
Human
o.ob
0.0
0.0
0.0
0.2
0.6
1.0
2.3
0.0
0.0
0.0
0.0
0.0
0.1
0.1
0.2
0.2
0.9
1.0
1.5
3.0
3.9
Mean Score (intact
Guinea Pig
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.1
1.7
cor
0.1
0.0
0.0
0.2
0.3
0.0
0.9
0.0
0.3
2.1
2.5
0.0
0.0
0.3
skin)
Rabbit
0.0
0.0
0.0
0.0
0.0
1.6
0.0
4.4
cor
c
cor
0.7
0.9
1.3
1.2
2.5
0.0
2.4
0.0
3.0
3.7
3.6
1.4
corc
1.0
aAdapted from Griffith and Buehler (1977)
bfiased on scale of increasing irritancy; mean score 0 to 8.0
cCorrosive.
15
-------
Table 1-8. Assessment of Irritation Mouse Eara
Reaction Category Score
No visible blood vessels or erythema. 0
Few blood vessels, barely visible. No erythema. 2
Main blood vessels visible on lower half of ear. 4
Slight erythema over lower third or base of ear.
Main blood vessels more obvious. Slight or generalized 6
erythema.
Main blood vessels extended to edge of ear; capillary 8
network extensively affected. Possible internal
hemorrhage; erythema more pronounced; loss of
suppleness of ear.
Pronounced blood vessels; extensive capillary network 10
evident; marked erythema; possible frilling of ear margin
Pronounced blood vessels; extensive capillary network 12
extending to ear margin; severe erythema; frilling and
thickening of ear margin; crusting evident.
All of the above plus possible necroses with extensive 14
crusting
afrom Uttley and Van Abbe, 1973
16
-------
In several reports from the Research Institute for Fragrance Materials
published in Food and Cosmetic Toxicology, over 90 essential oils were studied
in humans (48 hours, closed pat ch), rabbits (24 hours, occluded patch, intact
and abraded skin), and mouse (several hours, open epicutaneous). Over 50% of
the compounds were irritating in the rabbit but not in humans or the mouse;
17% of the compounds induced different responses in the human and the mouse
and 28% were nonirritating in all three species. In three instances, the
rabbit was predictive of the positive human response. Testing of the swine
using 41 of the essential oils resulted in findings similar to those for the
mouse, including, predicted nonirritation. Results of a limited number of
tests using the guinea pig resembled those for the rabbit.
Analysis of the preceding studies indicates that the rabbit, guinea
pig, and hairless mouse are useful in predicting irritant effects in man.
However, as in any toxicity study, no one species will be a perfect model for
all chemicals. The guinea pig, rather than being an alternative to the
rabbit, may actually be complementary as some chemicals detected as irritants
by one species are missed by the other. Other species that have not been
tested as frequently may also be useful for detecting skin irritation in man,
e.g. the hairless mouse. The objective is to discriminate quantitatively
among mild irritants with tests that are simple, reproducible, inexpensive,
and accurate. An approach to achieve this goal may be to use human volunteers
with attention to first screening out the moderate and severe irritants
through prior animal testing.
17
-------
4.0 TESTING PROCEDURES
The current OECD guidelines evolved from the testing procedures devel-
oped by Draize (1955) (Table 1-3). According to the OECD method, the test
chemical is introduced under gauze patches applied to the previously clipped
backs of at least three albino rabbits. The patches are held in place by non-
irritrting tape for 4 hours, and the animals are restrained, preventing access
to the patches. Reactions are evaluated visually for erythema and edema
(Table 1-2) 0.5 to 1, 24, 48, and 72 hours after the patch is removed. A
Primary Irritation Index (PII) obtained by averaging the 24- and 72-hour
reaction scores is used to rate the chemical as a nonirritant, a moderate
irritant, or a severe irritant (Table 1-2).
Modifications to commonly used testing methodologies have frequently
been made to reduce the variability and/or increase the sensitivity of patch
tests. Variations in testing procedures are outlined in Table 1-9. Major
areas of concern include: 1) the patch test unit; 2) occlusion; 3) use of ab-
rasion; 4) application of the test substance; 5) the application site; 6) the
duration of exposure and observation; and 7) enhancement of visual scoring.
Methodologies in humans and animals have been compared and studied for the
usefulness of specific testing procedures and for the ability to reliably de-
tect irritating substances.
4.1 Patch Test Unit
The accuracy and reproducibily of skin irritation tests depends on the
method of applying and securing the test substance. The size, thickness, and
patch material as well as the type of tape, volume of test material and the
method of occlusion can all affect results. Although occlusion generally
exaggerates the type of exposure that humans usually encounter, it must be
remembered that the purpose of the test is to detect potential irritants and
not necessarily to duplicate the response in man. Most procedures described
in the literature involve use of adhesive tape to secure the patch, but little
attention is given to the tightness of the fit. This can affect the degree of
occlusion.
According to Frosch and Kligman (1976, 1979), the standard patch test
has many flaws. Uniform exposure is hard to attain, reproducibility and accu-
racy are difficult because the test substance can escape, and, more important-
ly, there is a lack of sensitivity in detecting mild irritants. Recently much
attention has been given to the use of an occlusive chamber in humans to apply
the test substance. This device can more colsely control the surface area
exposed, the volume of sample in direct contact with the skin, the degree of
occlusion, and the amount of sample leakage.
The adhesion chamber technique for testing skin irritation was first
used by Rokstad (1940). In this method the test substance is placed in a cir-
cular depression chamber in the center of a celluloid plate. After positioning
the patch at the site of application, the chamber is secured with adhesive tape
while the skin is pressed up into the chamber.
Kurokawa et al. (1980) have reviewed the various types of patch test
units and have divided them into several categories, including the following.
18
-------
Table 1-9. Summary of Testing Procedures Used in Dermal Studies
Author
BENKE
et_ al_.
BJORNBERG
Method
patch
Cup tests
Patch Patch
Type Size
Webril .875 in
dia.
(Blohn, '60)
Occlusion
Method
yea; no
method
given
Chamber tests (Rokstand '40)
Open (Wedroff Dolgoff '35)
DAHL &
TRANCIK
FINKELSTEIN
£1 Si-
FINKELSTEIN
It *!
FROSH &
KLICMAN
FROSCH &
KLICMAN
GRIFFITH
& BUEHLER
JUSTICE
si 5l-
JUSTICE
et al.
patch
patch
patch
Duhring
chamber
chamber
patch
arm im-
mersion
patch
Al test N.S.
cotton 1.25 in x
flannel 1.25 in
cotton 1.25 in x
flannel 1.25 in
Uebril or 12 mm dia.
Curity
Webril 12mm dia.
N.S. N.S.
none none
gauze N.S.
Blenderm
tape
poly-
ethylene
6 elastic
bandage
poly-
ethylene
none
none
N.S.
none
elasto-
plast
Exposure
Time
4 hr.
2 hrs.
24 hrs
N.S.
24 hr.
5/17 hr.
daily
for 4-5
days
varies
with sub-
stance
tested
24 hrs.
than 6
hrs/day
4 days
Ix day
for 3 day
4 hr.
3 x day
2 hr. in-
terval
15 min.
each
18 to 24
hours
Observation
Time
H.S.b
24/72 hrs.
24/48 hrs.
immediate &
24/72 hrs.
24,26,26,30
72,96 hours
daily for
4-5 days
daily
36 hrs.
after test
finished
72 hr.
4,24,48
hours
2 hrs.
18 to 24
hours
Scoring
Evaluation Misc.
N.S.
visual controls
visual
visual
visual
visual
visual
visual
visual
visual
visual
visual
Type
Animal"
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
-------
Table 1-9. Summary of Testing Procedures Used in Dermal Studies
(Continued)
Author
KLICMAN
KLIGMAN
& WOODING
KUROKAWA
et al.
KUROKAWA
et al.
KUROKAWA
e_t al.
LANKAN
£t al.
LARSEN
MAI BACH &
EPSTEIN
MARZULU &
MAI BACH
MOTOYOSHI
£t Si'
ODOM &
MAIBACH
RAPPAPORT
et al.
Method
patch
patch
KI
chamber
Finn
chamber
Al test
patch
patch
patch
. patch
patch
spread
with
glass
rod
patch
Patch
Type
nonwoven
cotton
Webril
Webril
filter
paper
fitter
paper
filter
paper
nonwoven
cotton
Webril
N.S.
N.S.
Webril
lint
none
N.S.
Patch Occlusion
Size Method
1 in x 1 in Blende rn
1 cm x 1 cm Blcnderm
N.S. aluminum
N.S. aluminum
N.S. aluminum
foil &
poly-
ethylene
1 in x 1 in Blenderm
N.S. aluminum
backed
N.S. occlusive
dressing
N.S. blendera
15mm dia none
none none
N.S. N.S.
Exposure .
Time
daily for
10 days
until
ery. or
10 days min.
24 hr.
74 hr
24 hr.
daily
until irr-
ritation
observed
48 hrs.
7 to 12
days
21 days
cumulative
48 hr.
30 min
21 days
contin-
uous
Observation
Time
daily
each day
lhr/24 hr.
lhr/24hr.
lhr/24hr.
daily
48 or 72
hours
every 24 hr.
24 hr. than
daily
48 hr.
30 min
daily or
ev. 48 hrs.
Scoring
Evaluation Misc.
visual
visual
visual
visual
visual
visual
visual
visual controls
visual
visual
visual contact
uticaria
testing
Type
Animal9
MAN
MAN
'MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
MAN
-------
Table 1-9. Summary of Testing Procedures Used in Dermal Studies
(Continued)
N>
Author
SHELANSKI &
SHELANSKI
SKOG
SKOG
SHEENK
SMEENK
SULZBERGER
Si a_l .
SULZBERGER
et_ al.
BENKE
e_t al.
FINKELSTEIN
£t al_.
GRIFFITH
& BUEHLER
OPDYKE &
BURNETT
Method
repeated
insult
patch
rubbed
for 2
rain.
patch
arm im-
mersion
patch
patch
iramer.
patch
patch
immersion
Patch
Type
N.S.
N.S.
none
cotton
lint
none
gauze
linen or
cotton
none
cotton
flannel
N.S.
none
Patch Occlusion
Size Method
N.S. none
N.S. N.S.
none ^ none
16 mm. dia adhesive
tape
none none
3 in x 3 in vaseline
gauze
elastic
adhesive
1 cm x 1 cm etas to-
pat ch
none none
1 in dia. poly-
ethylene
N.S. N.S.
none none
Exposure
Time
I. Ev
48 hra.
for 30
days;
11.48 hrs.
N.S.
2 wks.
Iw
trtments
24 hr.
2x day
for 30
min. for
5 daya
24 hr.
24 hr.
4 hr.
16 hr.
4 hr.
4hrs.
each
for 3
days
Observation
Time
ev. 48 hrs.
phases
48 hrs.
24 hrs.
after removal
N.S.
24 hr.
N.S.
24 hr.
24 hr.
freq.
intervals
for 2 wks.
16 hr.
4,24,48
hours
72 hrs.
Scoring
Evaluation Misc.
visual Two
visual
gross
icro.
visual
visual
visual
visual
visual
visual
visual
visual
Type
Animal8
MAN
MAN
MAN
MAN
MAN
MAN
MAN
GP
GP
GP
GP
-------
Table 1-9. Sunmary of Testing Procedures Used in Denial Studies
(Continued)
Author
ROUDABUSH
et al .
SKOG
HOMBERGER
et al.
JUSTICE
£t_ al.
SCHMID
SCHMIDT
to & EVANS
UTTLEY &
VAN ABBE
BENKE
£l !
BROWN
BROUN
DRAIZE
FINKELSTEIN
et al.
GRIFFITH
& BUEHLER
Method
patch
rubbed
for 1
rain.
open
painting
open
mouse
ear
mouse
eat
patch
patch
drip
patch
patch
patch
Patch
Type
cellulose
none
none
none
none
none
none
felt
lint
none
gauze
cotton
flannel
N.S.
Patch Occlusion
. Size Method
1 in x 1 in "appro-
priate
sleeve"
none none
none none
none rubber
dam
none none
none none
none none
0.75 x 1 in elasto-
plast
2 cm x 2 cm poly-
ethylene
none none
1 in x 1 in tape; rub.
1 in. dia. poly-
ethylene
N.S. N.S.
Exposure
Time
N.S.
prob.
DRAIZE
daily
between
36 6 95
times
daily for
30 days
every 10
min. for
70 min.
3 x wk
for 8 wks
until
irritancy
daily for
4 days
24 hr.
7 days
4 1/2 wk.
5 day/wk
24 hr.
16 hr.
4 hr.
Observation
Time
N.S.
prob.
DRAIZE
daily
30 days
70 min.
3 x wk
24 hr
daily
24/72 hr.
daily/7 days
daily/5
day s /week
24hr/72hr
16 hr.
4,24,48
hours
Scoring
Evaluation Misc.
visual
viaual
hiitolog-
ical
histolog-
ical
histolog-
ical
visual
visual
visual
visual
visual sulfan
blue
and trypan
blue
visual standard
formal-
visual dehyde &
controls
visual
Type
Animal3
GP
GP
HRL MICE
MUS
MUS
MUS
MICE
RBT
RBT
RBT
RBT
RBT
RBT
-------
Table 1-9. Summary of Testing Procedures Used in Dermal Studies
(Continued)
Author
INGRAM &
GRASSO
MARZULLI
MAI BACH
MOTOYOSHI
et al.
STEINBERG
It Si-
to
U)
VINEGAR
WOLVEN &
LEVENSTEIN
HOTOYOSHI
et al .
MOTOYOSHI
et_ a\_.
Method
patch
open
applied
from
syringe
patch
open
open
patch
patch
patch
patch
patch
applied
from
syringe
Patch
Type
Melolin
open
none
gauze
none
none
gauze
gauze
gauze
Webril
N.S.
none
Patch
Size
1.5 cm x
1.5 cm
open
none
area
3 cm x 3 cm.
2 in x 2 in
none
none
5 cm .: 5 cm
5 cm x 5 cm
1 in x 1 in
2.5 cm x
2.5 cm
15mm dia.
none
area
3 cm x 3 cm.
Occlusion
Method
Blenderm
none
plastic
collar
elastic
bandage
none
saran
saran
elastic
bandage
rubber
dam
Blenderm
rubber.
cloth
adhesive
tape
plastic
collar
Exposure
Time
5hr./day
for 5 days
14 days
cumulative
24 hr. &
2nd 24 hr.
24 hrs.
24 hrs.
24 hrs.
7 x wk
for 3 wks.
24 hr
24/4/lhr
48 hr.
24 hr. &
2nd 24 hr.
Observation
Time
each day
for 5 days
24 hr. each
day for 15
days (16
readings)
24/48/72 hrs.
24hr./48hr.
24 hrs.
24 hrs.
daily
daily
24hr./72hr.
24/4/lhr
48 hr.
24/48/72hrs.
Scoring
Evaluation Misc.
visual
histolog-
ical
visual 6
skin thick
visual Evans
hiatolog- blue
ical
visual
visual
visual
visual
visual
visual
visual sulfan
blue
visual
histolog-
ical
visual
histolog-
ical
Type
Animal3
RBT
RBT
RBT
RBT
RBT
RBT
RBT
RBT
RBT
RBT
MINI
SWINE
GP
RAT
-------
Table 1-9. Summary of Testing Procedures Used in Denial Studies
(Continued)
Author Method
DAVIES patch
et al.
Patch
Type
gauze
Patch Occlusion
Size Method
1 in x 1 in rubber
cloth
Exposure
Tine
48 hrs.
Observation
Ti«e
48,72 hrs.
Scoring
Evaluation Misc.
visual
Type
Animal8
MICE, GP,
DOG, RBT,
PIG, BBOON,
MAN
aGP - Guinea Pig
.RBT - Rabbit
HRL - Hairless
BBOON - Baboon
MUS - Mouse
bHot Specified
-------
The standard cotton pad or gauze and adhesive tape has been the most
widely used and studied. In this example, a cotton pad, gauze, or porous
sheet is impregnated with or placed on the test substance and secured to the
skin with adhesive tape. A potential problem with this technique is that the
test substance may become contaminated with the adhesive material or leak onto
surrounding areas. In addition, the tape is in contact with the skin
proximate to the patch. These factors may influence the results of the test
and thereby decrease reproducibility and accuracy.
ADHESIVE TAPE
ADHESIVE AGENT
COTTON PAD
or
FILTER PAPER
The Al-Test method of the International Contact Dermatitis Research
Group uses a patch test unit that consists of polyethylene lined aluminum foil
on to which a filter paper disc is placed. This procedure separates the area
where the adhesive tape is placed from the test site and allows perspiration
to flow into and out of the paper disc. Perspiration between the polyethylene
sheet and the skin may influence the response.
ADHESIVE TAPE
ADHESIVE AGENT
ALUMINUM FOIL
POLYETHYLENE SHEET
FILTER PAPER DISC
25
-------
The Finn Chamber, a tray, was designed to eliminate the contamination
of the test substance by perspiration or the tape adhesive. The aluminum tray
(no polyethylene is used) is shaped so that a filter paper disc fits inside
which can be "sealed" to the skin by adhesive tape. Although this design
greatly reduces the chance of contamination, the close proximity of the
adhesive tape to the test area may alter the response.
ADHESIVE TAPE
ADHESIVE AGENT
ALUMINUM TRAY
FILTER PAPER DISC
The Kl-Chamber is a device that was designed by Kurokawa and colleagues
to eliminate the possible contamination of the test substance with perspiratio
or the adhesive. It consists of an aluminum tray which is inserted in a hole
in a porous protective paper sheet. The filter paper disc is then placed in
the tray. Thus the adhesive tape does not come into contact with the test
area.
ADHESIVE TAPE
ADHESIVE AGENT
PROTECTING SHEET
FILTER PAPER DISC
26
-------
Kurokawa et al (1980) compared the effectiveness of the KI chamber with
the Al-test and Finn chambers in humans exposed to vehicles and irritants.
They found that the KI chamber showed fewer false positive reactions with vehi-
cles and was superior to .the other patch test units in detecting water soluble
irritants.
Frosch and Kligman (1979) have also designed a patch test unit, the
Duhring Chamber, which is similar to the Finn chamber but has a larger inner
diameter. An elevated flange indents the skin in a circular pattern thus pre-
venting leakage of the test substance. A comparison of the two chambers showed
that the Duhring chamber produced reactions that were uniformly more intense.
The authors conclude that this increased sensitivity was due to the fact that
twice as much test agent can be applied to a given area of skin with,the Duhr-
ing chamber. On removing the chamber, an imprint or pressure ring on the skin
indicates that total occlusion was used during the test. Another advantage is
that the area of skin tested and the amount of test agent can be standardized.
4.1.1 Immersion
Immersion has been used in attempts to reduce the variability of test
results and increase the sensitivity of the test subjects. Benke et al.
(1977), Opdyke and Burnett (1965) and MacMillan et al. (1975) used an immer-
sion technique to evaluate the irritancy potential of aqueous solutions in the
guinea pig. Good agreement was found between this technique and the Rabbit
5-Day Dermal Irritation (Patch) Test (MacMillan et al., 1975). The technique
of immersion has not been validated in the rabbit.
"Human Arm Immersion" has been used to increase sensitivity to mild
irritants in humans (Polano, 1968; Smeenk, 1969; and Justice et al., 1961).
This technique, as with repeated patch tests, is based on the principle of
elicitation of response by repeated exposures of the same area to the test
material. The number of exposures required to achieve a prescribed level of
irritation in this sense amounts to a quantal response. This technique is
generally suitable for water soluble substances (soaps). The arm immersion
test method allows the standardization of the exposure conditions. Statistical
evaluation of the arm immersion technique has shown that it is reproducible
(Justice et al., 1961). Disadvantages are that it could induce systemic
toxicity if the test material were readily absorbed and it requires a large
amount of test material.
4.1.2 Occlusion
Steinberg et al. (1975), and Phillips et al. (1972) tested a variety of
compounds under different occlusive conditions (e.g., saran and stockinette,
elastic bandage, and both elastic bandage and stockinette). The type of occlu-
sion had a significant effect on the irritation response of the rabbit to the
test compound, thereby producing a change in its numerical score and the
irritation category.
27
-------
Gilman et al. (1978) demonstrated the effect of occlusions on dry and
moistened samples of commercial detergents in the rabbit. Occlusive patches
gave 1.5 to 10 times higher Primary Irritation Indices when compared to semi-
occlusive patches. The longer the patch was in place, the more severe the
response and the more marked the difference between the two patches. The
results are shown in the following table.
Primary Dermal Irritation With a Detergent in Rabbits
Dry Powder Paste (0.5 g + 0.1 ml)
Exposure Time (Hours)
4
8
16
24
Occluded
0.2
0.4
1.3
3.4
Semi-occluded
0.0
0.2
0.5
0.3
Occluded
0.0
0.3
2.0
7.1
Semi-occluded
0.0
0.05
0.25
1.3
The use of occlusive patches possibly puts the skin under duress, im-
pedes the normal functions of sweat evaporation, cooling, and respiration, and
interferes with evaporation of the test compound. As a result, skin permeabil-
ity and sensitivity are increased, which often leads to a "false positive"
reaction (Idson, 1969; Lanman et al., 1968; Rostenberg, 1961). This was
further illustrated by Phillips et al. (1972) who examined the occlusive and
nonocclusive (open) conditions of testing. A majority of the compounds that
caused some reaction under occlusive patch testing caused no reaction under
open conditions. Magnusson and Hersle (1966) studied occlusion to detect
subtle irritation. The use of the occlusive patch statistically decreased the
incidence of false negative responses with water-based irritants and petrolatum
bases by increasing the degree of irritation when compared to non-occluded
sites. /
Similiar observations suggest that adequate assessment of primary irri-
tants may require both open and occlusive tests if very high sensitivity is
required for special use and applications (Battista and Rieger, 1971; Skog,
1963; Rostenberg, 1961).
4.1.3 Conclusions on Patch Test Unit
In summary, the use of chambers to hold the test material in contact
with the skin in dermal irritation studies is a promising technique that
deserves further evaluation in animals. A number of investigators have con-
cluded that the chamber increases reproducibility. Since total occlusion
produces a substantial increase in sensitivity, substances that are non-
irritating by other methods may be weakly irritating when tested with a
chamber. Another advantage of the use of chambers is that the amount of
chemical and the test area can be standardized. Leakage from the patch site
is also decreased. The use of chambers may, however, increase costs in the
short run. Immersion is another promising human procedure which should be
given further consideration for use in animals.
28
-------
4.2 Abrasion
The standard Primary Irritation Index (PII) (Table 1-3) is derived from
scores equally weighted from intact and abraded skin. Abraded skin generally
gives a more severe reaction than intact skin because destruction of the stra-
tum corneum removes the barrier for penetration. The concentration of the
test chemical needed to produce an effect is, therefore, usually lower. Stan-
dardization of abrasion techniques is also believed to be difficult. For these
reasons, the National Academy of Sciences (1977) recommended against using
abraded skin, citing the data of Nixon et al. (1975). These data showed that
rank order classifications of irritation based only on intact skin usually
resembled those for abraded skin. Nixon and his coworkers (1975) stated that
most test materials gave irritation scores in animals, especially rabbits,
that were considerably higher than human scores. They attributed this dis-
crepancy to the much higher scores obtained from abraded skin. This is shown
clearly in Table 1-10. These authors came to the same conclusion as the
National Academy of Sciences on the testing of abraded skin. They explained
that the Draize test was originally designed to test topical drugs and
cosmetics where damaged skin may be present.
McCreesh and Steinberg (1977) found no differences in irritation when 5
materials were tested in rabbits on intact skin and skin abraded by 3 different
techniques. They also cited the finding of Nixon et al. (1975) that no corre-
lation existed between scores on abraded skin of rabbits and the potential for
a compound to be a primary.skin irritant in humans. Therefore, if there are
no differences between abraded and intact skin, or if abrasion gives an exag-
gerated response, abraded skin testing is an unnecessary step as test results
will be misleading.
A scarification (abrasion) technique was used by Frosch and Kligman
(1977) in human subjects to assess the irritancy of a large number of agents.
Having first experimented with scotch tape stripping, abrasion with hard
particles, pretreatment with anionic surfactants and the use of dimethyl-
sulfoxide (DMSO) to alter the stratum corneum, they were unable to demonstrate
any consistent reproducibility. The most satisfactory technique consisted of
scratching with a 30-gauge half-inch needle. Using the Duhring chamber (Frosch
and Kligman, 1979) sealed to the skin by a nonocclusive tape, the test agent
was applied once daily for 3 days. Daily readings were taken and reactions
graded from 0 to 4. The ratio of the mean irritancy thresholds on scarified
and normal skin was then determined and designated as the scarification index
(SI). Substances believed to be nonirritating produced no reaction, but most
irritants were detectable at much lower concentrations using scarification,
indicating that abrasion increases the sensitivity of the test. The authors
suggested that the increase in sensitivity and reproducibility should justify
the use of the scarification technique.
Frosch and Kligman (1977) reported that scarification (abrasion) greatly
increased sensitivity and reproducibility for the detection of weak irritants
in humans. They measured the lowest concentration at which the test substance
caused irritation. This was identified as the threshold concentration. They
recognized that abrasion may be inappropriate for strongly irritating sub-
stances but emphasized that a high threshold concentration (low score) with
29
-------
Table 1-10. Four-Hour Patch Test Results3
Test material
Metasilicate/carbonate
detergent granules
High carbonate detergent
granules
Low carbonate detergent
granules
Phosphate detergent granules
Enzyme detergent granules
Liquid detergent
Liquid cleaner
Pine oil cleaner
Household ammonia
Hypochlorite bleach
Liquid shampoo
Concentration
(w/v aqueous)
50%
50%
50Z
50%
50%
undiluted
undiluted
undiluted
undiluted
5.25%
undiluted
Animal
species
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Rabbit
Guinea pig
Human
Mean Scores''
Intact
6.8
0.0
3.0
0.9
0.0
0.0
0.7
o.i
0.0
1.2
0.2
0.0
1.3
0.0
0.0
2.4
0.8
0.1
3.0
0.3
0.2
3.6
2.5
1.0
1.4
0.0
1.5
1.0
0.3
3.9
3.7
2.1
0.9
Abraded
8.0
0.6
4.2
2.6
0.4
0.0
0.8
1.0
0.2
5.6
1.0
0.4
4.3
0.6
0.1
3.1
1.6
0.1
5.2
4.3
0.5
4.0
3.1
0.6
4.0
4.0
__
1.3
1.2
_
4.2
2.3
1.4
Irritancy
Judgement0
Corrosive
Negligible
Severe
Negligible
Slight
Negligible
Slight
Slight
Negligible
Moderate
Slight
Negligible
Moderate
Negligible
Negligible
Moderate
Slight
Negligible
Moderate
Moderate
Negligible
Moderate
Moderate
Slight
Moderate
Moderate
Moderate
Slight
Slight
Severe
Moderate
Moderate
Slight,
30
-------
Table 1-10. Four-Hour Patch Test Results*
(Continued)
Test material
Antiperspirant
Coconut soap
Isopropyl alcohol
Lemon juice
Table salt
Potassium hydroxide
Sodium metasilicate
Sodium carbonate
Sodium tripolyphosphate
Sulfuric acid
Acetic acid
Concentration Animal
(w/v aqueous) species
undiluted Rabbit
Guinea pig
Human
50% Rabbit
Guinea pig
Human
undiluted Rabbit
Guinea pig
Human
undiluted Rabbit
Guinea pig
Human
50% Rabbit
Guinea pig
Human
10% Rabbit
Guinea pig
Human
50% Rabbit
Guinea pig
Human
50% Rabbit
Guinea pig
Human
50% Rabbit
Guinea pig
Human
10% Rabbit
Guinea pig
Human
10% Rabbit
Guinea pig
Human
Mean Scores"
Intact
0.0
0.0
0.1
2.5
0.3
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
0.0
6.9
7.6
8.0
1.7
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.0
0.2
0.0
0.0
1.0
Abraded
1.0
0.0
0.5
3.1
0.8
0.0
0.0 .
0.0
0.8
1.0
0.0
0.3
0.5
0.0
0.8
7.0
7.6
8.0
3.2
1.5
0.1
2.0
0.5
0.0
1.8
0.1
0.0
0.2
1.7
0.1
1.1
Irritancy
Judgement0
Slight
Negligible
Negligible
Moderate
Slight
Negligible
Negligible
Negligible
Negligible
Slight
Negligible
Negligible
Negligible
Negligible
Negligible
Corrosive
Corrosive
Corrosive
Corrosive
Moderate
Corrosive
Slight
Negligible
Negligible
Negligible
Negligible
Negligible
Negligible
Negligible
Negligible
Slight
Negligible
Slight
31
-------
Table 1-10. Four-Hour Patch Test Resultsa
(Continued)
Test material
Sodium lauryl sulfate
CgCjQ fatty acids
Concentration Animal
(w/v aqueous) species
50Z Rabbit
Guinea pig
Human
undiluted Rabbit
Guinea pig
Human
Mean Scores'5
Intact
1.6
0.0
0.6
4.4
0.1
2.3
Abraded
2.6
0.8
1.1
4.7
0.8
Irritancy
Judgement0
Moderate
Negligible
Slight
Moderate
Slight
Moderate
aAdapted from Nixon et al. (1975).
bsum of mean erythema and edema scores (on a 0-4 scale) at 4, 24, and 48 hr.
clrritancy judgements for the human scores are according to the following scale (for
intact skin sites only): 0-0.4 « negligible; 0.5-1.4 a slight; 1.5-2.4 = moderate; 2.4
severe; substantial tissue destruction or irreversible change =» corrosive. For both
animal species, the scale is based on Pll scores, as follows: 0-0.4 » negligible;
0.5-1.9 - slight; 2.0-4.9 - moderate; 5.0-8.0 « severe; tissue destruction or
irreversible change = corrosion (Draize, 1959).
32
-------
abrasion is a guarantee of mildness no matter how the product is used. The
main objective of their work was to develop methods to detect mildly irritating
compounds and/or those causing irritation in a small fraction of the popula-
tion. Organic acids, antimicrobials, inorganic salts, and surfactants were
tested with and without abrasion. In every instance, irritation was detected
at a lower concentration on abraded skin (2.5- to over 100-times lower) than
on intact skin (Table 1-11).
In summary, the value of abrasion in dermal irritation testing depends
on the exact purpose of the test. Abrasion could be used to increase sensitiv-
ity in the testing of skin preparations or detergents where irritation in a
small percentage of the population is to be avoided whenever possible. When
measuring the corrosive potential of a chemical for a DOT shipping label where
the extra sensitivity is not required, abrasion would be seen as causing an
exaggerated response.
An initial test on intact skin should be performed. If no irritation
is noted and increased senstivity is required, the test could then be per-
formed on abraded skin. If a low score is again obtained, the mildness of a
product is further assured. The added sensitivity of the abraded skin test
should continue to be optional in the general case for industrial chemicals
but may be useful in more fully assessing irritation potential.
4.3 Application of Dry Test Substances
For animal experiments, most methods utilize 0.5 g or 0.5 ml on an inch
square area, as was originally recommended by Draize. Although the exact
method of application of solids and semi-solids is not clearly defined, the
sample usually is moistened before it is applied. Liquids are generally
applied undiluted but a dilute test substance may be justified based on the use
of the material in its final form. For solids or semi-solids, considerable
variation in technique is possible within the framework of the proposed guide-
lines. For example, a solid may be applied dry to premoistened skin, dry and
covered with a moistened patch, dry and "injected" beneath a preapplied wet
patch, dry or as a slurry. Sullivan et al. (1975) examined these variables and
found signficant differences in irritation. Depending on whether the material
tested was applied dry and subsequently moistened or applied as a slurry, re-
sponse ranged from no response to necrosis. McCreesh and Steinberg (1977),
however, reported that the different methods of application had no effects on
irritant ranking (although there were some differences in scoring and classi-
fication). They cited the findings of Steinberg et al. (1975) in which an
evaluation of 12 compounds showed similar irritancy when the results for appli-
cation to rabbit skin on a fixed area (2x2 cm) were compared with those for
direct application under or on a gauze pad. They concluded that the physico-
chemical nature of the test agent is more important than the method of
application.
Oilman et al. (1978) tested a number of detergents as a dry powder
(100%), aqueous paste (83%) and as aqueous suspensions (50, 25, and 10% w/v)
in rabbits. The degree of wetness was found to influence the degree of skin
irritation. The most severe skin reactions occurred when the detergent was
applied as a paste for 24 hours with an occluded patch.
33
-------
Table 1-11. Effect of Abrasion on Skin Irritation in Humans3
Threshold Concentration (%)
Agent
Acids
Oleic acid
Benzoic acid
Laurie acid
Linoleic acid
Antimicrobials
Formalin
Tricloaan
Benzalkonium chloride
Hexachlorophene
Inorganic Salts
Nickel sulfate
Aluminum chloride
Potassium iodide
Surfactants
Isostearamidopropyl
morpholine lactate
S tear ami dopropyl
dimethylamine lactate
Tr i ethanolamine
Triton X-100
Sodium lauryl sulfate
Solvent Normal Skin
ethanol
ethanol
ethanol
ethanol
water
ethanol
water
ethanol
water
water
water
water
water
ethanol
water
water
30.0
30.0
4.0
20.0
2.0
1.5
0.2
2.5
20.0
30.0
60.0
25.0
10.0
100.0
50.0
0.5
Abraded Skin
-
5.0
7.5
1.0
5.0
0.05
0.25
0.05
1.0
0.13
2.5
5.0
2.5
0.5
5.0
1.0
0.05
Normal/
Abraded
6
4
4
4
40
6
4
2.5
154
12
12
10
20
20
50
10
aAdapted from Frosch and Kligman (1977)
34
-------
The OECD guidelines specify that solid test substances should be mois-
tened sufficiently with water or, where necessary, a suitable vehicle, to
ensure good contact-with the skin. Similarly, other methods call for pre-
moistening solid test materials with water or an appropriate solvent.
Water probably should be the standard choice for moistening the test
agent. One reason for using water is to mimic the presence of perspiration.
To standardize the procedure for testing solids it would be advantageous to
choose one condition such as a slurry with water applied to the patch at a 1:1
ratio (w/v) or any other convenient concentration. Another alternative would
be to apply a dry powder and cover with patch soaked with 0.5 ml water. This
would alleviate some of the imprecision in applying solid substances. Stan-
dardization of the method for moistening solid test substances would increase
the ability of the test to assess the comparative irritancy of these materials.
Extra tests with other solvents could also be performed to simulate conditions
of use or exposure.
4.4 Application Site
The region of the body where the patch is applied can affect the results
of irritation studies. In humans, patches are usually placed on the arm or on
the back, while the abdomen and back are used most often in animals. The
Federal Hazardous Substance Act does not specify the region on the rabbit to
which the test substance should be applied, although a revision proposed in
1972 (by FDA) specified the back as the test site (FR No. 27, 27635-27636).
Vinegar (1979) examined the regional variation of primary skin irrita-
tion in rabbits. Thirty three common types of household items were tested
with a modified Draize procedure. The Primary Irritation Indices of the abdo-
men were significantly higher than those of the back. A possible explanation
of different skin thickness was offered. This study clearly demonstrated that
the site of application is a potential source of variation between laboratories
in skin irritation studies. Studies on the regional variation in skin irrita-
tion in humans were not identified in a search of the literature. However,
Maibach et al. (1971) found marked regional variation in the percutaneous
absorption of pesticides in man. Because the degree of irritation may be
related to the rate of percutaneous absorption, regional variation in skin
irritation probably also occurs in humans.
In order to minimize inter-laboratory variation a single skin site for
testing should be specified. In animals, the back has been used most frequent-
ly. This is advantageous since access by the animal to the patch is minimized
preventing resultant interference with the test site as well as ingestion or
inhalation of the test substance.
4.5 Exposure And Observation Period
The original Draize test and FHSA guidelines (Table 1-3) specified a 24-
hour exposure. The NAS recommended a 4-hour exposure based on a more realistic
simulation of the type of exposure expected in man. Similarly, the IRLG and
OECD guidelines specify a 4-hour exposure period. This does not preclude the
use of longer exposures when warranted. When very large numbers of people
35
-------
PII
4-hr Exposure
0.0
0.0
0.0
0.0
(See Table 1-2)
24-hr Exposure
4.7
3.9
3.4
might be exposed or when extended exposure is possible under anticipated condi-
tions of use, a longer exposure period may be desired. The original Draize
test, being intended for the study of cosmetic and topical agents, was designed
to detect mild irritation that might occur in a very small proportion of ex-
posed people. A substance that is not irritating after a 4-hour exposure may
be irritating after a 24-hour exposure. This was shown by Oilman et al. (1978)
for four dry detergent powders.
Primary Dermal Irritation Indices with Detergents in Rabbits
Sample
A
B
C
D
The authors considered these 24-hour irritation indices to be exaggerat-
ed, because even with misuse, exposures to detergents would be expected to be
less than 4 hours. An increase in sensitivity with prolonged exposure was also
demonstrated in humans by Kligman and Wooding (1967). Therefore, as is found
for abrasion, the use of an extended exposure period is an effective means of
achieving heightened sensitivity, when required.
While the current method for measuring skin irritation is usually satis-
factory for detecting severe irritants, it lacks the ability to differentiate
between a mild and a moderate human irritant (Phillips et al., 1972; Roudabush
et al., 1965; Steinberg et al., 1975, NAS, 1977), and may not even rate them
as irritating. Selectively increasing the exposure period in both animal and
human studies may help in the determination of comparative irritancy. To
obtain responses to mild and moderate irritants, extended periods of exposure
have been suggested (e.g., 21-day continuous closed patch test). With this
procedure in humans, the test material is applied to subjects under an occlu-
sive patch for 24 hours before the patch is removed for evaluation. After
scoring, the test material and patch are reapplied daily to the same site for
21 days (Steinberg et al., 1975). When this procedure was used, the authors
found that sensitivity increased to a point where it agreed with data for rab-
bits treated under a similar regimen. This protocol is an adaptation of the
repeated patch test for humans (Kligman, 1961), whereby the test material and
patch are reapplied for a maximum of 10 days or until inflammation appears.
A major benefit of the 21-day continuous closed patch test is that it
considerably reduces errors arising from subjective assessment of skin reac-
tions scored after a single exposure of 24 hours, and thus offers better
predictability of the irritant effects of test chemicals in man (Lanman et al.,
1968). The added sensitivity gained, however, is not without its drawbacks
and the 21-day test has been termed "economically heroic," (Steinberg et al.,
1975).
In the Rabbit 5-Day Dermal Irritation Test devised by MacMillan et al.
(1975), the test material is washed off after 4 hours of contact and the irri-
tation is scored 20 hours later, according to the Draize method. Application
36
-------
of the material to the same site and scoring of the irritation are performed
on 5 consecutive days. After removing the test substance, a period of 30-60
min. is allowed to elapse before scoring the site to minimize pressure and
hydration effects. Readings are also usually taken at 48 and 72 hours after
the patch was originally applied. The observation period is generally 7 days
because corrosive effects are best seen at this time (NAS, 1977). It may be
advisable to observe the animals for 2 weeks but practical considerations such
as cost and usefulness of information should be taken into account.
In conclusion, four hours appears to be an adequate minimum exposure
time for measurement of primary irritation from exposure to industrial chemi-
cals. This time interval is specified by the IBLG and OECD guidelines and is
compatible with DOT requirements. Many investigators may still choose to con-
tinue exposure for 24 hours or longer when increased sensitivity is required.
The extent of both the exposure and observation periods remains primarily a
matter of individual preference and need.
4.6 Enhancements to Visual Scoring
When the skin reaction to the standard patch test for skin irritants
(especially with mild irritants) is difficult to detect (because the erythema
is masked by the skin color of the animal), vital dyes or formaldehyde have
been applied to increase sensitivity and to enhance the visibility of the ery-
thema 1 response. Evans, Sulfan or Trypan blue are injected intravenously in
animals being patch tested; the dyes collect in areas where vasodilation has
occurred, coloring the skin within minutes after injection. This technique
has proven useful in identifying mild erythema which was undetectable by the
naked eye (Brown, 1971; Finkelstein et al., 1963; Wolven and Levenstein, 1967;
Motoyoshi et al., 1979).
37
-------
5.0 REPRODUCIBILITY AND SCORING
5.1 Reproducibility
Weil and Scala (1971) examined the reliability and reproducibility of
skin irritation testing. Even though many toxicology laboratories attained a
degree of internal consistency using their own unique experimental designs, no
information had existed on the consistency of results among laboratories.
Neither the results using the methods specified in the FHSA nor individualized
laboratory methods had been compared. In a similar study in 1967, Weil and
Wright examined the question of interlaboratory variability in acute oral tox-
icity testing. The assumption was that an oral LD50 determination required
fixed methodology in order to attain reliability and reproducibility. The
authors concluded that this was not the case but rather, competent laboratories
will generally be in good agreement even though methods vary.
A different conclusion was reached by Weil and Scala (1971) for dermal
irritation testing. In their study, a total of fourteen materials were
selected and aliquots of single lots of each were coded and sent to over 20
participating laboratories for blind testing generally using FHSA testing
methodologies. The results showed considerable variation between laboratories
and at the same laboratory among rabbits treated with the same substance. The
variability in scoring was marked in several areas including the range of
scores for the same substances, the range of animals with necrosis, the pre-
sence or absence of recovery during the 72 hour grading period for the same
substance, and the rank ordering of the substances for relative irritancy. One
laboratory had edema and erythema scores of zero for every rabbit for each of 9
materials. The other laboratories scored these same materials much higher, in
some cases, near the maximum possible score (23) for primary irritation.
Unlike FHSA (Table 1-3), the reference scoring system used by Weil and
Scala scored necrosis as well as erythema and edema in determining the primary
irritation score. Necrosis was scored as zero (no necrosis) for several com-
pounds at one group of laboratories while the same compounds caused necrosis
in a large percentage of the rabbits at several other laboratories. Recovery
(decreased irritation) was apparent at 72 hours in some laboratories, while
others reported much more severe reactions at 72 than at 24 hours. There was
some sharp disagreement between labs on the relative ranking of several sub-
stances. When the average of all laboratories' scoring was compared with
individual laboratory results, however, reference results correlated well in
20 of 22 laboratories and nonreference methods in 17 of 20, (in the other labo-
ratories, there was no correlation.) The average responses when rank ordered
by degree of irritation also agreed fairly well between reference and nonrefer-
ence methods. The greatest variation occurred among the lowest irritation
scores. This good correlation was not unexpected as the nonreference methods
were in most cases either the FHSA or Draize procedures.
A follow-up study carried out at two laboratories, revealed that one
rated compounds more severely than the other. This was considered by Weil and
Scala to be a possible explanation for many of the variations in scoring. A
steering committee member also performed the tests in each laboratory for com-
parison. There were apparent and very obvious differences in the application
38
-------
and wrapping procedures used. In both laboratories, readings were more severe
when the laboratory personnel applied the test substances in comparison with
the steering committee member. In one laboratory, the plastic wrapping was
applied tightly and completely occluded the patch, therefore enhancing irri-
tancy. Scoring in both laboratories came closer to the average scores for all
laboratories in the test program when rerun, but they were still different from
the "standard" results of the steering committee member. In one laboratory,
the severe scores were due to the use of a different technician the first time.
The steering committee member also obtained different readings in the two
laboratories.
Weil and Scala (1971) concluded that although the Draize procedure had
been in use in the various test laboratories for over 20 years, several labo-
ratories were outliers. Since the type of laboratory; i.e. consultant,
government, food, cosmetic, or industrial, had no relationship to its internal
or external variability, they felt that the Draize test should not be used to
classify materials as irritants when consistency was desired. Subjective
scoring seemed to be the main source of error with procedural variations also
contributing. As a solution to the problem the authors advised that training
courses and clinics be conducted frequently.
5.2 Other Scoring Methods
Inspection of Tables 1-1, 1-3, 1-8, and 1-9 show that nearly all studies
have utilized visual scoring. To minimize subjectivity and improve objectivity
of the standard test results, several investigators have used additional quan-
titative techniques to identify skin damage caused during testing more clearly.
Ingram and Grasso (1975) found a good correlation between the histologic and
visual evaluations of irritant induced changes. Histological examination was
used by Guillot and coworkers (1981) to evaluate irritation responses where the
color of the test agent (e.g., copper nitrate, orthovanillin) precluded visual
scoring. A scoring system based on histopathologic examination was also used
by Brown (1971). Other approaches, such as measurement of dermal respiration
and enzymatic activity, alterations in dermal collagens, changes in dermal pH,
elasticity, and electrical properties, have been discussed by Lansdown (1972).
A simpler quantitative procedure, which is much less dependent on expen-
sive equipment or limited resources (pathology), was originally described by
Kligman and Wooding (1967) in humans. The sole criterion of irritation in this
method is the presence or absence of erythema. The data are subjected to a
statistical analysis, similar to that used in determining an LD50, and the
values obtained can be used to assess the relative irritancy of materials.
Test agents are applied to a Webril patch which is occluded to the skin
by overlapping strips of impermeable plastic tape. A 1 square centimeter patch
is used based on the results of quantitative measurements which showed that
above 0.5 cm2, the size of the patch does not affect results. Mixtures con-
taining volatile substances (i.e., alcohol, acetone) at concentrations of 20%
or more are tested with a nonocclusive patch since the strong irritation pro-
duced by the volatile components masks the effects of any other ingredients.
In these tests, Webril patches (1 cm2) are held on the skin under slightly
larger squares of gauze and are fastened only along the edges with perforated
39
-------
tape, test materials are applied in a volume which loads the patch completely
without producing overrun (approximately 0.05 ml/cm2).
For weak irritants, an ITSO value (the number of days required to cause
50Z of the sampled population to develop a threshold irritant response) is
obtained per each irritant tested in a minimum of 10 subjects exposed continu-
ously for 10 days. Patches and irritants are applied once daily at the same
site. An end point is reached when positive erythema is noted. The number of
days required for this response is recorded. The logarithm of the cumulative
percentage of animals showing a reaction is plotted against days and the ITSO
is obtained from the resulting line.
For tests of strong irritants, exposure lasts only one day. Different
concentrations are used and the logarithm of the cumulative percentage of
animals showing a reaction is plotted against concentration. In this case, an
ID50 value (the concentration which produces an irritant response in 50Z of the
sampled population) is determined.
IT50 and ID50 values represent relative assessments of irritancy which
are most meaningful when a standard of reference is included in the test. A
similar approach was used by Justice et al. in 1961.
Steinberg et al. (1975) have suggested the use of a Threshold Irritation
Concentration (TIC) of test chemicals in this type of testing. This resulted
from their observation that a good correlation exists between the threshold
concentration and the corresponding irritation score. Nevertheless, the re-
sults of repeated patch testing could be misleading in distinguishing between
products that cause primary irritation and the general effects of repeated
patching, sometimes termed "skin fatigue".
5.3 Recommendations
Standardization of scoring procedures combined with improved training
guidelines can minimize variation in subjective scoring. FDA's Proposed
Revision of the test for Primary Skin Irritants (37 CFR 244, p.27635) des-
cribed the first step in such training efforts as an "Illustrated Guide for
Grading Dermal Irritation." Thus far, only a guide for eye irritation has
been published (formerly available from the Consumer Product Safety
Commission). It is strongly recommended that a series of photographs or
slides depicting various grades of irritation be produced. The reproductions
could be evaluated and "graded" by a suitable panel of toxicologists. Ulti-
mately a guide, much the same as that originally developed by the FDA for eye
irritation, could be made available for public dissemination. This would
greatly facilitate the training of personnel in dermal irritation studies and
could help standardize the scoring. Photographs of the various degrees of oc-
clusion would also be valuable. The validation of the quantal response method
in animals described by Kligman and Wooding (1967) should also be pursued as
this less subjective method would markedly reduce the sources of error out-
lined by Weil and Scala (1971). Solicitation of specific information on the
parameters used in dermal irritation studies by laboratories is also suggested.
40
-------
6.0 CONCLUSIONS
The rabbit is the species most widely used for dermal irritation test-
ing although the guinea pig shows similar sensitivity and is more economical
to house. Both species do not completely predict human response, however, as
moderately and minimally irritating compounds may show either stronger or
weaker response. In many cases, the rabbit and guinea pig results taken
together more accurately predict the human response; compounds misgraded by
one species are more accurately detected by the other. The final measurement
of human potential exposure must be tested in man. The hairless mouse has
received attention recently as an alternative test species. Both the guinea
pig and the hairless mouse seem to deserve further study.
The use of chambers is becoming more common in human testing. These
devices show several advantages over the standard guaze patch. Leakage from
the test site is decreased and the degree of occlusion, amount of chemical and
area of exposure are more effectively controlled when chambers are used. No
one chamber design seems to be superior to the others at this time. The cham-
ber has not been validated in animals; this action is strongly recommended.
Immersion has also been used successfully in humans and appears promising but
needs to be examined further in animals.
Occlusion of a patch test site affects the sensitivity of the skin to
irritants as does abrasion. There is a difference in viewpoint whether this
is an increase in sensitivity or an exaggerated response depending on how the
results of the test are used. Neither full occlusion nor abrasion is recom-
mended for general testing but should be allowable modifications if more
sensitivity is required by the testing organization.
Solid materials are less easily handled than liquids and the method of
application can affect the degree of irritaion. Water is the most common sol-
vent used for moistening solids.
The site of application can also affect results, the abdominal region
being more sensitive than the back. The dorsal region is preferred as it is
easily accessible to the animal handler but not to the animal.
Four hours appears to be an adequate minimum exposure time for the
large majority of compounds that would require testing. Increasing the time
of exposure to a compound will increase skin sensitivity._ Investigators,have
used periods up to 21 days to enhance effects of very mild irritants.
Scoring, as it is currently practiced, is a rather subjective proce-
dure. At present, there are no standard study sets available for training as
there are for eye testing. Several approaches to measure quantal responses
have been described for human testing but need further evaluation in animals.
41
-------
7.0 REFERENCES
Battista, G.W. and M.M. Rieger. 1971. Some Problems of Predictive Testing,
J. Soc. Cosmet. Chem. 22:349-359
Benke, G.M., N.M. Brown, M.J. Walsh, et al. 1977. Safety Testing of Alkyl
Polyethoxylate Nonionic Surfactants. I. Acute Effects, Fd. Cosmet.
Toxicol. 15:309-18
Bjornberg, A. 1974. Skin Reactions to Primary Irritants and Predisposition
to Eczema, Br. J. Dermatol. 91:425-7
Bjornberg, A. 1975. Skin Reactions to Primary Irritants in Men and Women,
Acta Derm. Venerecol. 55:191-4
Brown, V.K.H. 1971. A comparison of Predictive Irritation Tests with
Surfactants on Human and Animal Skin, J. Soc. Cosmet. Chem. 22:411-20
Dahl, M.V., and R.J. Trancik. 1977. Sodium Lauryl Sulfate Irritant Patch
Tests: Degree of Inf1annation at Various Times, Contact Dermatitis
3:263-6
Davies, R.E., K.H. Harper; S.R. Kynoch. 1972. Inter-species Variation in
Dermal Reactivity, J. Soc. Cosmet. Chem. 23:371-81
Draize, J.H., G. Woodard and H.O. Calvery. 1944. Methods for the Study of
Irritation and Toxicity of Substances Applied Topically to the Skin and
Mucous Membranes, J. Pharm. Exp. Therap. 82:377-419
Draize, J.H. 1959. Dermal Toxicity. In: Appraisal of the Safety of Chemi-
cals in Food, Drugs and Cosmetics. Assoc. of Food and Drug Officials of
the United States. Austin, Texas p. 46
Draize, J.H. 1955. VIII. Dermal Toxicity, Food Drug Cosmet. Law J.
10:722-32
Finkelstein, P., K. Laden, and W. Miechowski. 1963. New Methods for
Evaluating Cosmetic Irritancy, J. Invest. Dermatol. 40:11-4
Finkelstein, P., K. Laden, and W. Miechowski. 1965. Laboratory Methods for
Evaluating Skin Irritancy, Toxicol. Appl. Pharmacol. 7:74-8
Frosch, P.J. and A.M. Kligman. 1977. The Chamber-Scarification Test for
Assessing Irritancy of Topically Applied Substances. In: Cutaneous
Toxicity. V.A. Drill and P. Lazar, Eds. Academic Press, N.Y. pp.
127-154
Frosch, P.J. and A.M. Kligman. 1979. The Duhring Chamber: An Improved
Technique for Epicutaneous Testing of Irritant and Allergic Reactions,
Contact Dermatitis. 5:73-81
42
-------
Oilman, M.R., R.A. Evans, and S.J. DeSalva. 1978. The Influence of
Concentration, Exposure Duration, and Patch Occlusivity Upon Rabbit
Primary Dermal Irritation Indices. Drug and Chemical Toxicity 1:391-400.
Gloxhuber, C. 1976. Testing of Sunbath Preparations in Hairless Mice,
J. Soc. Cosmet. Chem. 27:399-409
Golberg, L. 1975. Safety Evaluation Concepts, J. AOAC 58(4):635-644
Gordon, B.I., and H.I. Maibach. 1969. Adhesive Tape Anhidrosis, Arch.
Dermatol. 100:429-31
Griffith, J.F., and E.V. Buehler. 1977. Prediction of Skin Irritancy and
Sensitizing Potential by Testing with Animals and Man, Cutaneous Toxic.,
(Proc. Conf.) 3rd, 155-73
Guillot, J.P.; Caillard, L.; Gonnet, J.F.; Clement, C. 1981. Chemicals -
Ocular and Cutaneous Local Tolerance "Cosmetic", A.F.N.O.R. and O.E.C.D.
Protocols. Institut Francais de Recherches et Essais Biologiques Service
de Tolerance Locale.
Homberger, F., A. Tregier, J.R. Baker, et al. 1961. The Use of Hairless
Mice for Study of Cosmetics, Proc. Sci. Sect. Toilet Goods Assoc. 35:6-11
Idson, B. 1969. Primary Irritation Testing, Toxicol. Appl. Pharmacol.
Suppl. 3, 84-9
Interagency Regulatory Liaison Group (IRLG; March 1981) Testing Standards and
Guidelines Work Group. Draft IRLG Guidelines for Acute Test for Primary
Skin Irritation.
Ingram, A.J., and P. Grasso. 1975. Patch Testing in the Rabbit Using a
Modified Human Patch Test Method. Application of Histological and Visual
Assessment, Br. J. Dermatol. 92:131-42
Justice, J.D., J.J. Travers, L.J. Vinson. 1961. The Correlation Between
Animal Tests and Human Tests in Assessing Product Mildness, Proc. Sci.
Sec. Toilet Goods Assoc. 35:12-7
Kastner, W. 1977. Species Dependence of the Skin Compatibility of Cosmetic
Ingredients, J. Soc. Cosmet. Chem. 28:741-54
Kligman, A.M. 1969. Evaluation of Cosmetics for Irritancy, Toxicol. Appl.
Pharmacol. Suppl. 3, 30-44
Kligman, A.M. 1961. Quantitative Testing of Chemical Irritants. In: The
Evaluation of Therapeutic Agents and Cosmetics. T.H. Sternberg and V.D.
Newcomer, eds. New York: McGraw Hill Book Co. pp. 186-192
Kligman, A.M., and W.M. Wooding. 1967. A Method for the Measurement and
Evaluation of Irritants on Human Skin, J. Invest. Dermatol. 49:78-94
43
-------
Kurokawa, M., H. Iwamoto, I. Hasegawa. 1980. Kl-chamber patch test unit,
J. Soc. Cosmet. Chem. 31:97-104
Lanman, B.M., W.B. Elvers, C.S. Howard. 1968. The Role of Human Patch
Testing in a Product Development Program, Proc. Joint Conf. Cosmet. Sci.,
Toilet Goods Assoc. 135-45
Lansdovn, A.B.C. 1972. An Appraisal of Methods for Detecting Primary Skin
Irritants, J. Soc. Cosmet. Chem. 23:739-72
Larsen, W. 1977. Perfume Dermatitis, Arch. Dermatol. 113:623-626
McCree«h, A.H., and M. Steinberg. 1977. "Skin Irritation Testing in
Animals" In: Advances in Modern Toxicology, Dermatotoxicology and
Pharmacology, Edited by Marzulli, F.N.; Maibach, H.I., 4:193-210
MacMillan, F.S.K., R.R. Rafft, W.B. Elvers. 1975. "2. A Comparison of the
Skin Irritation Produced by Cosmetic Ingredients and Formulations in the
Rabbit, Guinea Pig, and Beagle Dog to that observed in the Human". In:
Animal Models in Dermatology. H.I. Maibach, ed. Churchill Livingstone,
N.Y. pp. 12-22
Maibach, H.I., R. J. Feldman, T.H. Milby, W.F. Serat. 1971. Regional
variation in percutaneous penetration in man; pesticides. Arch. Environ.
Health, 23(3):208-211.
Maibach, H.I., and U.L. Epstein. 1965. Predictive Patch Testing for
Sensitization and Irritation. Amer. Perfum. Cosmet. 80:55-56
Ma gnuason, B., K. Hersle. 1966. Patch Test Methods. 3. Influence of
Adhesive Tape on Test Response, Acta Derm. Venereol (Stockholm) 46:275-78
Mathias, C.G.T., and H.I. Maibach. 1978. Dermatotoxicology Monographs I.
Cutaneous Irritation: Factors Influencing the Response to Irritants,
Clinical Toxicology 13:333-346
Marzulli, F.N., and H.I. Maibach. 1975. The Rabbit as a Model for
Evaluating Skin Irritants: A Comparison of Results Obtained on Animals
and Man Using Repeated Skin Exposures, Food Cosmet. Toxicol. 13:533-40
Motoyoshi, K., Y. Toyoshima, M. Sato, et al. 1979. Comparative Studies on
the Irritancy of Oils and Synthetic Perfumes to the Skin of Rabbit,
Guinea Pig, Rat, Miniature Swine, and Man. Cosmet. Toiletries, 94:41-8
National Academy of Sciences. 1977. Principles and Procedures for
Evaluating the Toxicity of Household Substances. National Academy of
Sciences, Washington, D.C., 130pp.
Nixon, G.A., C.A. Tyson, W.C. Wertz. 1975. Interspecies Comparisons of
Skin Irritancy, Toxicol. Appl. Pharmacol. 31:481-90
44
-------
Odom, R.B., and H.I. Maibach. 1977. Contact orticaria: A Different Contact
Dermatitis. In: Advances in Modern Toxicology, Vol. 4, Marzulli, F.N.
and H. I. Maibach, eds. pp. 441-454.
Opdyke, D.L., and C.M. Burnett. 1965. Practical Problems in the Evaluation
of the Safety of Cosmetics, Proc. Sci. Sect. Toilet Goods Assoc. 44:3-4
OECD Guidelines for Testing of Chemicals. Acute Dermal Irritation/Corrosion.
Organization for Economic Cooperation and Development. OECD Publications
and Information Center, Suite 1207, 1750 Pennsylvania Ave., N.W.,
Washington, D.C. 20006
Phillips, L., M. Steinberg, H.I. Maibach, et al. 1972. A Comparison of
Rabbit and Human Skin Response to Certain Irritants, Toxicol. Appl.
Pharmacol. 21:369-382
Polano, M. 1968. In Interaction of Detergents and the Human Skin, J. Soc.
Cosmet. Chem. 19:3
Rapaport, M., D. Anderson, V. Pierce. 1978. Performance of the 21 Day
Patch Test in Civilian Populations, Cosmet. Toilet. 93:29-31
Rostenberg, A. 1961. Methods for the Appraisal of the Safety of Cosmetics,
Drug Cosmet. Ind. 88(5):592-86
Rostenberg, A., and C.M. Solomon. 1968. Jean Henri Fabre and the Patch
Test, Arch. Dermatol. 98(2):188-90
Roudabush, R.L., C.J. Terhaar, D.W. Fassett, et al. 1965. Comparative
Acute Effects of Some Chemicals on the Skin of Rabbits and Guinea Pigs,
Toxicol. Appl. Pharmacol. 7:599-65
Schmid, 0. 1970. Animal Experiments on Skin Tolerance, J. Soc. Cosmet.
Chem. 21:835-843
Schmidt, R.J., and F.J. Evans. 1980. Skin Irritant Effects of Esters of
Phorbol and Related Polyols, Arch. Toxicol. 44:279-89
Schwartz, L., and S.M. Peck. 1944. The Patch Test in Contact Dermatitis,
Public Health Rep. 59:546-57
Shelanski, H.A., and M.V. Shelanski. 1953. New Techniques of Patch Tests,
Drug Cosmet. Ind. 73:186
Skog, E. 1963. Irritant Effect of Industrial Hand Cleaners. A Comparative
Investigation on Guinea Pig and Human Skin, Arch. Environ. Health 7:682-5
Smeenk, G. 1969. The Influence of Detergents on the Skin, Arch. Klin. Exp.
Dermatol. 235:180-191
45
-------
Steinberg, M., W.A. Akera, M. Weeks, A.M. McCreesh, and H.I. Maibach. 1975.
1. A Comparison of Test Techniques Based on Rabbit and Human Skin
Responses to Irritants with Recommendations, Regarding the Evaluation of
Mildly or Moderately Irritating Compounds. In: Animal Models in
Dermatology. H.I. Maibach, ed. Churchill Livingstone, N.Y. pp. 1-11
Sullivan, J.B., J.C. Strausfeurg, and R.W. Kapp, Jr. 1975. "A Comparative
Study of Dermal Reaction Using the Intact Rabbit Skin". Abstracts of
Papers for the Fourteenth Annual Meeting of the Society of Toxicology,
Williamsburg, Va., pg. 87-88
Sulzberger, M.B., R.L. Baer, A. Kanof, and C. Lowenberg. 1946. Methods for
the Rapid Evaluation of the Beneficial and Harmful Effects of Agents
Applied to the Human Skin. J. Invest. Dermatol. 7:227-238
Uttley, M., N.J. Van Abbe. (1973). Primary Irritation of the Skin: Mouse
Ear Test and Human Patch Test Procedures, J. Soc. Cosmet. Chem. 24:217-27
Vinegar, M.B. (1979). Regional Variation in Primary Skin Irritation and
Corrosivity Potentials in Rabbits, Toxicol. Appl. Pharmacol. 49:63-69
Weil, C.S., and R.A. Scala. 1971. Study of Intra- and Interlaboratory
Variability in the Results of Rabbit Eye and Skin Irritation Tests,
Toxicol. Appl. Pharmacol. 19:276-360
Weil, C.S., and Wright. 1967. Intra- and Interlaboratory Comparative
Evaluation of Single Oral Test, Toxicol. Appl. Pharmacol. 11:378-388
Wolven, A., I. Levenstein. 1967. Techniques for Evaluating Dermal
Irritation, J. Soc. Cosmet. Chem. 18:199-203
46
-------
DERMAL SENSITIZATION
1.0 SUMMARY -
Dermal sensitization or contact dermatitis is a delayed allergic re-
sponse to a substance applied to the skin in which the clinical manifestations
are similar to those observed in dermal irritation, i.e., erythema and edema.
The guinea pig is the animal model used most frequently for contact
dermatitis testing. The most common procedures employed for determining dermal
senzitization in the guinea pig can be divided into three groups according to
the method of administration of the potential allergen.
o Intradermal injection - used in the Draize and the Freund Complete
Adjuvant Tests.
o Topical application - used in the Open Epicutaneous Test and the
Closed Patch Test.
o A combination of intradermal injection and topical application -
used in the Maximization, Optimization, Split-Adjuvant and the
Footpad Tests.
These test procedures are reviewed and evaluated for their efficacy as predic-
tive tests.
Comparative data from various sources indicate that all the test
procedures except that of Draize have some potential for detecting "weak"
sensitizers. The identification of weak sensitizers is extremely important in
the testing of cosmetics and toiletries. However, no one test is suitable for
testing all compounds and wide variation in the data from different sources
using the same test procedure, compound, and concentrations indicates the need
for a more thorough and systematic evaluation and validation of the test proto-
cols. Test procedures requiring intradermal injections are inappropriate for
use on finished products such as those containing emulsifiers, bacteriostatic
agents and formaldehyde.
The Maximization test is one of the better validated tests for determin-
ing weak sensitizers but the Optimization, Open Epicutaneous and the Closed
Patch tests appear to be viable alternatives. More published evidence, how-
ever, is needed to confirm the reliability of the latter three test methods.
In humans, the Maximization test procedure was found to be more sensi-
tive than Schwartz-Peck, Shelanski-Shelanski and the Draize tests, especially
for detecting "weak" allergenic reponses. Some problem with variation in re-
sults exists due to the different modifications of the Maximization test that
have been used for human testing. Therefore, this procedure needs to be stud-
ied further. The results of the Maximization test indicate that substances
which sensitize guinea pigs also sensitize humans. The quantitative agreement
between the human and guinea pig response to a variety of test chemicals is
also good.
47
-------
A practical skin sensitization testing program for a wide variety of
materials, such as industrial chemicals, pesticides, new drugs, paints and
coatings, toiletries and cosmetics should use both the guinea pig and man as
test subjects. Positive (strong sensitization) results in the guinea pig would
suggest the unsuitability of the substance where use exposure is likely. A
weak or negative response would indicate the need for further characterization
and testing in small groups of human subjects using the Maximization test.
48
-------
2.0 INTRODUCTION
2.1 Objective
The objective of this survey is to characterize, compare, and evaluate
the eight most common dermal sensitization testing procedures employed in the
guinea pig with the goal of determining which of the test methods is most pre-
dictive of the human sensitization response. At present these eight methods
are approved for use by the OECD (1981) guidelines for measuring dermal sensi-
tization.
The eight procedures described are the Draize, Freund's Complete
Adjuvant, Open Epicutaneous, Closed Patch, Maximization, Optimization, Split-
Adjuvant and Footpad Tests.
2.2 Scope
This report evaluates tests for identifying potential allergens. In
some instances, however, compounds appear to act more subtly and require re-
petitive contact or special environmental conditions (increased temperature
and humidity, occlusion, provocation, etc) to produce a response. Thus, no
single test or group of test will correctly identify all potential allergens
under all circumstances.
Scoring and grading charts are included when necessary to illustrate
differences in criteria and test results and the correlation among findings of
individual investigators or test laboratories. Data from different procedures
are used to compare the efficacy (or lack of it) of the procedures for detect-
ing grades of sensitization; e.g., strong, moderate or weak allergens.
Test procedures used in human sensitization are reviewed briefly. The
review is limited to those procedures for which comparable data were found in
the guinea pig. The comparative data were used to show the similarities and
differences in allergenic responses between the human and the guinea pig under
similar experimental conditions and to illustrate the predictability and re-
producibility of the human response from animal experiments.
The comparative data from a diagnostic test on humans from different
geographic areas (the North American Contact Dermatitis Group and the Inter-
national Contact Dermatitis Research Group) were included to illustrate the
limitations involved in extrapolating data from test animals to humans or
results from small localized test populations under closely defined environ-
mental conditions to the general population. These regional differences have
not been thoroughly studied.
While specific biochemical measurements and complementary tests have
also been used to provide information on the sensitization potential of test
materials, this report examines the methods which are more commonly performed.
49
-------
2.3 Definitions
o Allergens are conventionally defined as substances that will induce
innunologically dependent immediate or delayed reactions such as
erythema, edema, and vesiculation, in subjects that have been pre-
viously exposed.
o Freund's Complete Adjuvant is a mixture of killed bacteria and
mineral oil containing detergent. This is conventionally used to
emulsify allergens in water.
o Sensitization reactions are frequently more severe and more persis-
tent than irritation reactions, and sensitizers can be graded from
"weak" to "strong" based on the number of subjects that become
sensitized, rather than on the severity of the skin reaction.
A satisfactory predictive test for sensitization must (1) be able to
detect weak sensitizers, (2) show a good correlation between positive and neg-
ative sensitizers in the test and known human sensitizers and non-sensitizers,
(3) be reproducible, and (4) should be quick, easy to perform, and economical.
2.4 The Sensitization Test
A typical sensitization test involves exposing the subject animal to the
test substance in order to induce a response to subsequent exposures; this
phase is the induction phase (sensitizing). After a rest period of about 2
weeks (during which time the test subject develops the ability to respond to
the test material) the subject is re-exposed. This is the challenge phase.
The reaction during the latter phase is taken as evidence of sensitization only
if no reaction in the control subject without induction is seen.
2.5 Methods for Sensitization
The salient features of the sensitization process are as follows:
(1) When a simple chemical substance (hapten) is applied to the skin
it reacts with certain skin components. Two types of proteinconjugates
- mobile and immobile - are formed at the site of application. The
mobile forms are aUergenic and penetrate to regional lymph nodes where
after a suitable incubation period, a complicated immunologic process
results in the production of sensitized lymphocytes. The immobile forms
remain vn situ for some time and react with the sensitized lymphocytes
that are produced on reapplication of the hapten. This interaction
results in macroscopic dermal/epidermal alterations typical of contact
dermatitis (leucocyte chemotaxis, vasodilatation and increased
vasopermeation).
Contact dermatitis in humans is characterized by itching, erythema,
erythema, edema and vesiculation, and sometimes purpura and necrosis
whereas in the guinea pig the main features of sensitization are
erythema and edema. Characteristic histological features in humans
include spongiosis, exocytosis, vesicles, bulla formation, pustules,
50
-------
necrosis, acantholysis of the epidermis and perivascular infiltration,
eosinophilic leucocytes, dilatation of the lymphatic vessels and blood
capillaries and edema of the dermis (Nater and Hoedemaeker, 1976).
(2) A second requirement for skin sensitization is that the potential
allergen must gain entrance into the body. To enhance penetration of
the sensitizer, the skin site must be clipped, shaved or pretreated
(generally with sodium lauryl sulfate), irritated, excoriated (with
sand paper), stripped (with tape) or frozen.
A mixture of dead mycobacteria in paraffin oil is often used in
the guinea pig for its adjuvant properties. Mycobacteria in oil and
picrylated stromata, for example, can be included in an injection by
using an emulsifier, such as Freund's Complete Adjuvant.
(3) When the skin is tested for hypersensitivity, the results obtained
may differ due to the route of administration of the test material
(topical application or intradermal injection). The topical route
(epicutaneous) is the natural route of entry of most contact allergens,
but the intradermal route offers a more rapid entry to the lymphatic
system and a higher concentration can be achieved by this method than
by the epicutaneous route. The reactivity of the skin may vary with
the season and the test results may be affected by the degree of
intactness, hydration, contact pressure and occlusion.
(4) Other factors of importance during dermal sensitization testing
are the physiochemical properties of the vehicle, concentration of the
test substance, and duration of the exposure. Additionally, genetic
background, age, pregnancy and general state of health of test animals
are important variables (Magnusson and Kligman, 1977; Coenraads et al.,
1975).
2.6 Experimental Animal
The albino guinea pig is used for dermal sensitivity testing because it
most closely resembles man in its response to sensitizers (See Section 6.0).
Young adult animals (300-500 gm) of both sexes and various strains (Hartley,
Pirbright, or Himalayan white-spotted) with proven allergenic aptitude are
generally used. In many studies, a positive control group of animals is
exposed to a known sensitizer in order to demonstrate allergenic aptitude.
51
-------
3.0 PREDICTIVE TEST METHODS FOR SKIN SENSITIZATION USING GUINEA PIGS
In the cutaneous sensitivity tests developed in the last few years for
the guinea pig, an attempt has been made to improve the correlation of results
with human skin sensitivity compared to the intradermal test of Draize (1965).
As noted above the most common procedures currently used in the guinea
pig for testing materials for contact dermatitis can be classified into three
groups according to the method used to administer the allergen. They are -
o Group I, Draize Test and the Freund's Complete Adjuvant Test which
use the intradermal route,
o Group II, The Open Epicutaneous Test and Buehler's Closed Patch Test
in which the allergen is applied topically; and
o Group III, which includes the Kligman-Magnusson guinea pig
Maximization Test, the Optimization Test of Maurer, the Maguire
Split-Adjuvant Test and the Footpad Technique of Roudabush, all of
which use both the intradermal and epicutaneous routes.
The details of these test procedures are summarized and compared in
Table 2-1.
3.1 Draize Test
This test is based on the classical Landsteiner technique (Landsteiner
and Jacobs, 1935) as modified by Draize (Draize 1965).
Animals; Twenty guinea pigs each are used in the test and control
groups for each test material.
Test Material; The test material is injected intradermally as a 0.1%
solution (suspension or emulsion if solid or powder) in physiological
saline, paraffin oil, or propylene glycol.
Induction; Induction is achieved by a series of 10 intradermal
injections (one every alternate day) of the test material (0.05 or 0.1
ml) into different sites on the shaved anterior flank of the guinea
pig. Skin reactions are read 24 hours after each injection.
Challenge; Challenge is performed 14 days after the tenth intradermal
injection on the contralateral flank of the animal on a site correspond-
ing to the site of the first injection. Control animals receive the
same treatment with 0.05 ml of the l.OZ test solution given intrader-
mally. Readings are taken 24 and 48 hours later on shaved skins to
determine the intensity of erythema and size of fedema of the test reac-
tion. The reactions of all animals to the first intradermal injection
(0.05 ml) are compared with reactions at challenge and with those in
control animals. When a large variation is observed between the reac-
tions within the same group, the mean values for induction and challenge
phases of the test and control animals are compared.
52
-------
Table 2-1. Predictive Tests For Guinea Pig Skin Sensitization*
Test
Freund's Complete
Draize Adjuvant
Open Epicutaneoun
Closed Patch
Maximization Optimization
Split Adjuvant
Footpad
NO. ANIMALS
Test/Vehicle
Control
20 per group
8-10 per concen-
tration
6-8 per concentra-
tion
10-20 per group
and negative
controls
20-25 per con-
centration
20 per group
10-20 per group
10 per
group
INDUCTION
Test Substance,
amount or
concentration
0.05 or 0.1 ml
of 0.1Z solution
suspension or
emulsion
0.1 ml of 5-501
in FCA
0.1 ml undiluted or
concentrations of
30, 10, 3, 1Z or
lower
0.5 ml MIC
0. 1 ml varied
concentrations
alone and in
FCA
0.1 ml of 0.1-
10Z test sub-
stance in FCA
0.2 ml ointment 0.05 ml
or 0.1 ml liquid of l.OZ
(varied concen- (W/V)
trations) and in FCA
0.1 ml FCA
Ul
Co
Vehicle
Skin Site
No. Intradermal
Injections
Physiological Water, acetone,
saline, paraffin Ethanol PG,
oil or PG petrolatum
Anterior flank
(shaved)
10; on alternate
days; observe
24 hrs after
each injection
No. Epicutancous NA
Applications
(Open.).
Flank (shaved)
5; on alternate
day8; observe
24 hrs after
each injection
NA
Same
Flank (clipped)
NA
Ethanol,
acetone, tetra-
propylene ben-
zene sulfonate
Flank
NA
21; consecutively to NA
same site or 5
times a week for
4 weeks
Water, paraffin Physiological
oil, peanut
oil, PG
Various solvents
Shoulder
(clipped)
6| 3 pairs made
simultaneously
on day 0
NA
Flank and back Back (shaved)
9; 3 pairs made
over 3wk; the
last 6 being
made together
with FCA
NA
1
FCA only, prior
to 3rd induction
patch
NA
Front
foot
pad
1
NA
Type Patch
NA
NA
NA
7/8x1" webril
occluded with
elastoplast
coverlet
2x4 cm filter NA
paper qccluded
with imper-
meable plastic
adhesive tape
secured with
adhesive
bandage
2x2 cm; filter
paper occluded
with impermeable
plastic adhesive
tape secured with
adhesive bandage
-------
Table 2-1. Predictive Tests For Guinea Pig Skin Serialization8
(Continued)
Freund's Complete
Test Draize Adjuvant Open Epicutaneous
No. Patches HA HA NA
Patch Duration NA HA NA
(hrs)
REST (days) 14 11 0
CHALLENGE
Test substance, 0.05 ml of l.OZ 0.05 ml NIC and 0.025 ml MIC and
amount and solution or HNIC HNIC
concentration suspension
Skin Site Contralateral Same Sane
flank
No. Intradermal 1; observe 2
Injections days
No. Epicutaneous NA 2; on days 21 and Same
applications 35
(open)
Patch Type NA NA NA
No. Patches NA NA NA
Closed Patch
3
days 0, 7 and
14
6
14
0.5 ml MNICC
Flank
(shaved, both
aides)
NA
Same as
induction
1
Maximization
1
1 week after
injection
48
14
0.1 ml MNICC
Flank
(shaved)
HA
2x2 cm filter
paper - -same
as induction
1
Optimization
NA
NA
14
0.1 ml of 0.1-
10X solution
Flank
(untreated
fresh site; .
shorn)
I) Test material
without adjuvant
NA
2x2 cm filter
paper covered
by occlusive
plaatic foil
1
14 days after
ID challenge
Split Adjuvant
4
48
after each
application
10
0.1 ml of varied
concentrations
Back (clipped)
NA
NA
Same as
induction
1
Footpad
NA
HA
7
0.3 ml
of IX
solution
in fat:
dioxanet
acetone
(1:2:7)
solvent
system
lower
back
(clipped)
1
Same
NA
-------
Table 2-1. Predictive Tests For Guinea Pig Skin Sensitization9
(Continued)
Freund's Complete
Test Draize Adjuvant Open Epicutaneous Closed Patch Maximization Optimisation Split Adjuvant Footpad
Patch Duration 24 24 6 24 24 24 24
(hrs.) (open); observe (open) observe observe 18 hrs observe 2 days observe 24 hrs observe 2 days (open)
3 days 3 days later later wash
site
observe
3 hrs
later
3Abbreviations;
FCA Freund's complete adjuvant
HA = Not applicable
PG Propylene glycol
MIC = Minimal irritating concentration
MNIC Maximal nonirritating concentration
Reference for test methods:
Draize - Draize, 1965
Freund's Complete Adjuvant - Klecak et al., 1977
Open Epicutaneous - Klecak et al., 1977
Closed Patch - Buehler and Griffith, 1975
Maximization - Magnusson and Kligman, 1969
Optimization - Maurer et al., 1975a
Split Adjuvant - Maguire, 1973
Footpad - OECD, 1981
clf test agent is a nonirritatnt, the test site is pretreated with sodium lauryl sulfate in petrolatum 24 hours prior to challenge to enhance
penetration of test material.
-------
If the challenge values are substantially higher than the erythema/edema
values noted during induction or in control animals, the substance is
considered to have produced sensitization. The degree of sensitization
is proportional to the increase in the final reading compared to the
mean of the readings following the 10 original doses.
Evaluation and Critique; The Draize Test is easy to perform, economi-
cal, and useful as a screening method for detecting "strong" and
"moderately strong" sensitizers. However, the route of application
(intradermal) does not parallel normal human exposure; the induction
concentration fixed at 0.1% does not relate to the use concentration;
reaction readings are not easily quantifiable; it is not appropriate to
detect "weak" or "moderately weak" sensitizers and cannot be used for
testing many finished bacteriostatic products, due to the presence of
irritating bacteriostats, stabilizers, pigments, and other ingredients.
Because of its limitations, the Draize Test has been frequently modified
or replaced by other testing techniques in an attempt to improve the
sensitivity (Prince and Prince, 1977). In several instances, the
sensitivity of this test has been enhanced by simply increasing the
number of injections and raising the dose of the test material.
3.2 Freund's Complete Adjuvant Test
The Freund's Complete Adjuvant test (Klecak et al., 1977) is a variant
of the intradermal test method. It is a semiquantitative test method in which
the sensitizing concentration and the minimal eliciting concentrations (i.e.,
epicutaneous) are determined by the investigator.
Animals; Eight to 10 guinea pigs each are used in the test and control
groups at each concentration of the test material.
Test Material; For induction, the test material is incorporated in
Freund's Complete Adjuvant so as to give a final concentration range of
5-50Z. For the challenge, the test material is diluted, emulsified or
suspended in an appropriate vehicle (water, acetone, ethanol, propylene
glycol, petrolatum).
Induction; Induction is achieved by intradermal injection of 0.1 ml of
varied concentrations of the test material in Freund's Complete Adjuvant
into the shaved flank of the guinea pig skin on_alternate days (5 times
in all) in a 3 x 2 cm area. The control animals receive 0.1 ml of
Freund's Complete Adjuvant alone.
Determination of the Threshold Concentrations; One day prior to start
of challenge, varied concentrations (3-100%) of the test agent are
topically applied in 0.025 ml portions to the clipped left flank skin
of four untreated guinea pigs (2 cm2 area) simultaneously. The
application site is left uncovered, and skin reaction is read 24 hours
later. The maximal nonirritating concentration (MNIC) and the minimal
irritating concentration (MIC) are determined. The minimal irritating
concentration is defined as the lowest concentration causing mild
56
-------
erythema in 25% of the treated animals; maximal nonirritating concen-
tration is defined as the highest concentration which induces no
macroscopic reaction in any animal.
Challenge; On the 12th day after the last intradermal injection, i.e.
on day 21 and again 14 days later on day 35, 0.025 ml of the minimal
irritating concentration and maximal nonirritating concentration are
applied to the contralateral flank of the test and control animals. The
application is made over a 2 cm^ skin area which is left uncovered.
Reactions are read at 24, 48, and/or 72 hours after the application.
The test material is considered allergenic when 1/8 of the animals test-
ed show a positive reaction to the maximal nonirritating concentration
used at challenge.
Evaluation and Critique; This test is technically simple and economi-
cal, but it is not adequate for testing finished products and often
gives false positive results with materials found later to be negative
in the Maximization test in humans. It is, therefore, useful as a
screening test in the guinea pig but is a poor predictor of human
response (Klecak et al., 1977).
3.3 Open Epicutaneous Test
The open epicutaneous (no patch) test has been proposed for testing the
skin irritant and allergenic capacity of chemical agents intended for use in
cosmetics, perfumes, and dermatological products (Klecak et al., 1977). It
has not been tested on industrial chemicals.
Animals; Six to eight guinea pigs each are used in 1 to 6 test groups
along with 1 control group.
Test Materials; The test materials are used undiluted or dissolved,
suspended or emulsified at concentrations of 30, 10, 3, and 1% or lower
in a suitable vehicle (acetone, water, ethanol, propylene glycol,
petrolatum, etc.). Application volumes are held constant.
Determination of the Threshold Concentrations; One day prior to induc-
tion a test group is used to determine the maximal nonirritating and
minimal irritating concentrations of the test material using the method
described under Freund's Complete Adjuvant test-challenge.
Induction; Induction is achieved by application of 0.1 ml portions of
undiluted test agent and its progressive dilutions to the clipped flank
skin (8 cra^) of the guinea pig. Application is repeated daily for 3
weeks or 5 times weekly for 4 weeks using the same site. The applica-
tion site is left uncovered and the reaction is read 24 hours after
each application. The maximal nonirritating and minimal irritating
concentrations are determined by the all or none criterion. When the
reaction is strong, the application site is changed.
Challenge; Challenge is made on day 21 (on the day of last induction)
and 35 (14 days after last induction). A 0.025 ml portion of the
57
-------
minimal irritating and maximal nonirritating concentration of test agent
is applied to the contralateral flank skin (2 cm*); the application is
left uncovered, and the reaction is read after 24, 48, and/or 72 hours.
Vehicle treated or untreated (negative control) guinea pigs are treated
likewise and the reactions read at similar intervals. The minimal irri-
tant concentration of the test agent is used to confirm the biological
activity determined before starting the induction and to exclude false
results due to instability of the agent.
This procedure permits determination of the minimal sensitizing concen-
tration necessary to induce allergenic contact hypersensitivity and the
minimal eliciting concentration necessary to cause a positive reaction.
A concentration is considered allergenic when at least 1/8 of the
animals of a group show a positive response.
Evaluation and Critique; The open epicutaneous method of testing
employs several concentrations of the test material at one time in
contrast to most of the other procedures. It is realistic and appears
to be in accord with the current needs of the pharmaceutical and cosme-
tic industries in that (1) substances are tested by repeated uncovered
topical application, which parallel use conditions, and tests are easy
to perform; (2) the test material is used in decreasing concentrations
to establish a dose response curve; and (3) the test is scored on an
all-or-none basis, considering the use concentration.
The open epicutaneous method for besting for allergenicity is suitable
for testing mixtures and finished products as no injection is involved.
3.4 Closed Patch Test
Buehler (1964, 1965), and Griffith and Buehler (1977) have presented
several predictive tests using the guinea pig which have introduced the use of
a patch in the application of substances.
The experimental evaluation of skin sensitization in guinea pigs
according to these authors (Buehler and Griffith, 1975) is as follows:
Animals; Ten to twenty guinea pigs each are used in the test, vehicle
control, and negative control groups. The vehicle controls are some-
times omitted.
Test Materials; The test agent is diluted, emulsified, or suspended in
a suitable vehicle (ethanol, acetone, tetrapropylene benzene sulfonate
etc.). The concentration used during induction is one which causes
skin irritation and is obtained from the concentration of use.
Induction; Induction is evoked by application of 0.5 ml portion of the
test material at the minimal irritating concentration to the flank skin
and held in contact by an occlusive patch for 6 hours. The vehicle
control group is treated likewise except only the vehicle is applied.
During the exposure, the animals are immobilized in a special re-
strainer. The patching is repeated on days 7 and 14.
58
-------
Challenge; Fourteen days later, the animals are challenged using the
6-hour occlusive patch test of the induction phase. The maximum non-
irritating concentration is used. The patch is applied to shaved flank
skin on both sides in the test group but only on the left side in the
two control groups. The test group is challenged with the test material
in the vehicle, the vehicle control group with the vehicle alone, and
the negative control group (not pretreated in the induction phase) with
the test material in the vehicle.
Eighteen hours after patch removal, the flanks are depilated and the
reaction is read. Readings are again taken at 48 and 72 hours after
patching. The reaction is graded on a scale of 1 (slight erythema) to
3 (marked erythema). The results are expressed in terms of the inci-
dence and severity of response. Incidence is derived from the number
of animals showing response at 24 or 48 hours divided by the number
tested. Severity is calculated from the sum of the test grades divided
by the number of animals tested.
Evaluation and Critique; The closed patch test of Buehler and Griffith
(1975) has all the essentials of a predictive test: the applicaton is
by the topical route; finished products can be tested as such; the in-
duction concentration can correspond to user concentration; the vehicle
chosen can be similar to the end formulation and the sensitivity of the
method is comparable to the repeated insult patch test for humans. A
subjective factor involved in the evaluation of the severity of the
response could bias the conclusion.
Comments; There are numerous variants of this test in existence today
but their use has been described as limited (Maurer et al., 1975a).
3.5 Maximization Test
Magnusson and Kligman (1969, 1970) and Magnusson (1975) developed a
procedure in the guinea pig for identifying contact allergens in which a
deliberate attempt is made to "maximize" the chance of inducing sensitization
without regard to realistic skin exposure. This test combines the use of the
intradermal injection and the patch. It is considered to be the most sensitive
test currently in use. Results from these tests have shown a high degree of
correlation with similar tests in humans (Kligman, 1966c) which are described
in Section 5.4.
Animals; Twenty to 25 guinea pigs each are used in the test and control
groups.
Test Material; The test material is applied intradermally and epicuta-
neously during induction and epicutaneously at challenge. For
injection, water-soluble test agents are dissolved in the water phase
and emulsified in Freund's complete adjuvant; oil-soluble or insoluble
materials are dissolved or suspended in the adjuvant (a mixture of
paraffin oil and an emulsifier with mycobacteria). Paraffin and peanut
oil, or propylene glycol are used to dissolve or suspend water insoluble
materials which are to be injected without adjuvant. When solids are
59
-------
used for topical application they are ground to fine powder and incorpo-
rated in petrolatum. Liquid materials are applied neat (if not irritat-
ing) or in dilutions in petrolatum or water. If the test agent is an
irritant, a concentration which causes a weak to moderate inflammation
is selected for use.
Induction; Induction is achieved in two steps. On day zero, 3 pairs
of intradermal injections are made simultaneously to a clipped skin site
on the shoulder (3 injections on either side of the midline) within a
2 x 4 cm area. Injections are: (1) 0.1 ml Freund's Complete Adjuvant
alone, (2) 0.1 ml test material alone, and (3) 0.1 ml test agent in
Freund's Complete Adjuvant. Control animals are treated likewise except
the test material is omitted. On day 7 a filter paper patch (2x4 cm)
spread with a thick, even layer of the test agent (or saturated, if
liquid) is applied over the same shoulder area (after clipping) and
secured by an impermeable plastic adhesive tape and bandage (occlusion).
The exposure is for 48 hours.
If the test agent is a nonirritant, the test site is pretreated with
sodium lauryl sulfate in petrolatum 24 hours prior to patching, to en-
hance penetration. Otherwise, the allergen is applied at concentra-
tions which produce irritation. Control groups are treated similarly
with the test agent.
Challenge; Challenge is performed on the shaved flanks after a further
2-week period (day 21). Occlusive patches (2x2 cm) spread or satu-
rated with the maximum nonirritant concentration of the test agent are
applied for 24 hours to the right (patch with vehicle) and left (patch
with test agent) sides. Kero and Hannuksela (1980) used aluminum cham-
ber units instead of the patches, and because of the small size of the
units, were able to perform all the challenges on the same flank.
Readings are made 24 and 48 hours after the removal of the patches and
cleansing of the test site. Reactions are scored on a 4-point scale
with 1 (one) representing mild and 4 representing extreme redness and
swelling. The important statistic in the maximization test is the
frequency of sensitization and not the intensity. The rating is based
on the percentage of animals sensitized; test substances are assigned
to one of five classes:
MAXIMIZATION GRADING IN THE GUINEA PIG
Sensitization Grade Classification
Rate (%)
0-8
9-28
29-64
65-80
81-100
I
II
III
IV
V
Weak
Mild
Moderate
Strong
Extreme
(Magnusson and Kligman, 1969)
60
-------
Evaluation and Critique; The Magnusson-Kligman (1969, 19.70, 1975)
Maximization method of testing virtually eliminates the false negative
results of the Draize (1965) technique. It is an excellent procedure
for identifying "weak" contact allergens. The method is, however, less
adequate for predicting sensitization to finished products as intra-
dermal injection of irritating components is involved.
The treatment with either Freund's Complete Adjuvant or the occlusive
bandage may lower the threshold level for skin irritation. As such, a
challenge concentration of the test agent, which in untreated animals
is found to evoke no response, might be active in an animal treated
with adjuvant in an occlusive dressing. In this situation, a false
positive response would be recorded. To exclude false positive results,
the control group should be treated the same way as the animals in the
test group.
Comments; Several variations of the Maximization test procedure have
been tried (Fahr and Schulz, 1976) and found to be less sensitive than
the Magnusson-Kligman procedure described above.
3.6 Optimization Test
Maurer and his colleagues (Maurer et al., 1975a, 1975b, 1978, 1980) have
recently developed an alternative method to the Maximization Test involving
additional Freund's Adjuvant and have called it the Optimization Test.
Animals; Twenty guinea pigs each are used in the test and control
groups.
Test Material: The test materials are used in varied concentrations
(0.1-10%).?7ater-soluble substances are dissolved in 0.9% NaCl and
mixed with Freund's Complete Adjuvant; substances soluble in oil are
dissolved in Freund's Complete Adjuvant and mixed with 0.9% NaCl. The
1:1 mixture of adjuvant and saline are prepared shortly before adminis-
tration.
Induction; Induction is achieved over a 3-week period. First week;
0.1 ml of the test substance is injected intradermally in to the flank
and back (day 0), and into the back twice (days 2 and 4). Twenty-four
hours later, the sites are chemically depilated (5 min) and 3 hours
later the reactions are assessed. The two largest diameters of the ery-
thematous reaction in vertical alignment are measured and the skin-fold
thickness (mm) determined with a skin-fold gauge. From these values
the individual "reaction volume" (microliter) is calculated for each
animal and each reaction. Second and third weeks; intradermal injec-
tions of 0.1 ml of the test agent in Freund's Complete Adjuvant are made
into the nuchal skin as for the first week.
Challenge; Challenge is performed in two steps.
First challenge; Fourteen days after the last induction dose, the
test material is injected in the same dose and volume as in the first
61
-------
week of induction (without Adjuvant) at a fresh site on the untreated
flank. Twenty-four hours later, the reaction is measured and the reac-
tion volume determined as during the first week of induction.
Assessment! The average extent of the reaction to the first four induc-
tion doses (first week) and the standard deviation are calculated for
each animal. The mean and standard deviation of the four induction
doses are then added to give an individual threshold value for each
animal. If the reaction volume at challenge exceeds the corresponding
threshold, value, the animal is considered to be sensitized. The number
of positive animals in each group is counted and the significance of
the differences between the treated and control groups is assessed by
the Fisher Exact test.
Second challenge; A further epidermal challenge is administered
fourteen days after the first challenge. The test substance in the
appropriate vehicle (to give the maximum nonirritant concentration) is
spread uniformly on a patch (2.0 x 2.0 cm) of filter paper, which is
applied to a shorn, untreated site, covered with occlusive plastic and
left in place for 24 hours. Twenty-one hours after patch removal, the
site is chemically depilated and 3 hours later the extent of erythema
and skin-fold thickness is determined.
Assessment; The presence of clearly discernible reddening of the reac-
tion site is taken as indication of an allergic reaction. The signifi-
cance of the difference in the number of positive animals in the treated
and control groups is assessed by the Fisher Exact test.
Evaluation and Critique; The Optimization Test appears to be more ef-
ficient than the Draize Test in detecting sensitizers, and comparative
studies show that there is good agreement between the Optimization Test
and the Maximization Test and between both tests and experience in man.
In the Optimization Test the element of subjectivity is significantly
reduced by using the reaction volume as the main evaluation criterion.
Also the measurement of reactions occurring during the first week of
induction, the calculation of standard deviations and the comparison in
the same animal help assure that the effective reaction is objec-
tively assessed.
With reference to the importance of the two challanges the authors indi-
cate that "the first intradermal challenge (with the same dose and volume of
substance that is used in the initial phase of induction) is usually appro-
priate to elicit a positive reaction if sensitization has taken place. The
second epidermal challenge (under occlusion), serves only as an added precau-
tion that takes further kinds of affinity of the test substance for the skin
into consideration." (Mauer et al 1975a)
3.7 Split-Adjuvant Test
In a series of articles published between 1972-1975, Maguire and his
coworkers (1972, 1973, 1975) described an adjuvant technique that amplifies
the sensitization process in the guinea pig to allow detection of
62
-------
"weak" and "moderately weak" contact allergens in humans. This technique de-
rives from the split-adjuvant test procedure (Maguire and Chase, 1967, 1972)
in which the allergen and Freund's Complete Adjuvant are administered sepa-
rately and together with the test material (Maguire, 1973) rather than as an
emulsion. It is based on the observation of Magnusson and Kligman (1970) that
intradermal injection of Freund's Complete Adjuvant beneath a site where a
topical allergen has been applied for challenge, greatly potentiates the
sensitization.
Anamals! Ten to twenty guinea pigs each are used in the test and
control groups.
Test Material! The test material is used at varied concentrations as
solutions or suspensions in appropriate solvents.
Induction; Induction is achieved in four steps: (1) on day zero, 0.2
ml of ointment (or 0.1 ml of liquid) of the test agent is applied
topically to the shaved skin of the back and this is covered with filter
paper and occluded with tape; (2) a second application is made under
occlusion 48 hours later (day 2); (3) on day 4, the patch is removed
and 0.1 ml of Freund's Complete Adjuvant is injected intradermally twice
into the sensitizing site followed by a topical application of the test
substance under occlusion; and (4) on day 7, the patch is removed and
the test material reapplied under a closed patch, which is finally
removed on day 9.
Challenge! Challenge is performed on day 20 by a closed patch test
using 0.1 ml of the test agent; the patch is applied to the clipped
skin of the back. Twenty-four hours later (day 21), the patch is re-
moved and readings are made. Further readings are made at 48 and 72
hours (day 23 and 24). Retesting at a different site for a second or
third time occasionally will bring out borderline earlier readings.
The intensity of the reaction is classified as follows:
0 * Normal skin
^ * Very faint, nonconfluent pink
+ * Faint
++ * Pale pink to pink, slight edema
Pink, moderate edema
Pink and thickened
+++++ = Bright pink, markedly thickened
Sensitization is assessed by comparing the number, intensity, and dura-
tion of skin reactions in the test and control groups.
When repeated contact applications are made during induction, the degree
of sensitivity of the animal to low concentrations of the allergen rises
stepwise with the concentration range. This heightening of reactivity
to the allergen represents an anamnestic response in the area of delayed
hypersensitivity and contrasts sharply with relatively high antibody
titers found when chemical allergens are incorporated into Freund's
63
-------
Complete Adjuvant and administered as a single injection (combination
technique of Chase, 1954).
Evaluation and Critique; The Maguire Split Adjuvant Test method has
all the features of a predictive test. Finished preparations can be
tested as such and other substances can be tested with regard to the
concentration of use. The repeated exposure and the number of patches
applied during induction qualifies this procedure as a test for detect-
ing weak sensitizers. It could also be used for screening of numerous
chemicals, in which the dose dependence of presumed allergenic proper-
ties is to be checked, but this is cumbersome. The use of a different
but still subjective scoring method is less appropriate than the more
quanta! methods developed recently. To exclude false positive results,
it is important that the control animals be pretreated with Freund's
Complete Adjuvant.
Comments; Several variations of the Maguire Split Adjuvant Test tech-
nique exist; however, their use is limited (Fahr and Schulz, 1976).
One variant involving sensitization through intraperitoneal injection
of pre-formed stromata conjugates in Freund's Complete Adjuvant can
only be used with chemicals that couple with red blood cell stromata
(Chase, 1954, 1967).
3.8 Footpad Test
The footpad technique for evaluating sensitization potential in the
guinea pig (OECD 1981) is as follows.
Animals; Ten guinea pigs each are used in the test and control groups.
Test Material; The material is incorporated into Freund's Complete
Adjuvant to give a l.OZ mixture (w/v). It is stirred gently at room
temperature for 3 hours and is allowed to settle for a few minutes
prior to being used.
Induction; Induction is achieved by injection of 0.05 ml of the test
mixture into the front footpad of the guinea pig, which has not been
previously exposed to the test material by any route. Controls are
injected with Freund's Complete Adjuvant instead of the test material.
Challenge; One week later, 0.3 ml of a 1.0% mixture of the test agent
in a solvent system of guinea pig fat:dioxane:acetone (1:2:7) is applied
to the clipped skin of the lower back of the guinea pig. If a l.OZ
solution produces moderate irritation, a 0.1% solution is used. Con-
trols are challenged similarly.
Twenty-four hours later, the area is depilated, washed (with warm tap
water, 37°C) and three, hours after this the reaction is scored under
fluorescent light for erythema and edema. The reaction is graded on a
scale of 1 (slight erythema) to 4 (dark red, with hemorrhagic areas
accompanied with swelling and increased skin temperature). The degree
of swelling of the skin is determined by lifting the skin fold (about
64
-------
1 cm in length), feeling it (between thumb and forefinger), and grading
on a scale of 1 (slight, just detectable) to 3 (marked, difficult to
pick up a fold). The individual scores of the two reactions for each
animal are added and the mean value derived. The means of test animals
are then compared with those of the controls which are similarly
derived.
The difference in the reaction values between the control guinea pigs
(primary irritation) and the test guinea pig is considered to be a
measure of the degree of skin sensitization.
Preparation of Guinea Pig Fat; The following method has consistently
yielded adequate product:
1. Strip fat from large, obese guinea pig.
2. Freeze.
3. Grind frozen fat (kitchen-style meat grinders).
4. Add acetone to ground fat (10:1, acetone:fat). Stir and heat
over a hot water bath.
5. Filter (Whatman #4).
6. Add activated charcoal and stir (warmed).
7. Filter through Whatman filter #4 and Supercel. Pour into
beaker.
8. Freeze (fat will solidify on the bottom of beaker).
9. Decant acetone and discard.
10. Gently warm fat and vacuum distill off acetone.
11. Pour warm fat into vials (use 1 vial per group of
sensitizations).
12. Freeze all vials and use as needed (frozen fat will remain
acceptable for at least a year).
Depilatory; The preparation and use of the following depilatory is
suggested. Take 6 parts soluble starch, 6 parts talc, 6 parts barium
sulphide, and 2.7 parts of granular nonirritant anionic surfactant.
Add the ingredients in the order listed, mix well, and add cold water
to make a viscous paste. Apply to clipped skin of guinea pig for 4
minutes. Rinse well with tap water.
Evaluation and Critique; No data are available in the open literature
on this technique to enable adequate evaluation of_its predictive
nature.
Comment; At least one variation of this procedure is reported in the
literature (Loomis, 1978) which differs sufficiently to warrant some
explanation. Induction is achieved by topical applications on days 0
and 2 to the depilated back of the guinea pig, and reactions are read
24 and 48 hours after the first treatment only. After a rest period of
3 weeks, a challenge dose is applied to the depilated shoulder skin and
reactions are read at 24 and 48 hours. The reaction scores made at
induction and challenge are averaged for each group and compared. If
the challenge scores are 2-4 times the induction scores, the test
material is considered a moderate sensitizer; if the challenge score is
4-7 times higher, the agent is considered a strong sensitizer.
65
-------
4.0 COMPARATIVE RESULTS OP SENSITIZATION TESTS IN THE GUINEA PIG
The sensitivities of different induction methods have been widely inves-
tigated with the view to determine the most appropriate mode of induction based
on objective analysis and scoring of the results.
The effectiveness of different methods of sensitization is illustrated
below with reference to various concentrations of a single compound or fixed
concentrations of different compounds by three different routes of administra-
tion. The closed patch technique is more sensitive than the intradermal or
epicutaneous route as sensitization was detected at lower concentrations or at
a higher incidence with this technique.
Comparison of Methods for Induction of Sensitization in the Guinea Pig
Concen
Compound tration
l-Chloro-2,4-dinitrobenzene
p-Phenylenediamine HC1
Tetrachlorosalicylanilide
Thioglycerol
Monobenzyl ether of hydroquinone
Formalin
Potassium chromate
0.01
0.05
0.10
0.25
0.5
2
1
14
5
5
1
Closed
Patch
40-50
100
100
80
60
60
30
10
Incidence
Intradermal
0
30
0
0
0
0
10
10
Z
Epicutaneous
0
70
a
aNot tested by this method
Data are from Griffith and Buehler, 1977.
The relation between dose and effects in contact allergy has been
studied experimentally and by field survey. Results of these studies indicate
a dose-dependent relation between induction and sensitization by intradermal
as well as topical application (Griffith and Buehler, 1977, Matsushita and
Aoyama 1980). The table below shows the responses to intradermal and epicu-
taneous (Closed patch) challenges in the guinea pig with different amounts of
two unsaturated sulfones having 12 or 16 carbon atoms. The results show that
the use of Freund's Adjuvant not only lessens the amount of sulfone required
to induce sensitization, but also alters the relative sensitization potential
of the Cj2 versus C^g sulfone as compared with the epicutaneous results.
The above results suggest that caution should be exercised in assigning
relative sensitizing activity to topical agents that are tested intradermally
with Freund's Complete Adjuvant when the two methods (intradermal or epicu-
taneous) are used as screening tests prior to human sensitization test.
66
-------
Sensitization with 1-Alkeny1-1,3-sulfones in the Guinea Pig
% Response to Sensitization, nmolea
Induction Intradermal (in FCA)Epicutaneous (Closed
Challenge Challenge Patch)
Compound
nrnole
7.1
71
710
71
710
7100
C12
C16
200
20
2
200
20
2
100
100
80
100
93.3
40
100
78.6
50
100
86.6
20
73.3
46.6
6.6
73.3
0
0
60
26.6
13.3
0
0
0
86.6
53.3
40
6.6
0
0
92.9
71.4
64.3
40
6.6
6.6
aPercent animals sensitized, nanomoles in challenge dose.
Data adapted from Griffith and Buehler, 1977.
bpCA « a Freund's Complete Adjuvant.
Maurer et al. (1975a, b), Magnusson (1975), and Magnusson and Kligman
(1970) compared the sensitization potentials of several compounds in 3 dif-
ferent test methods (Draize test, Maximization test, Optimization test) and
made the following observations (see Table 2-2):
(1) Potent allergens (e.g., dinitrochlorobenzene, DNCB) are readily
assessed by the Draize test but this test is not sensitive enough to
detect the so-called "weak" allergens (e.g., Penicillin G, formalin,
ethylaminobenzoate).
(2) The known allergens (DNCB, Penicillin G, formalin, etc.) give
significantly positive results with both the Maximization test and the
Optimization test while the essentially non-allergenie (e.g., peni-
cilloylpolylysine, paraffin, physiological saline, 5% ethanol) do not
respond.
(3) "Weak" allergens responded equally well to both Maximization test
and the Optimization test but not to the Draize test (e.g., formalin,
CPY-1, CPY-2, Pencillin G, ethylaminobenzoate).
(4) Maximization test and the Optimization test sensitization rates
are similar when the concentrations used for induction are the same
(e.g., Penicillin G).
(5) Nickel sulfate experiments gave positive results with both the
Optimization test and the Maximization test but no animals were sensi-
tized with the Draize test. A higher percent of animals were sensi-
tized by the intradermal than by the epicutaneous route, and the
67
-------
Table 2-2. Comparative Results of Senaitization Test In Guinea Pig*
Draize Teat
Optimization Teat
Concentration
Teat
Material
WK3C
PPI.C
Penicillin G
Ethylamino-
benzoate
Formalin
CPY-1C
CPY-2C
Nickel
Sulfate
aData compiled
Sensiti-
zation
Hat*
20/20
0/20
0/20
.
7/204
0/20
0/20
l/20d
0/20
0/20
0/20
0/20
fro* Maurer
r
Induc-
tion
H)C
0.1
6x10
M
0.1
0.3
0.1
2.0
0.1
0.1
'0.1
0.1
0.1
et al.
^Number of poaitive animals/total
cAbbreviationat
Challenge
ID
0.1
-5 6*10-5
M
0.1
0.3
0.1
2.0
0.1
0.1
0.1
0.1
0.1
1975a,b;1978
EC
0.1
50xlO~5
M
10
10
5
5
2«
0.5
0.5
0.1?
0.58
*
Maximization Test
Concentration Z
Sensitization"
Rate
ID
20/20
0/20
0/20
0/20
0/20
10/20
20/20
19/20
17/20
20/20
20/20
EC
20/20
0/20
10/20
15/20
0/20
9/20
10/20
17/20
16/20
12/20
7/20
Induction Challenge
ID
0.1 '
6xlO~5
M
0.3
3.0*
0.1
2.0
2.0«
5
5*
0.1
0.1
EC
1.0
SOxlO"5
M
5.0
5.0
25
25
25
5
5
1.0h
1.08
EC
0.01
50xlO~5
M
10
10
5
5
5
2«
2
0.5
0.5
0.5
0.5
Senaiti-
zation
Rate
20/20
0/20
14/19
20/20
1/20
6/20
7/25
9/20
16/20
20/20
20/20
0/20
7/20
number of animals.
ID intradermal
EC epicutaneoua occlusive patch
DNCB l-chloro-2,4-dinitrobenzene
PPL penicilloylopolylysin
Penicillin G benzyl penicillin potassium salt
CPY-1 l-(3-chlorphenyl)-3-phenyl-pyrazoline
CPY-2< l-(3-chlorphenyl)-3-(4 chlorphenyD-pyrazoline.
dMagnu'mon and Kligman, 1970, and Magnusson, 1975.
*EC (occluded); 22 in water on shaven skin.
fEC (open); 0.1Z in 70S ethanol on shaven skin.
SEC (occluded); 0.5Z in water on depilated skin.
hEC (occluded); 0.5Z in water on shaven skin.
68
-------
sensitization rate was greater when the compound was applied to the
open shaven skin than when it was applied to the depilated skin
occluaively in both the Optimization test and the Maximization test.
(6) Increasing the penetration of the test substances applied to the
skin by pretreatment (croton oil or sodium lauryl sulfate application)
(Table 2-3), use of occlusive dressings or by administration of high
concentration of test compound has much less influence .on the effici-
ency of induction than has stimulation of the immune system by means of
an adjuvant (e.g., ethylaminobenzoate which depends solely on the in-
tradermal induction concentration to attain successful sensitization in
the maximization test, Table 2-2).
Kero and Hannuksela (1980) compared the sensitization results of vari-
ous concentrations of neomycin and propylene glycol in separate groups of
guinea pigs by the Maximization test, Open Epicutaneous Test and the Chamber
test methods (Table 2-4), and found no difference in the rate of sensitization
between the Maximization Test and the Chamber Test with petrolatum and pro-
pylene glycol. In the Open Epicutaneous Test, neomycin in propylene glycol
sensitized more readily (45% response) than in petrolatum (25Z response). The
results from all three methods agreed well and were not significantly different
from each other. In the Open Epicutaneous Test the influence of the vehicle
was more apparent than in either of the other two tests. The authors recommend
the Open Epicutaneous Test over the Maximization Test because it is a more
"natural" situation for contact sensitivity.
Benzocaine sulfamylon cream and cinnamic aldehyde were tested by the
Maximization test, Split Adjuvant test, Open Epicutaneous test (modified
Maguire test) and Draize test (Prince and Prince, 1977). In this study the
Draize test method was again found to have the lowest sensitivity. The authors
graded the 4 tests in the following descending order of sensitivity: Split
Adjuvant; Maximization Test and Open Epicutaneous Test; Draize Test.
A comparison of the results for 5 compounds (allyl isocyanate, thio-
glycerol, paraphenylenediamine HC1, potassium chrornate and formalin) tested
for allergenic potentials by the Draize and closed patch test showed the
closed patch method to be clearly superior (Buehler, 1965).
Potassium dichrornate, nickel sulfate and sodium zirconium lactate are
established allergic sensitizing agents in man. The potassium and nickel com-
pound produce a superficial eczematous reaction, the zirconium compound induces
a deep nodular granuloma associated with epithelial cell infiltration. Turk
and Park (1977) tested all three salts by the Maximization test, Split Adjuvant
test and the "Polak" test and found that it took longer to sensitize the guinea
pig to zirconium than to chromium or nickel. All three protocols produced a
delayed hypersensitivity-like reaction to zirconium. The best protocol for
chromium and zirconium sensitization was the "Polak" method and the most effec-
tive method for nickel was the Split Adjuvant test (Table 2-4). The reaction
with all three metals was often transient, which was evident in the fact that
animals that failed to show sensitivity on an earlier challenge would develop
sensitivity at a later one. Cross reactivity was also noted between zirconium
and chromium. It is apparent that the Maximization Test is not highly sensi-
tive to metal salts.
69
-------
Table 2-3. Percent of Animals Reacting Per Group in the Optimization Test,
Maximization Test and the Epidermal Testa
Induction
Optimization Group
Maximization Group
Epidermal test
Epidermal test +
Appl.
20
75
5
55
Challenge (lOug DNCB/Appl.!
Intensity of Erythema (%)
1 Appl. 2
55
100
45
45
)
Appl. 3
90
100
45
95
irritation with
croton oil
Epidermal test +
irritation with
SLS
Optimization Group
Maximization Group
Epidermal test
Epidermal test +
irritation with
croton oil
Epidermal test +
irritation with
Sodium lauryl Sulfate
Increase in skin thickness (%)
20
5
5
50
68
85
63
10
55
63
100
100
90
10
85
100
aData from Maurer et al. 1978.
70
-------
Table 2-4. Comparison of Six Protocols For Development of Delayed Hypersensitivity
to Seven Compounds3
Induction
Compound (Cone. %)
Neomycin 30
(in pet.)d
Neomycin 30
(PG/70%)
Neomycin 30
(PG/70%)
Benzocaine 5
(in pet.)
Sulfamylon 5
(emulsion)
Cinnamic 2
aldehyde
(in pet.)
Potassium 1 mg
dichr ornate
Nickel 1 mg
sulfate
Sodium 1 mg
zirconium
lactate
Challenge
(Cone. %)
\
20
20
(in pet.)
70
(in water)
5
5
2
0.3 mg
0.33 mg
0.33 mg
MT
7/20
6/20
0/20
6/20
9/20
16/20
0/6
0/6
0/6
Positive Reaction at Challenge
(.# of animals/ total;
OET CT SAT DTC PTd
5/20 5/20
9/20 6/19
0/20 0/19
11/20 17/20 3/20
8/20 16/20 2/20
15/20 20/20 4/20
3/6 3/12
2/5 0/6
0/6 2/4
aData compiled from Kero and Hannuksela 1980 (Neomycin); Prince and Prince 1977
(Benzocaine, Sulfamylon and Cinnamic aldehyde); Turk and Parker 1977 (metals).
^Abbreviations - pet. = petrolatum; PG = propylene glycol; CT = Chamber test;
MT - Maximization test; OET = Open Epicutaneous test; SAT = Split Adjuvant Test
ctopical induction without irritation, with adjuvant (foot pad injection).
dPolak Test:
Day 0: 4 footpad injections of 0.1 ml of emulsion with 2 mg/ml of metal salts
in Freund's complete adjuvant. In addition 0.1 ml of emulsion was
injected into the nape of the neck (total dose 1 mg).
Day 14: Intradermal challenge by 25 micrograms of metal salt in 0.1 ml saline
into shaved flank. This was repeated weekly for 13 weeks.
71
-------
Klecak et al. (1977) selected 32 fragrances known to be allergenic to
humans and tested them concurrently on separate groups of guinea pigs by the
Draize test, Maximization test, Open Epicutaneous test and Freund's Complete
Adjuvant test (Table 2-5). Twenty five of the 32 compounds were allergenic by
one or more of these tests and 22 of these were detectable by the Open Epicu-
taneous test and 21 by the other tests. Thus 4 allergenic compounds were ex-
clusively detected by the Open Epicutaneous test and 3 others solely by one or
more of the three intradermal tests. On the basis of the number of positive
responses, the Open Epicutaneous test was considered by the authors to be
superior to the other three tests. The Draize test was again found to be the
least sensitive of the four test methods.
Conclusions;
A review of the comparative data from various sources indicates that
except for the Draize test all of the methods discussed above have some poten-
tial for detecting the so called "weak" sensitizers. No one test method
appears to be suitable for testing of all compounds, and significant variations
in results have been observed from different sources using the same test
method, compound, concentrations (Table 2-2, e.g., Penicillin G, Maximization
test).
Based on the published evidence, the guinea pig Maximization test is
one of the better tests for detecting the weaker sensitizers, the use of which
must be avoided in cosmetics and toiletries. The test virtually eliminates the
false negative results seen with the Draize test but does not appear to be ade-
quate for predicting sensitization to finished products. The Optimization
Test, Open Epicutaneous Test and the Closed Patch Test may be viable alterna-
tives but the published evidence on their reliability is insufficient. All
three can be used on finished products. The Closed Patch test involves a sub-
jective evaluation of the sensitivity, but allows testing of the actual user
concentration. The Open Epicutaneous test is also flexible in this regard.
Several of the commercial and industrial test labs contacted used both the
Maximization and Closed Patch test with success.
The aim of animal testing is to establish to what extent a particular
substance has the potential for acting as a skin sensitizer in humans. The
tests reveal that a chemical possesses immunologic capabilities, but the per-
cent of animals sensitized does not necessarily indicate the probable incidence
of human sensitization. A negative result with a sensitive test method pre-
dicts with a reasonable degree of certainty that the test material will not be
a sensitizer. On the other hand, this does not mean that the substance will
never sensitize anyone but rather that the risk of sensitization to humans is
low.
72
-------
Table 2-5. Allergenicity of 32 Incriminated Compounds For Humans and Their
Allergenicity For the Guinea Pig By Four Test Methods3
Groupc
Id
II
III
IV
Total #
of Cmpds.
7
18
4
3
DT
pos
0
7
0
1
aData from Klecak et al. 1977.
b Abbreviation s - DT = Draize Test;
Epi cutaneous Test; FCA » Freund's
b
neg
7
11
4
2
pos
0
15
0
3
MT
neg
7
3
4
0
MT » Maximization
Complete Adjuvant
OET
pos neg
0
18
4
0
Test;
Test;
7
0
0
3
OET
pos
FCA
pos
0
17
0
3
neg
7
1
4
0
« Open
= positive;
neg = negative.
cGroup I - Acetophenone Group III - Amyl salicylate
Benzophenone Benzyl salicylate
Diethyl phthalate Bromo styrol
Dimethyl-benzyl carbinol Methyl salicylate
Dimethyl benzyl phthalate Group IV - Benzaldehyde
Hydroxycitronellal Cinnamic alcohol
Thymol Vanillin
Group II - Benzyl alcohol
Benzyl cinnamate
Carvacrol
Cinnamic aldehyde
Citral
Citronellal
Cuminic aldehyde
Geraniol
Helitropin
Isoeugenol
Limonene
Methyl cinnamate
Methyl heptine carbonate
Methyl octine carbonate
Phenylacetaldehyde
Phenyl-ethyl salicylate
3 phenyl-propionaldehyde
10-Undecenal
<*The compounds are subdivided into 4 groups according to their allergeni-
city in the 4 animal tests, namely: Group I, not sensitizing in any test;
Group II, sensitizing in the OET and in one or more of the other tests;
Group III, sensitizing exclusively in the OET; Group IV, not sensitizing in
the OET but sensitizing in one or more of the other tests.
73
-------
5.0 HUMAN SKIN SENSITIZATION TEST PROCEDURES
The human patch test has evolved from a single patch test (Schwartz,
1960), to the Prophetic Patch Test (Schwartz and Peck, 1944), the Repeated
Insult Patch Test (Shelanski and Shelanski, 1953; Draize, 1965; Marzulli and
Maibach, 1973, 1974; Maibach and Epstein, 1965) and finally to the Maximization
test of Kligman (1966a,c). The human patch test should never, however, be used
prior to screening a compound for activity in guinea pigs.
The procedures currently used for the Maximization test require repeated
occluded patches during induction (10 patches, 48 hours each, same site)
followed by a 2-week rest period, and a challenge. There are several varia-
tions which include the use of provocative chemicals (sodium lauryl sulfate)
(Kligman 1966d), special skin preparation (freezing) (Epstein et al., 1963),
high concentrations of test substance during induction (Marzulli and Maibach,
1974), special patches (Magnusson and Hersle, 1965) and increased number of
test subjects (Kligman, 1966c). Methods of historic and current interest are
summarized in Table 2-6.
The following are the most frequently used procedures for testing sen-
si tizati on in humans.
5.1 Schwartz and Schwartz-Peck Test
The Schwartz (1960) and Schwartz-Peck tests (1944), described in Table
2-6 and later modified to include a 6-day induction period under occlusive
conditions, is useful in testing polymeric materials which contain small
amounts of low molecular weight sensitizing or irritant chemicals which are
released from the polymer slowly. The Schwartz (1960) and the Schwartz-Peck
test have certain inherent defects. False positives may occur since border-
line primary irritants can sometimes produce reactions which can be confused
with sensitization when only one patch reading is made; but more important are
the false negatives which result from the single application of small amounts
of the test substance, which is often inadequate to produce sensitization ex-
cept in the case of strong allergens. The modified Use Test (see below)
recommended by these authors was probably a recognition of this inadequacy.
5.2 Shelanski-Shelanski Test
Because the original Schwartz-Peck test with one 48 hour induction ex-
posure was ineffective in testing cosmetics, Shelanski and Shelanski (1953)
recommended multiple insults, involving 10-15 24-hour treatments on alternate
days. This repetitive application to the same site gives a higher yield of
sensitized subjects but tends to magnify the irritant properties of the test
substance. The resulting "skin fatigue" due to repeated irritation under oc-
clusive conditions could make the differentiation between irritation and sen-
sitization difficult.
*
5.3 Draize Test (and modifications)
Marked by multiple insults at induction (10-15 treatments of 24 hours
each), this test is extensively used for determining human sensitization
74
-------
Table 2-6. Predictive Patch Tests For Human Skin Seneitiration
Test
No. Subject
INDUCTION
Test sub-
stance, amount
or concen-
tration
Vehicle
Skin Site
Type Patch
No. Patches
Patch
Duration
(hours)
Schwartz
200
Liquid or
powder (on 1"
fabric)
None
Arm, thigh or
back
Cellophane
covered with
2"x2" Elasto-
plast
1
72
Shelanski-Shelanski Draize
Schwartz-Peck Repeated Insult Repeated Insult
200 200 100 males, 100
females
Liquid8 Proportional to area 0.5 ml or 0.5 g
(saturating V of ultimate use; liquid
4-ply) gauze or powder
Petrolatum or None NSC
corn oil
Arm or back Arm or back
1" non-water Same 1" sq. band aid8
proof cellophane
covered with 2"
adhesive plaster
1 10-15 10
27, 72 or 96 24 alternate days; 24 alternate days
same site
Draize Kligman
Modified "Maximization"
200 25
0.2 ml or 0.2 g 1 ml 5Z SLS followed
(high cone.) by 1 ml 25Z test
material
Petrolatum Petrolatum
Arm Forearm or calf
1" sq. band aid"; 1.5" sq. Hebril
no perforations occluded with Blend-
em" held in place
with perforated
plastic tape
10 5 same site
48 - 72 24 hrs. SLS followed
10 days by 48 hr.
test material for
each of 5 inducing
applications
"Maximization"
Modifi.ed
25
1 ml 1Z SLSb
(24 hr) follow-
ed by 0.3 ml
0.3 g test
material
Petrolatum
Forearm
2 cm Uebril
occluded with
Blenderm"
held in place
with perforated
tape
7
24 hrs. SLS
followed 10
days by 48
hr. test
material for
each of 7
inducing
applications.
No patch for
24 hrs. between
each of 7
inducing
applications
REST (Days)
7-10
10 - 14
14 - 21
10 - 14
14
10
10 - 14
-------
Table 2-6. Predictive Patch Teats For Human Skin Senaitization
(Continued)
Shelanski-Shelanaki Draize Draize
Test Schwartz Schwartz-Peck Repeated Insult Repeated Insult Modified
CHALLENGE
Test sub- Same as Same as Same as induction Sane as induction MNICC
stance, amount induction induction
or concen-
tration
Kligman
"Maximization"
0.4 ml 10Z 8L8 (1 hr.)
followed by 0.4 ml,
101 test material
"Maximization"
Modified
1 ml 21 SLS
Oi hr.)
followed by
0.3 ml or
0.3 g test
material
Skin Site Same site Any site NS
especially thin
keratin
New skin site
New skin lite
Lower back or forearm Upper back
Type Patch
No. Patches
Patch
Duration (hrs)
Same aa
induction
1
72
observe 10
days
Same as Same as induction
induction
1 1
48 48
observe 3 days Reaction read
after patch
remova I
Same aa induction Same as induction
1 1
(rechallenge if
necessary)
24 72
Same . Same
1" aq. patch. Same
a induction
1
48
reaction read
.soon after patch
removal and at 72
and 92 hrs
Same as
induction
1
48
Same
sModified for solids, powders, ointments and cosmetics. Concentration, amount, area and site of application are important in evaluating results. Authors
recommend cosmetics be tested open (Schwartz and Peck, 1944).
bSodium lauryl sulfate (SLS) pretreatment is used to produce moderate inflammation of the skin. SLS is mixed with test material when compatible. SLS is
eliminated when test material is a strong irritant.
°Abreviations
MNIC = Maximal nonirritating concentration
NS ° Not Stated
-------
(Marzulli and Maibach, 1973, 1974, 1976a,b; 1980). Maibach and Epstein (1965)
found that the sensitivity of the test could be increased by raising the con-
centration of the test substance.
Subject - 200 adult volunteers (who sign an FDA approved consent form
before participation).
Test Material - The test material is used in graded concentrations (0.2
- 0.5 g) and at actual use concentrations (1/100 of the concentration
producing a 0.1% response). The vehicle is generally petrolatum.
Induction - Sensitization is achieved by topical application of the test
material in 0.2 ml portions to the skin of the upper lateral part of
the arm. The site is covered with an occlusive patch (Band AidR with
perforations) for 48 or 72 hours. Usually 10 epicutaneous applications
are made successively to the same site.
Challenge - Challenge is performed 14 days after the last sensitizing
application when the maximum nonirritating concentration of the test
material is applied under occlusion to a fresh skin site for 72 hours.
Reactions are assigned one of 4 grades, (1) erythema, (2) erythema with
induration, (3) vesiculation, and (4) bulla formation.
Evaluation - The data collected over the years indicate that substances
that are not strong sensitizers are not likely to give false positive
reactions by this test, even when high concentrations are used at in-
duction, provided that a non-irritant concentration is used for the
challenge.
5.4 Maximization Test
The Maximization Test was first reported in 1966 (Kligman, 1966a,b,c,d),
modified several times and updated in 1975 (Kligman and Epstein, 1975). It
now takes into account the large variations and susceptibilities that exist
between skins of different human populations and includes a pretest exclusion
of'subjects who are non-reactors to the test materials, hypersensitive to
sodium lauryl sulfate or who are already sensitized by previous exposure to the
chemical. A basic premise of the Maximization test is that the exposure be
deliberately intensified to increase the chances of detecting weak allergens.
Subject - 25 adult volunteers (who sign an FDA-approved consent form
before participation).
Test Material - The test material is 25% by weight in petrolatum (i.e.,
25 gm compound to 75 gm petrolatum). If the agent is an irritant at
this level, lower concentrations are used. If the agent is non-
irritating it is used undiluted.
Pretesting - Before induction begins, all members of the test group are
patch-tested (with the tested materials as they will be used at chal-
lenge - see below) to eliminate hypersensitive people and obtain a base-
line reading for each material on each subject. The degree of reaction
77
-------
is recorded. This reaction must be appreciably exceeded following
challenge before the patch reaction is considered allergic.
Induction - Induction is achieved by delivering 1.0 ml of 5% aqueous
sodium lauryl sulfate to a Webril patch (1.5" square) and fastening
this occlusively to the arm (with Blenderm tape) held in place with
perforated plastic tape for 24 hours in order to produce a moderate
inflammatory response. The same site is treated on the following day
with a 48-hour (251 test material) occlusive patch with the test
material. The 48-hour allergen patches are repeated for a total of
seven exposures each, with no patch for 24 hours between each of the 7
inducing applications.
Challenge - Challenge is performed with 2Z sodium lauryl sulfate for 30
minutes followed by a 48-hour occlusive patch with the test material to
the skin of the upper back. Reaction is read soon after patch removal
and at 72 and 96 hours after patching.
The reactions are graded on a scale of I to V based on the number of
subjects sensitized as shown below:
HUMAN MAXIMIZATION TEST GRADING SCALE
Sensitization
Numbers5 Grade Classification
0-2/25 I Weak
3-7/25 IT Mild
8-13/25 III Moderate
14-20/25 IV Strong
21-25/25 V Extreme
aKligman, 1966c.
Evaluation - The maximization procedure is useful in identifying very
low-grade sensitizing chemicals following prescreening in the guinea
pig. It is primarily designed to yield allergenicity ratings for in-
dividual substances, not complex mixtures, finished products or formu-
lations. The number of substances tested by this method by the Research
Institute for Fragrance Materials suggests that it is the most widely
used human test method. A major problem with this procedure lies in
the interpretation of false positive reactions resulting from "toxic"
effects due to factors other than the test material (e.g., infection,
pressure, miliaria, drug reactions, food allergies) (Kligman and
Epstein, 1975).
It should be noted that the Maximization Test does not directly assess
safety in use (except when negative) and it does not predict the incidence of
sensitization in a population of users. A better estimate of sensitization.
potential might be obtained with 100-200 volunteers than with 25.
78
-------
6.0 APPLICATION OF PREDICTIVE TESTS TO KNOWN CONTACT ALLERGENS
The validity of human and animal test methods for sensitization is de-
termined by their ability to detect compounds which pose hazards to man. The
most effective test procedures are those which provide reliable information on
the degree of response to be expected.
6.1 Human Tests
The result of.ten "moderately strong allergens" (whose allergenic activ-
ity was confirmed medically) tested by the Schwartz-Peck, Draize and Shelanski-
Shelanski methods on groups of 200 volunteers are summarized in Table 2-7. Two
of the ten allergens were detected by the Schwartz-Peck test, one by the Draize
Test and four by the Shelanski-Shelanski procedure. None of the three test
procedures detected penicillin G, streptomycin, neomycin and benzocaine
(Kligman, 1966a).
When the Maximization Test was applied to those compounds, penicillin G
and streptomycin produced grade I (weak) and grade II (mild) reactions at
equivalent doses, while thephorin, tetrachlorosalicylanilide and monobenzyl
ether of hydroquinone gave grade II and III reactions with one-half to I/20th
the concentrations used in the Schwartz-Peck, Draize and Shelanski-Shelanski
Tests (Kligman, 1966b). The Maximization Test in humans is not only sensitive
but highly reproducible as is evident from the data in Table 2-8. The repro-
ducibility is extremely high at both ends of the scale; upon repetition, weak
(grade I) and strong (grade V) allergens are likely to be assigned same rating.
There is greater variability in the mid-zone (grades II-IV), however the shift
is rarely more than one grade class (e.g., penicillin G; chloroquine
diphosphate).
6.2 Guinea Pig Tests
When the system of grading of allergenic response in the human Maximi-
zation Test was extended to the guinea pig a good correlation was found between
the results of the two tests (Klecak et al., 1977). Of the 22 sensitizing sub-
stances that were tested using this grading system in the human and the guinea
pig, ten were within the same rating level; 7 were within a single grade level
and 5 were within two grade levels of discrepancy. Seven weak allergens (e.g.,
vioform, vanillin, cinnamic alcohol, eugenol, geraniol, heliotropin, limonene)
were recognized by the guinea pig test but not by the human test (Table 2-9).
In contrast to the above findings a comparison between the results of
other tests, e.g., the human Repeated Patch Test and the guinea pig Closed
Patch Test showed a wide variation in response between the two systems (Table
2-10) (Griffith and Buehler, 1977; Marzulli et al., 1968; Marzulli and Maibach,
1974).
79
-------
Table 2-7. Predictive Patch Tests In Humans
oo
o
Compound
Penicillin G
Streptomycin
Neomyc in
Benzocaine
The ph or in
Ammoniated Mercury
Tetrachlorosalicylanilide
Monobenzyl ether of
hydroquinone
Furacin
Butyn-Sulfate
Concentration3
%
1.0
1.0
.0
2.0
5
10
0.2
20
0.2
2.0
Schwartz-
Peckbb
Test
0/200
0/200
0/200
0/200
2/200
0/200
2/200
0/200
0/200
0/200
Draize
Testb
0/200
0/200
0/200
0/200
2/200
0/200
0/200
2/200
0/200
0/200
Shelanski-
Shelanski
0/200
0/200
0/200
0/200
6/200
2/200
9/200
9/200
0/200
0/200
Maximization
Testc
4/22d
8/22
-------
Table 2-8. Reproducibility of Maximization Test Grades In Humansa
Induction Sensitization
Concentration Test No.
Substance %d 12
Ammonia ted
mercury
p-Aminobenzoic
acid
ApresolineR
BenzocaineR
Chloroquine
diphosphate
Hexachlorophene
Monobenzyl ether
of hydroquinone
Penicillin 6
Tetrachloro-
salicylanide
Tetramethyl-
thiouram-disulfide
(Thiran)
25
«
5
25 d
25 d
25
25
25 d
5
25
13/25
0/23
25/25
5/24
9/24
0/24
22/22
16/23
24/24
4/22
11/24
0/24
24/24
3/24
6/25
1/25
25/25
12/23
22/23
1/23
Ratesb
3
15/25
0/24
25/25
5/22
11/23
0/23
24/25
13/25
24/25
1/25
Grades0
Test No.
1 2 3
III III IV
III
V V V
II II II
III II HI
III
V V V
IV III III
V V V
II I I
aData taken from Kligman 1966c
Number of sensitized subjects/total
cGrades: I - Sensitization rate; 0-2/25; weak
II - Sensitization rate; 3-7/25; mild
III - Sensitization rate; 8-13/25; moderate
IV - Sensitization rate; 14-20/25; strong
V - Sensitization rate; 21-25/25; extreme
^sodium lauryl sulfate provocative test
81
-------
Table 2-9. Grade* of Allergenic Potency By cbe Maximization Tests la Humans and Guinea Pigs
Induction0
Concen-
tration
(topical)
Substance Z
Acrylic Monomer
Aluminum Chloride
Apresolin*&
AtabrineR
Ben>ocaineR
Formalin
Hexachlorophene
Lano lin
MalathionR
MarfanilR
Mereapto-
benzothioazole
Mercuric Chloride
Monobenzyl ether
of hydroquinone
Heomycin
Nickel Sulfate
Penicillin G
Potassium
dichromate
Sodium lauryl
sulfate
Streptomycin
Sulfathiacole
Tetrachloro-
salicylanilide
Turpentine
Tween 80
Vioform*
5
25
5
25
25
5*
25
25
10
5
25
1.0
25
ye
5"
5
1
5
10
25
1
25
25
25
Guinea Pig*
Challenge0
Concen-
tration Positive
Z Z.
10-
2
1
10
5
2«
1.5
15
20
20
15
0.1"
23
25«
0.5«
10
O.le
0.5
0.5«
10
le
20
20
5
84
0
80
90
28
80
0
0
54
100
40
32
50
72
55
100
75
0
72
36
72
64
0
20
Graded
V
I
IV
V
II
IV
I
I
III
V
;.
Ill
III
III
I
III
V
IV
I
IV
III
IV
III
I
II
Induction0
Concen-
tration
Z
25
5.08
258
258
5
258
258
258
258
2.0
10
258
108
258
2.0
10
258
258
5.08
SO
258
258
Human
Challenge0
Concen-
tration
Z
10
5.0h
10h
10°
1.0
10
10h
10h
10h
0.05
5
10h
2.5h
10h
0.25
0.1
10h
10h
0.5h
20
5.0
10"
Positive
Z
0
100
78
22
72
0
0
100
38
92
92
28
48
67
100
0
80
4
88
72
0
0
Grade4
HD*
I
V
IV
II
IV
I
I
V
ND
III
V
V
II
III
IV
V
I
IV
I
IV
IV
I
I
Results from Magnusson and Kligman 1969
bResulcs from Kligman 1966c,d
cVehicle is petrolatum
^Maximization Grading from Kligman 1969
Grade I - Sensitizatlon rate, 0-8Z; weak
Grade II - Sensitizacion rate, 9-281; mild
Grade III - Sensitization rate, 29-641; moderate
Grade IV - Sensitization rate, 65-801; strong
Grade V - Sensitization rate, 81-1001; extreme
eVehicle is water
%D * not done
8?retreatment of skin with sodium lauryl sulfate
hSLS provocative test
^-Vehicle is 70Z ethanol
82
-------
Table 2-10. Sensitization Rate In Guinea Pig Closed Patch Test and
Human Repeated Insult Patch Testa
Substance (Vehicle)
Benzoyl peroxide/
1.0% sulfur (PG)
Tetrachl or osa li cyl an i li de
(TCS) (1% ethanol)
Dithioquaternary Ammonium
Cmpd (DA) aqueous surfactant
Sulfonyl Compound
Hydroxylamine Sulfate
l-(3-Chlorophenyl)-3-phenyl-
2-pyrazole (CPP)
Ethanol
Neomycin (petrolatum)
Benzocaine (petrolatum)
Hexachlorophene (petrolatum)
Bithionol (petrolatum)
Furacin (petrolatum)
Human
Concen-b Sensiti-
tration zation
I I
10
0.05
0.2
0.5
0.05
(aqueous
detergent)
1.0
(aqueous
detergent)
50
5
10
0.5
20
0.2 lc
40.5
19.0
10.9
7.5
3.9
2.9
6.4
1.5C
(2.6)d
1.0C
(3.0)d
1
Oc
(0)d
lc
(3)d
Guinea
Concen-b
tration
%
10
1.0
1.0
(1% ethanol)
0.5
15
(aqueous
detergent)
0.05
(1% ethanol)
80
5
10
10
20
0.15e
Pig
Sensiti-
zation
%
42.1
80
100
0
0
0
66.6
0
0
0
0
0
aData compiled from Griffith and Buehler 1977; Marzulli et al. 1968,
Marzulli and Maibach 1974.
^Concentrations are for induction and challenge.
cValues without sodium lauryl sulfate.
dValues with Sodium lauryl sulfate.
elnduction (0.15%) and intradermal (0.15%) and topical (20%) routes for
challenge.
-------
7.0 USE OF HUMAN TESTS
Essentially, three types of tests (predictive, diagnostic and use) are
required to evaluate skin sensitization potential. The Predictive test is
needed to identify allergic substances. The procedures used in predictive
tests include the Draize Test (Marzulli and Maibach, 1976b) or the Maximization
Test (Kligman, 1966c). In the Research Institute for Fragrance Materials'
Monographs on Fragrance ingredients, over 350 different ingredients were tested
for sensitization potential in humans. Greater than 99.5% of the tests are
now run using the Maximization procedure. The Diagnostic test is required to
determine which substances are actually posing dermatologic problems and the
Use test provides information on the safety of ingredients in a particular
combination for a specific use. This information is partially derived from
consumer complaints and injury reports.
Comparative diagnostic and predictive data in humans for 15 compounds
are given in Table 2-11. In the Diagnostic test, wide geographic areas are
tested with a standard screening kit to evaluate possible racial, ethnic, cli-
matic and genetic variations in response. In this test, a preparation is ap-
plied to a clinical patient's skin under an occlusive patch for 48 hours. The
skin is evaluated for evidence of erythema, edema or more severe skin changes
occurring at 24, 48 and 72 hours after patch removal. Substances, concentra-
tions and methos are standardized and the data are collected centrally,
analyzed and evaluated in terms of a sensitization index (% of positive skin
reactions). The differences in the response between the North American Contact
Dermatitis Group (NACDG) and the International Contact Dermatitis Research
Group (ICDRG) could be due to climatic or genetic differences or simply to the
types of patients being tested (e.g., occupationally exposed, patients with
intractable eczema, etc.). Consistently higher values were obtained in the
Predictive test than in the diagnostic tests. This could have resulted from
pretreatment with sodium lauryl sulfate, sodium lauryl sulfate provocative
patches or high con- centrations of the chemicals used.
The results displayed in Table 2-11 permit the following judgements:
(1) When a sensitization frequency in Diagnostic test exceeds that of the Pre-
dictive test (e.g., parabens), individuals are being identified who cannot
tolerate a widely used product. (2) When the Diagnostic and Predictive test
sensitivities show a high frequency, the substance is a strong sensitizer
(e.g., p-phenylenediamine) and (3) when the Diagnostic and Predictive test
frequencies are low, the Predictive test is accurately forecasting a low
allergenic potential, not a false negative reaction.
The results of the Use tests are more difficult to evaluate since the
products are generally confined to use at low concentrations and at varying
frequencies. For example, formaldehyde (aqueous solution), which is recog-
nized as a strong sensitizer and according to the Cosmetic Product Registry of
the FDA (1972) is used in about 5% of 8,000 preparations, has failed to sensi-
tize large numbers of the general population primarily because it is used in
low concentrations as a preservative in shampoos and other products. Most of
these preparations are rinsed off after use and do not remain in contact with
the skin for long periods. On the other hand, the use of formaldehyde as a
nail hardener is accompanied by significant injury to sensitive nails and
adnexal tissues.
84
-------
Table 2-11. Evaluation of Some Conmon Seneitizers By Diagnostic Test and Predictive Test
CD
Ln
Diagnostic Test8
Compound0
Nickel Sulfate
Potassium
dichromate
Thimerosal
p-Phenylene-
diamine
Ethyl enediamine
Neomycin
Sulfate
Bcnzocaine
Ammoniated
mercury
Mercaptobenzo-
thiazole
Formalin
(aqueous)
Wool wax
alcohol
Thiram
Parabens
mixture^
Dibucaine MCI
Cyclomethycaine
Sulfate
Total Tested
Concen-d
tration
I
2.5
0.5
0.1
1.0
1.0
20
5
1.0
2
2
30
2
15
1.0
1.0
NACDGb
Numbers
of
Reactors
131
91
91
98
85
71
54
65
58
43
37
50
38
32
23
1,200
\
lie
8
8
8e
7
6e
5
5
5e
4
3
4e
3e
3
2
Concen-d
tration
*
5.0
5.0
1.0
20
5
2
2
30
2
15
ICDRGb
Numbers
of
Reactors Z
321 6.7
318 6.6
237 4.7
__
176 3.7
192 4.0
_ __
99 2.0
169 3.5 .
127 2.6
97 2.0
91 1.9
4,824
Predictive Test8
Concentration
Z
Induc-
tion
10
2
0.2
1.0
5
25
20
5
25
10
20
25
25
5
5
10
25
1.0
5.0Mf
+1.25P
Chall-
enge
2.5
0.25
2.5
1.0
1.0
10
20
5
10
10
10
10
10
1.0
1.0
1.0
10
1.0
sane
~
Total
No.
25
23
24
88
61
25
42
186
23
173
99
25
24
25
52
102
25
309
98
1,419
Reactors
Z
48
100
29.1
53.4
8.0
28 «
16. 6*
1.6
22 f
1.2
6.0
52*
37. 5f
72*
7.7
7.8
Iftf
0.3
1.0
~
Type8
of Test
MT
MT
MT
DT
DT
MT
MT
MT
DT
DT
MT
MT
MT
DT
DT
MT
DT
DT
- -
Diagnostic test data taken from Marzulli and Maibach, 1974, 1976b; Predictive test data taken from Kligman, 1966c
(maximization test; MT) and Marzulli and Maibach, 1973, 1974; (Draize test; DT)
bData from the North American Contact Dermatitis Group (NACDG) - 1200 male and female, black and white subjects and from
the International Contact Dermatitis Research Group (ICDKG) - 4824 male and female, white subjects.
CA11 compounds prepared in petrolatum, except formalin.
dSame concentration of test substance was used for induction and challenge.
eNACDG va ICDRG, significant at 95Z level.
fPretreatment of skin with Sodium lauryl Sulfate and Sodium lauryl Sulfate provocative test.
BMethyl, ethyl and propyl parabens in equal amounts in the diagnostic test; methyl (M) and propyl (P) parabens in unequal
amounts in the predictive test.
-------
Two important considerations must be kept in mind when identifying po-
tential human allergens. One is the size of the test group which should be
small enough to be logistically feasible in the laboratory and large enough so
that the results are valid for the general population; the other is the ability
to predict the outcome of the test under conditions of use.
The complex mathematical problem of extrapolating from small test popu-
lations to the general population has been reviewed by Henderson and Riley
(1945). Using the generally accepted 95% confidence limit, a finding of no
skin reaction in a test population of 200 random subjects indicates that 22 of
every 1,000 members of the general population may react and at the 99% confi-
dence level, 15 of every 1,000 may react. If the test population is 100, up
to 30 of every 1,000 members of the general population may react (95% confi-
dence). Conversely, 1 (one) positive reactor out of every 200 subjects on test
indicates that a population of 10,000 subjects contain from 1 to 275 sensitized
individuals (95% confidence level). Thus, at least 30,000 subjects must be
studied to be assured of no risk (i.e., 0.01% maximum permissible percentage
of reaction in the population at 95% confidence limit). Obviously, this is a
cumbersome task, while extrapolation is uncertain.
86
-------
8.0 CONCLUSIONS
A review of the comparative data from various sources indicates that the
guinea pig is the most widely used animal model for detecting skin sensitiza-
tion potential. Except for the Draize test, all other procedures reviewed have
some potential for detecting "weak" sensitizers. No one test method appears to
be suitable for testing of all compounds, because significant variations in
results have been observed from different sources using the same test method,
identical compound, and same concentrations, (Table 2-2, e.g., Penicillin G,
Maximization Test).
The Maximization Test using guinea pigs appears to be one of the better
tests for detecting the weaker sensitizers that must be avoided in drugs, cos-
metics and toiletries. Lesser agreement between guinea pigs and humans was
noted for industrial chemicals and pesticides. The test virtually eliminates
the false negative results seen with the Draize Test, but does not appear to
be adequate for predicting sensitization to finished products. Application of
this test to other chemical classes, such as industrial chemicals, drugs, and
dyes, would require extensive validation in guinea pigs with follow-up testing
in humans.
The Optimization, Open Epicutaneous and the Closed Patch Tests may be
viable alternatives to maximization but the published evidence on their ef-
ficiency is sparse. All three of these tests, however, can be used on finished
products. The Closed Patch Test involves a subjective evaluation of the sensi-
tivity, but allows testing of the actual user concentration. The Open Epicuta-
neous Test is also flexible in this regard. Several of the commercial and
industrial test labs contacted used both the Maximization and Closed Patch Test
with success, but no statements were made as to which one is a superior method.
When the tests reveal that a chemical possesses immunelogic capabili-
ities, the percent of animals sensitized does not necessarily indicate the
probable incidence of human sensitization. A negative result is predictive and
indicative of relativly low hazard. More precise predictions of the potential
for human sensitization will only be obtained by extensive clinical testing.
87
-------
9.0 RECOMMENDATIONS
Identification of a preferred, single or pair of methods would require
a thorough validation with industrial chemicals, pesticides and consumer pro-
ducts, including drugs and cosmetic and fragrance ingredients. Costs for the
individual methods vary but the two most widely used differ by only approxi-
mately 15% making either method nearly equivalent on this basis.
When testing a compound for sensitization, the use of a known sensitizer
as a positive control, such as dinitrochlorobenzene, is recommended to aid in .
scoring and personnel training. Proper handling is necessary to prevent inad-
vertent sensitizatioo of laboratory staff. If positive controls are not used,
then the guinea pig strain must be specified and the stock's continued sensi-
tivity should be checked periodically, as it is possible to breed insentive
animals that may be used in these tests.
Careful validation of the method chosen by the individual investigator
is also strongly advised, because there may be class differences in a chosen
method's specificity.
A satisfactory skin sensitization program for industrial chemicals, new
drugs, pesticides, paints and coatings, toiletries, and cosmetics should in-
volve testing in the guinea pig and if possible, in man. The substance should
be tested first in the guinea pig to determine whether it is a low grade or
potent sensitizer. Strong positive results with the guinea pig clearly in-
dicate that the substance is not suitable for human use under uncontrolled
conditions; moderately positive results, in many cases, would preclude the use
of the substance in food and cosmetic products and general industrial exposure;
a negative result could indicate a need for further characterization and
testing.
The next step in a complete evaluation of sensitization or of weak and
negative sensitizers might involve the use of provocative procedures (high con-
centrations, abrasions, use of sodium lauryl sulfate, etc.) in a small group of
human subjects to determine if the individual compounds or formulated product
is likely to sensitize at all. If results are negative, the use test should be
done; if the results are positive, a standard human Patch Test without adjuvant
(Draize Test) should be run on a suitable population. The combined findings
will reveal whether a more expanded Use test is necessary.
88
-------
10.0 REFERENCES
Battista, G.W., and M.M. Reiger. 1971. Some Problems of predictive testing.
J. Soc. Cosmetic Chenu 22:349-359.
Buehler, E.V. 1964. A new method for detecting potential sensitizers using
the guinea pig. Toxieol. Appl. Pharmacol. 6:341-
Buehler, E.V. 1965. Delayed contact hypersensitivity in the guinea pig.
Arch. Dermatol. 91:171-177.
Buehler, E.V., and Griffith, J.F. 1975. Experimental skin sensitization in
the guinea pig and man: In Animal Models in Dermatology. Ed. H.
Maibach, pp. 56-66.
Chase, M.L. 1954. Experimental sensitization with particular reference to
picryl chloride. Int. Arch. Allergy App. Immunol. 5:163-191.
Chase, M.L. 1967. Hypersensitivity of simple chemicals. The Harvey Lecture
Series 61 169-203, Academic Press, N.Y.
Coenraads, P.I., E. Bleuraink, and J.P. Nater. 1975. Susceptibility to
primary irritants. Contact Dermatitis 12:377-381.
Draize, J.H. 1965. Appraisal of the safety of chemicals in food, drugs and
cosmetics. In Dermal Toxicity, p. 48. Austin, Texas. Assoc. of Food
and Drug Officials of the U.S., Texas State Dept. of Health.
Epstein, W.L., A.M. Kligman, and I.P. Senecal. 1963. Role of regional lymph
nodes in contact sensitization. Arch. Dermatol. 88:789-792.
Fahr, H., U. Noster, and K.H. Schulz. 1976. Comparison of guinea pig
sensitization methods. Contact Dermatitis 2:335-339.
Griffith, J.F., and E.V. Buehler. 1977. Prediction of skin irritancy and
sensitizing potential by testing with animals and man. In Cutaneous
Toxicity. V.A. Drill and P. Lazar, Ed., Academic Press, 155-173.
Henderson, C.R. and E. C. Reilly. 1945. Certain statistical considerations
in patch testing. J. Invest. Derm. 6:227-230.
Kero, M., and M. Hannuksela. 1980. Guinea pig maximization test, open
epicutaneous test and chamber test in induction of delayed contact
hypersensitivity. Contact Dermatitis 6:341-344.
Klecak, G., Gelciek, H., and J.R. Frey. 1977. Screening of fragrance
materals for allergenicity in the guinea pig. 'i. Comparison of four
testing methods. J. Soc. Cosmetic Chem. 28:53-64.
Kligman, A.M. 1966a. The identification of contact allergens by human
assay. I. Critique of standard methods. J. Invest. Dermatol.
47:369-374.
89
-------
Kligman, A.M. 1966b. The identification of contact allergens by human
assay. II. Factors influencing the induction and measurement of
allergenic contact dermatitis. J. Invest. Dermato1. 47:375-392.
Kligman, A.M. 1966c. The identification of contact allergens by human
assay. III. The maximization test. A procedure for screening and
rating contact sensitizers. J. Invest. Dermatol. 47:393-409.
Kligman, A.M. 1966d. The SLS provocative patch test. J. Invest. Dermatol.
46:573-589.
Kligman, A.M., and W. Epstein. 1975. Updating the maximization test for
identifying contact allergens. Contact Dermatitis 1:231-239.
Landsteiner, K., and J. Jacobs. 1935. Studies on sensitization of animals
with simple chemical compounds. J. Exp. Med., 61:643-656.
Loomis, T. 1978. Toxicology testing methods. In Essentials of Toxicology,
pp. 229, Lea and Febiger, Phila.
Magnusson, B. 1975. The relevance of results obtained with the guinea pig
maximization test. In Animal Models in Dermatology. Ed. H. Maibach, pp.
76-83. Edinburgh, Churchill Livingstone.
Magnusson, B., and K. Hersle. 1965. Patch test methods. I. Comparative
study of six different types of patch tests. Acta Dermato-Venerol.
45:123-128.
Magnusson, B., and A.M. Kligman. 1969. The identification of contact
allergens by animal assay. The guinea pig maximization test. J. Invest.
Dermatol. 52:268-275.
Magnusson, B. and A.M. Kligman. 1970. Identification of contact allergens.
In: Allergic Contact Dermatitis in the Guinea Pig. Springfield, 111.
Thomas, pp. 102-124.
Magnusson, B., and A.M. Kligman. 1977. Factors affecting contact
sensitization. Advances in Modern Toxicology 4:289-304.
Maguire, H.C. 1972. Mechanism of intensification by Fruend's complete
adjuvant of the acquisition of delayed hypersensitivity in the guinea
pig. Immunol. Commun. 1:239-246.
Maguire, H.C. 1973. The bioassay of contact allergens in the guinea pig.
J. Soc. Cosmet. Chera. 24:151-162.
Maguire, H.C. 1975. Estimation of the allergenicity of prospective human
contact sensitizers in the guinea pig. In Animal Models in Dermatology.
Ed. H. Maibach, pp. 67-75, Edinburgh, Churchill Livingstone.
90
-------
Maguire, H.C., and M.W. Chase. 1967. Exaggerated delayed-type
hypersensitivity to simple chemical allergens in the guinea pig. J.
Invest. Dermatol. 49:460-468.
Maguire, H.C., and M.W. Chase. 1972. Studies on the Sensitization of
animals with simple chemical compounds. XIII. Sensitization of guinea
pigs with picric acid. J. Exp. Med. 135:357-375.
Maibach, H.I., and W.L. Epstein. 1965. Predictive patch testing for
allergic Sensitization in man. Toxicol. and Appl. Pharmacol. Suppl. 2:
139-43.
Marzulli, F.N., T. Carson, and H.I. Maibach. 1968. Delayed contact
hypersensitivity studies in man and animals. Proc. Joint Conf. Cosmet.
Sci., Washington, DC. pp. 107-122.
Marzulli, F.N., and H.I. Maibach. 1973. Automicrobials: Experimental
contact skin Sensitization in man. J. Soc. Cosmet. Chem. 24:399-421.
Marzulli, F.N., and H.I. Maibach. 1974. The use of graded concentrations
in studying skin sensitizers: Experimental contact Sensitization in
man. Food Cosmet. Toxicol. 12:219-227.
Marzulli, F.N., and H.I. Maibach. 1976. A contact allergy: Predictive
testing in man. Contact Dermatitis 2:1-17.
Marzulli, F.N., and H.I. Maibach. 1976b. Effects of vehicle and
elicitation concentration in contact dermatitis testing. Contact
Dermatitis 2:325-329.
Marzulli, F.N., and H.I. Maibach. 1980. Contact allergy: Predictive
testing of fragrance ingredients in humans by Draize and maximization
methods. J. Environ. Pathol. Toxicol. 3:235-245.
Matsushita, T., and K. Aoyama. 1980. Dose response relationship in delayer
type contact sensitivity with maneb: an experimental model of "Simple
Chemicals". Ind. Health 18:31-39.
Maurer, T., E.G. Weirich and R. Hess. 1980. The optimization test in Che
guinea pig in relation to other predictive Sensitization methods.
Toxicology 15:163-171.
Maurer, T., P. Thomann, E.G. Weirich, and R. Hess. 1975a. The optimization
test in the guinea pig. A method for predictive evaluation of contact
allergenicity of chemicals. In Agents and Actions 5/2:174-179.
Birkhauser Verlag Basel.
Maurer, T., P. Thomann, E.G. Weirich, and R. Hess. 1975b. The optimization
test in the guinea pig. A method for predictive evaluation of the
contact allergenicity of chemicals. International Congress Series
376:203-208.
91
-------
Maurer, T., P. Thomann, E.G. Weirich, and R. Hess 1978. Predictive
evaluation in animals of the contact allergenic potential of medically
important substances. I. Comparison of different methods of inducing
and measuring cutaneous sensitization. Contact Dermatitis 4:321-333.
Nater, J.P., and J. Hoedmeaker 1976. Histological differences between
irritant and allergic patch test in man. Contact Dermatitis 2:247-253.
OECD 1981. Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals. Skin Sensitization. OECD
Publications and Information Center, Suite 1207, 1750 Pennsylvania Ave.,
N.W., Washington, DC 20006.
Prince, H.N., and T.G. Prince 1977. Comparative guinea pig assays for
contact hypersensitivity. Cosmet. Toilet. 92:53-58.
Schwartz, 1960. Twenty-two years experience in the performance of 200,000
prophetic patch tests. South Med. J. 53:478-483.
Schwartz, L., and S.M. Peck 1944. The patch testing in contact dermatitis.
Public Health Rep. 59:546-557.
Shelanski, H.A., and H.V. Shelanski 1953. A new technique of human patch
tests. Proc. Sci. Sect. Toilet. Goods Assoc. 19:46-49.
Turk, J.L., and D. Parker 1977. Sensitization with Cr, Ni and Zr salts and
allergic type granuloma formation in the guinea pig. J. Invest.
Dermatol. 68:341-345.
92
-------
PHOTOTOXICITY
1.0 Summary
Phototoxicity and photoallergy testing are in an evolving status and
well-defined proven tests are not available. Yet these important toxic proper-
ties should not be ignored when assessing the toxicity of photoactive com-
pounds. The action spectrum for photosensitization is normally similar to the
absorption spectrum of the photoactive compound. Methods for phototoxicity
testing in animals and humans are summarized in Tables 3-3 and 3-4 and methods
for photoallergy testing in guinea pigs are summarized in Table 3-5. Rabbits,
hairless mice, guinea pigs and miniature swine are presently acceptable animal
models for phototoxicity testing. The guinea pig is the only animal model
presently acceptable for photoallergy testing. Since extrapolation of animal
photosensitivity data to humans is often difficult, human testing is recommend-
ed for compounds to which humans will be exposed in the presence of sunlight.
Human testing of a substance should be conducted only after testing in animals
for both photosensitivity and systemic toxicity.
93
-------
2.0 PHOTOSENSITIVITY
Photosensitivity has not generated a great deal of interest outside of
the cosmetic field and no testing has been mandated by regulation. Guidelines
on this subject are being prepared by the OECD.
The involvement of light should be considered whenever the toxicity of
light-absorbing chemicals is being assessed. Photosensitivity refers to both
photoallergic and phototoxic (nonimmunelogic) light-induced skin responses.
Phototoxicity can be considered the light-induced counterpart of primary
irritation and photoallergy is the light-induced counterpart to contact
dermatitis. Table 3-1 shows a summary of the similarities and differences
between phototoxic and photoallergic mechanisms.
Selected studies in humans and guinea pigs indicate that many compounds
can be both phototoxic and photoallergic. Most compounds which have been
reported as photoallergic in humans can be shown to be phototoxic in guinea
pigs provided the concentration is sufficiently high and the appropriate wave-
length of exciting light is used (Barber and Shalita, 1977).
Chemicals with phototoxic and photoallergic potential have several
chemical features in common. Most are dicyclic and tricyclic aromatic com-
pounds that fluoresce e.g., phenanthrene (Harber and Shalita, 1977). The
absorption spectrum of both phototoxic and photoallergic chemicals is usually
in the ultraviolet (UV) range, but may extend into the visible region. Listed
in Table 3-2 are examples of phototoxic and photoallergic agents.
The conditions prevailing in the skin at a particular site or time can
influence the susceptibility of the skin to photoallergy (Harber and Shalita,
1977). These conditions include: 1) both the quality and location of the
photosensitizer on the skin; 2) the capacity of the chemical to penetrate the
skin through percutaneous absorption as well as trauma such as abrasion or
sunburn; 3) the pH, the presence of enzymes and solubility conditions at the
site of exposure; 4) the durations and intensity of exposure to activating
radiation; 5) the depth of penetration of the activating radiation; 6) tht
humidity and ambient temperature; 7) thickness of the horny layer of skin; 8)
the presence and degree of pigmentation; and 9) the immunologic state of the
subject or laboratory animal. These above conditions may also affect photo-
toxicity with the exception of #9.
94
-------
Table 3-1. Comparison of Fhototoxic and Photoimmunologic Reaction3
Reaction
Fhototoxic
Photoimmunologic
Reaction possible on first exposure
Incubation period necessary-first
exposure
Chemical alteration of photosensitizer
Covalent binding with carrier
Clinical changes
Flares at distant previously involved
sites possible
Persistent light reaction can develop .
Cross-reactions to structurally related
agents
Broadening of cross-reactions following
repeated photopatch testing
Concentration of drug necessary for
reaction
Incidence
Action spectrum
Yes
No
No
No
Usually like
sunburn
No
No
Infrequent
No
High
Usually relatively
high (theoretically
100Z)
Usually similar to
absorption
Passive transfer No
Lymphocyte stimulation test No
Macrophage migration inhibition test No
No
Yes
Yes
Yes
Varied morphology
Yes
Yes
Frequent
Possible
Low
Usually vary
(but theoretically
could reach 100%)
Usually higher wave
length than absorp-
tion spectrum
Possible
Possible
Possible
aAdopted from Earher and Baer, 1972.
95
-------
Table 3-2. Phototoxic and Photoallergic Chemicals3
Phototoxic Compounds
Photoallergic Compounds
Acridine
Anthracene
Chlorothiazides
Coal Tar
Pesticides
Salithion
Cyanophos
Dichlorvos
Chiorothaionil
Maneb
Paraquat
Phenanthrene
Psoralens
Pyridine
Sulfonamides
Sulfonylureas
Bithional
Chloropromazine
Fentichlor
Griseofulvin
Halogenated salicylanilides
4-chloro-2-hydroxybenzoic
acid N-m-butylamide
6-Methylcoumarin
Promethazine
aAdopted from Barber and Shalita, 1977 and Horiuchi et al., 1978
96
-------
3.0 PHOTOTOXICITY TESTING
3.1 Animal Models
Testing for phototoxicity (light induced irritation) has not developed
to the point that specific tests including animal models have become standard-
ized. Under appropriate test conditions, several species can be utilized for
phototoxicity testing (Marzulli and Maibach, 1970). The rabbit and the hair-
less mouse appeared to be more sensitive to 5-methoxypsoralen than the guinea
pig. Miniature swine were less reactive but "stripping" of the skin with cel-
lophane tape enchanced responsiveness; the squirrel monkey appeared resistant.
The hamster showed histological changes due to phototoxicity; however, these
were not apparent on gross examination. Forbes et al. (1977) have tested a
number of fragrance raw materials using both sunlight and different laboratory
light sources on the skin of hairless mice, humans and miniature swine. The
hairless mouse skin was the most sensitive, the reactions in humans and swine
were qualitatively and quantitatively similar.
Dose; Dosing should be on a microgram or milligram-per-square centi-
meter basis, simplifying the extrapolation to dosing in humans (Maibach and
Marzulli, 1977). The chemical can be delivered to the skin with a micro-
pipette. Following application, the animals are exposed to ultraviolet light
from a high-output source. Most responses to phototoxic chemicals are elicited
by high intensity, U.V. light above 310 nm. One high dose normally is admin-
istered, since no situations have been reported in which a compound has been
negative at high dose and positive at lower doses. Each animal may be used as
its own control. Control should include negative (the vehicle), positive (a
known relevant phototoxic chemical such as methoxypsoralen), and a chemically
treated site which is not irradiated. Experience suggest that most chemicals
that are phototoxic by cutaneous exposure will produce toxicity in the majority
of the animals; therefore, small groups of from 4 to 10 animals are sufficient
for this testing.
Response; Phototoxic response is generally elicited quickly; therefore,
for a positive effect, the site should be irradiated within 30 minutes to 2
hours after the chemical application. Examination and grading are performed
12 to 24 hours later.
Specific methods for phototoxicity testing in laboratory animals have
been published by Marzulli and Maibach (1970), Alkin et al. (1979), and Forbes
et al. (1977) and are sumnarized in Table 3-3. The three methods are similar
but vary in specific details. One method uses intraperitoneal injection rather
than skin application. The rabbit and hairless mouse are found to be sensitive
species for phototoxicity screening studies. In one study the swine skin sen-
sitivity compared favorably with human skin. For the chemicals tested, the
hamster and monkey were poor animal models. Further test development must
occur before a standarized screening test can be recommended.
3.2 Human Testing
Extrapolation of animal phototoxicity data to humans is difficult. In
these cases, human testing may be necessary if the basic systemic toxicity data
97
-------
Table 3-3. Phototoxicity Testing in Animals
Animal Model
Site Preparation
and Application
Light Exposure
Observation and Scoring*
Reference
vo
00
Hairless Mice
CF-1 Swiss Mice
Hartley guinea
pigs
Hairless mice
Miniature swine
Rabbits
Hairless mice
Mice
Hamsters
Squirrel monkeys
Guinea pigs
Intrapertioneal Injection
(25-50 mg/kg)
20 micro 1 test material
on 2 cm* on back skin
Hair was removed by clipping
and 0.05 ml of test material
applied.
30 minutes after
drug treatment;
Blacklight 16 hrs.
2 cm above mice
10 cm above guinea
pig
30 minutes after
application; a. sun
(glass filtered)
30 min. b. compact-arc
xenon lamp-2 minutes
c. long-arc Xenon lamp-
40 minutes d. black-
light-60 min.
5 minutes after
application
Hanovia
"Inspectolite"
90% UV (between 300 -
400 mm) 3000 microwatts/
cmr at a distance of
10 cm. Irradiated
20-30 minutes.
6 to 18, and 48 hours
post-irradation
Scoring 0-4
4,24,48,72 and 96 hours
Scoring + or -
(not graded)
Akin et al. (1979)
Forbes et al. (1977)
24 and 48 hours
Scoring + or -
(not graded).
Marzulli and Maibach
(1970)
-------
are available. Human tests can be safely performed because only small test
areas are exposed to compounds found relatively safe in animals. The greatest
hazard to the experimental subject is the possible development of a small area
of dermatitis, which should heal promptly. Before any test involving humans,
fully informed consent of the subject must be obtained. The experimental pro-
cedure in humans resembles that used with animals; however, because human skin
is less permeable than that of most laboratory animals it is usually necessary
to make the skin more permeable by removing most of the stratum corneum with
repeated cellophane tape stripping. The control site is stripped in the same
manner. The dose should be administered to the test site in one small appli-
cation.
Phototoxicity testing procedures in humans have been published by
Marzulli and Maibach (1970), Kaidbey and Kligman (1978), and Forbes et al.
(1977) and are summarized in Table 3-4. A major difference is the time between
application and light exposure. The ideal time interval between application
and light exposure hasn't been determined. Further research is required before
specific tests can be recommended.
99
-------
Table 3-4. Phototoxicity Testing in Humans
Site Preparation
and Application
Light Exposure
Observation and Scoring
References
o
o
Topical application
on white volunteers.
(Details not given).
White volunteers
(ages 21-28) -
50 1 of test
material applied
topically
Volunteers, skin of
forearm was tapped
stripped and 0.05
ml of test material
applied.
30 minutes after application:
a. Sun (glass filtered)-
30 min.
b. Compact-arc xenon lamp -
2 min.
c. Long-arc xenon lamp - 40
min.
d. Blacklight - 60 min.
6 hr. after application Xenon
solar simulator, flux was
120 m w/can 2 for 8.5 min.,
if no reaction up 14 min.
(28.5 Joules/cm2).
5 minutes after application
Hanovia "Inspectolite"
90% UV (between 300-400 nm)
300 watts/cm2 at a
distance of 10 cm.
Irradiated 40 minutes.
4,24,48,72, & 96 hours
Positive (+) or
negative (-)
Forbes et al. (1977)
Immediately after
irradiation, 24 and
48 hours. Positive
or negative (no
grading).
24 and 48 hours
Scoring + or -
(no grading)
Kaidbey and Kligman
(1978)
Marzulli and Maibach,
(1970)
-------
4.0 PHOTOALLERGY TESTING
4.1 Animal Model
In contrast to the several species used for phototoxicity testing, the
guinea pig is considered to be the only reliable model for studying the immuno-
logic reactions of photoallergy. Harber and Shalita (1977) described a method
using guinea pigs and irradiating with either fluorescent "Sunlamp" tubes or
"Blacklight" fluorescent tubes. The procedure consists of topical exposure
and irradiation performed three times during a seven day period. Three weeks
after the last sensitizing exposure, the animals are challenged by topical
application to two sites on an area not previously exposed to the chemical.
Thirty minutes later, one of the sites is exposed to non-erythrogenic radia-
tion. All sites are scored and interpreted 24 hours later.
Other groups have published methods using the guinea pig model (Harber
et al., 1967, and Vinson and Borelli, 1966). These two methods are summarized
in Table 3-5 and are shown to be very similar; however, the rest period between
the induction phase and challenge was 7-10 days versus 21 days. The ideal
time for challenge has not been established.
4.2 Human Testing
Kaidbey and Kligman (1980) have developed a photomaximization test for
identifying photoallergic contact sensitizers. This test consists of applying
the test chemical to 25 subjects under occlusive patch for 24 hours followed
by exposure to three minimal erythema doses (MED) of solar radiation. The
irridiation consists of six exposures, twice weekly for 3 weeks. The subjects
are challenged 10-14 days later. An occlusive application is made at a new
site for 24 hours followed by exposure to 4.0 joules/cm2 of UV A irradiation
(lamp intensity, 27.0 nW/cm2). Controls include a treated non-irradiated
site and an irradiated vehicle-treated site. The reactions are evaluated 48
and 72 hours after irradiation.
101
-------
Table 3-5. Photoallergy Testing in Guinea Pigs
Induction Phase
Challenge
Light Exposure
Observation
and Scoring
Reference
o
N>
Skin clipped and depilated.
0.1 ml test material applied
3 times during 7-day
period.
Skin clipped on upper
dorsal area and 0.05 ml
test material applied
daily for 5 days.
3 weeks after induction
0.05 ml applied to
previous unexposed area.
After rest period of
7-10 days 0.05 ml test
agent applied.
30 minutes after application.
Sunlamp IxlO7 ergs/cm2
Blacklight 3xl08 ergs/cm2
Used successively.
Blacklight only for
challenge.
Time after
not given.
Sunlamp at
18 in. for
after each
Same after
dose.
application
distance of
15 minutes
application.
challenge
24 hours after
irradiation.
Scoring 0-4
24 hours after
irradiation.
Scoring 0-3
Harber et al.
1967
Vinson and
Borselli, 1966
-------
5.0 CONCLUSIONS
Phototoxicity and photoallergy testing are in an evolving status and
well-defined proven tests are not available. Yet these important toxic proper-
ties should not be ignored when assessing the toxicity of photoactive com-
pounds. The action spectrum for photosensitization is normally similar to the
absorption spectrum of the photoactive compound. Methods for phototoxicity
testing in animals and humans are summarized in Tables 3-3 and 3-4 and methods
for photoallergy testing in guinea pigs are summarized in Table 3-5. Rabbits,
mice, hairless mice, guinea pigs and miniature swine are presently acceptable
animal models for phototoxicity testing. The guinea pig is the only animal
model presently acceptable for photoallergy testing. Since extrapolation of
animal photosensitivity data to humans is often difficult, human testing is
recommend-
ed for compounds to which humans will be exposed in the presence of sunlight.
Human testing of a substance should be conducted only after testing in animals
for both photosensitivity and systemic toxicity.
103
-------
6.0 REFERENCES
Akin, F.J., A.P. Rose III, T.W. Chamness, and E. Marlow, 1979.
Sunscreen protection against drug-induced phototoxicity in animal
models. Toxicol. Appl. Pharmacol., 49, 219-224.
Forbes, P.O., F. Urbach, and R.E. Davis. 1977. Phototoxicity resting of
fragrance raw materials. Fd. Cosmet. Toxicol., 15, ,55-60.
Harber, L.C., .and R.L. Baer. 1972. Pathogenic mechanisms of drug induced
photosensitity. J. Invest. Dermatol., 58, 327-342.
Harber, L.C., and A.R. Shalita. 1977. Immunologically mediated contact
photosensitivity in guinea pigs. Adv. Mod. Toxicol., 4_, 427-439.
Harber, L.C., S.E. Targovnik, and R.L. Baer. 1967. Contact
photosensitivity patterns to halogenated salicylanilides in man and
guinea pigs. Arch. Dermatol., 96, 646.
Horiuchi, N., S. Ando, and A. Suzuki. 1978. Nippon Noson Agakkai (Jpn. J.
Rural Med.), 27(3), 450-451.
Raidbey, K.H., and A.M. Kligman. 1978. Identification of topical
photosensitizing agents in humans. J. Invest. Dermatol., 70, 149-151.
Kaidbey, K.H., and A.M. Kligman. 1980. Photomaximization test for
identifying photoallergic contact sensitizers. Contact Dermatitis, £,
161-169.
Maibach, H. I., and F.N. Marzulli. 1977. Phototoxicity (photoirritation)
of topical and systemic agents. .Adv. Mod. Toxicol., 4, 211-223.
Marzulli, F.N., and H.I. Maibach. 1970. Perfume phototoxicity. J. Soc.
Cosmet. Chem., 2_1^, 695-715.
Vinson, L., and V.F. Borselli. 1966. A guinea pig assay for the
photosensitizing potential of topical germicides. J. Soc. Cosmet. Chem.,
17, 123.
104
-------
SYSTEMIC DERMAL TOXICITY
1.0 SUMMARY
Dermal toxicity testing is performed to determine whether a substance
can be absorbed in quantities sufficient to produce systemic effects, as well
as the nature of such effects. Some of the factors that influence the degree
of irritation produced by an agent will also influence its systemic toxicity.
These include characteristics of test agents such as pH and lipid/water solu-
bility and specific.test procedures, such as abrasion of the skin and the
methods used to apply test substances. A dermal study alone will rarely be
sufficient to completely characterize the toxic effects of an agent, as it
provides information on the effects produced by only one route of exposure.
The OECD guidelines suggest testing the rat, rabbit or guinea pig. With the
IRLG guidelines, preference is given to the rabbit. The data evaluated indi-
cate that the rat is a more appropriate species to study systemic effects
after dermal exposure. This is primarily because much of the available
toxicity data resulting from tests by more conventional routes of exposure
have been obtained from the rat. If LD50 values from different routes are
compared in the rat, the relative rate of percutaneous absorption of a series
of compounds can be estimated.
A comparison of LD50 values for rabbits and rats shows that, in more
than 75% of the documented cases, the LD50's varied by less than a factor of
four, with neither species clearly showing greater sensitivity. It has also
been shown that the LD50 values were similar whether a rabbit was used for 24
hours or a rat was used for 4 hours.
Dermal toxicity studies of longer duration are limited in their prac-
ticality and cost-effectiveness. Animal restraint and patch attachment are
necessary for long periods. The assessment of systemic effects may also be
complicated by irritation or infection that can result from long term appli-
cation of tne test material. In addition, systemic effects can be adequately
determined by administration of the agent by other routes which are cost-
effective. Likewise, extensive clinical chemistry measurements and histopath-
ology studies should be selectively performed, depending on the intended use
of the substance. Inclusion of these additional measurements can be appro-
priate, however, in tests carried out by other routes.
When dermal toxicity studies are performed, particular attention should
be paid to the size of the patch, the area of skin in relation to the size of
tne animal, the degree of occlusion, and the concentration and amount of test
substances to allow development of consistent data.
105
-------
2.0 INTRODUCTION
The Testing Guidelines of the Organization for Economic Cooperation and
Development (OECD, 1981) for dermal toxicity include three tests that differ
in terms of the duration of exposure, numbers of animals and extent of
clinical chemical and histopathologic evaluation. These three tests are the
Acute, Repeated Dose (21/28 day), and Subchronic (90 day) Dermal Toxicity
Studies. The EPA originally proposed similar guidelines under the Federal
Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Toxic Substance
Control Act (TSCA). Public comment was solicited on these guidelines and has
been tabulated and examined by the EPA. No public comments were available for
consideration from the OECD and Interagency Regulatory Liaison Group (IRLG,
1981), which also have prepared guidelines.
Although a great number of comments were received, many of the issues
raised concerning the FIFRA and TSCA guidelines are being resolved by adopting
the OECD guidelines. Several issues still remaining to be clarified include
choice of species, exposure time, frequency of bleeding for hematology and
clinical chemistry, washing of insoluble compounds from the skin following
exposure, application of solid materials, use of abraded skin, application
procedures, extent of histopathology and clinical chemistry requirements,
difficulty in dose quantitations, and the circumstances or conditions that
dictate the need for long-term dermal testing. Some issues covered by public
comments are accounted for in the IRLG proposed test protocols but are not
covered by the OECD. To facilitate discussion, the various test methods are
compared for acute dermal LD50 in section 4.0 and subchronic dermal toxicity
in section 5.0.
Little information is available on comparisons of dermal LD50's, method-
ologies, and interspecies variation in toxicity. Since the skin of the rabbit
and guinea pig is usually more permeable to chemical substances than human
skin, care must be taken when using dermal toxicity data in animals to predict
potential adverse effects in man. In some respects, dermal toxicity studies
are analogous to inhalation studies. In neither cases is a fixed amount of
test substance administered to the test species as it is in an injection or an
intubation study; but instead the animal is exposed to a concentration of the
test substance. The actual dose received is a function of the amount of test
substance absorbed through the skin or lung tissue. For inhalation, the con-
centration of the chemical in the chamber is determined; for dermal toxicity,
the amount of material applied to a given surface area or the amount applied
per unit of body weight is the usual expression of dose.
106
-------
3.0 FACTORS INFLUENCING ABSORPTION AND TOXICITY
The extent to which a chemical is absorbed through the skin is deter-
mined by the physical and chemical properties of the test material and the
structural characteristics of the skin at the site of application.
3.1 Properties of Test Agents
Physicochemical properties of the test agent have the greatest influ-
ence on whether it .penetrates the stratum corneum of the epidermis which is
the major barrier of the skin. Lipid soluble compounds are normally absorbed
better than water soluble compounds. However, amphoteric compounds such as
DMSO with both hydrophobic and hydrophilic groups usually demonstrate the
greatest absorption. Other factors such as pH, pK (degree of ionization),
temperature, humidity, and molecular size will also influence penetrability.
Absorption can also take place through the follicular and sweat ducts. Inad-
vertent inhalation of a volatile test agent may contribute to the development
of toxicity, compounding the difficulty of accurately determining the actual
dose given via the dermal route (Casarett and Doull, 1980).
Although the skin is generally an effective barrier against the pene-
tration of a variety of chemical agents, many substances do penetrate to some
degree. Some of the better known examples of agents that can cause percutan-
eous toxicity are: organophosphate insecticides, chlorinated hydrocarbon
insecticides, solvents, some aromatic nitro and amino compounds, organometallic
compounds and steroids.
Because the stratum corneum acts as a barrier to passive diffusion,
steady penetration rates are proportional to the concentration gradient across
the membrane. Quantitative measurements of dermal toxicity are therefore
dependent on the amount and concentration of the test agent, and the size of
the test area in relationship to the total surface area or weight of the
animal. For example, a given weight of test substance applied to 12 of the
total surface area of an animal could be non-toxic, whereas, if it were
applied to 102 of the surface, toxicity might result as total uptake would be
proportional to surface area. The same dosage calculated on a mg/kg basis
could give disparate results. These variables should be controlled rigidly so
that valid conclusions and comparisons can be drawn from toxicity studies. As
with dermal irritation studies, control of the patch test unit (patch size,
material, tape, degree of occlusion.) is critical in keeping variability in
dermal toxicity studies to a minimum.
3.2 Species Differences
Bartek et al. (1972) compared the percutaneous penetration of 6 test
chemicals in rats, rabbits, miniature swine, and man by measuring the percent
of radioactivity excreted in urine for 5 days after treatment with labeled
compounds. The agents studied were haloprogin, N-acetylcysteine, cortisone,
testosterone, caffeine, and butter yellow. The percent of the dermal dose
(4 ug/cm^) divided by the percent of an i.v. dose recovered in the urine was
used to estimate the amount of test material absorbed through the skin. The
107
-------
data presented in Figure 4-1 indicate that skin permeability increased in the
following order: man, miniature swine, rat, and rabbit. The swine, there-
fore, had permeability characteristics closest to man. With the exception of
caffeine, there was a good correlation between the ether/water partition
coefficients and percutaneous absorption, implying that this could be used to
predict the absorption potential of a compound.
110
, 100
3
u
. oc
ff
t
-------
Table 4-1. Acute Dermal LD50 (ml/kg) of Test Compounds in the Rabbit
and Guinea Pig
Rabbit, Guinea Pig, (Abdomen) Guinea Pig, (Back)
Compound abraded skin intact skin abraded skin
Acetone
Benzene
Carbon tetrachloride
Butylamine
Aniline
Butoxyethanol
Acrylonitrile
Para th ion
9.4
9.4
9.4
1.5
0.82
0.68
0.28
0.07
9.4
9.4
9.4
0.58
1.29
0.23
0.46
0.60
9.4
9.4
9.4
1.5
2.15
0.30
0.84
0.80
Adapted from Roudabush et al (1964)
In a number of cases the rabbit LD50 parallels the rat L050 and in many
instances they agree quite well. Weil et al. (1971) showed for 33 unspecified
compounds that the rat LD50 after 4 hours correlated very well with the rabbit
LD50 after 24 hours. When data from the Registry of Toxic Effects of Chemical
Substances (RTECS) data base for 72 compounds is considered (Appendix) the
rabbit dermal LD50 differs by a factor of two or less in 42 instances (greater
than 65%) and differs by three or less in a total of 58 cases (greater than
80%) and overall by a factor of five or less in 63 cases (87%). Most recent
price quotes for acute dermal toxicity determinations indicate that a rabbit
test is 2.5 to 3 times more costly than a similar protocol in rats. In addi-
tion, the smaller size of the rat would allow testing of less material, an
important consideration if the compound under test is part of a small pilot
batch. These facts and the argument made by the National Academy of Sciences
(1977) support the use of the rat over the rabbit. "Although there is always
risk in extrapolation from animals to humans, it is usually safe to presume
that substances with lower dermal LD50s in animals will be potentially more
toxic to humans than those with higher LDSO's. On the other hand, predictions
of dermal versus oral toxicity in humans are more difficult, especially if the
dermal and oral measurements are made in different animal species. Therefore,
there is an important advantage in having both tests done with the same
species, e.g., the rat."
3.2.1 Recommendations
In summary, the results of the preceeding investigations indicate that
no one animal species will accurately predict the dermal effects of all classes
of chemicals in humans. Although the guinea pig and rabbit are generally more
sensitive than man, a reasonable approximation of expected toxicity can be
obtained by use of either species. Tne rat also appears to be a valuable test
animal for dermal studies because the availablity of toxicity data by other
routes of exposure will provide information on the relative rate of percutan-
eous absorption. Use of monkeys is impractical for routine testing. The
109
-------
choice of species for dermal toxicity studies, therefore, should not be
rigidly defined.
3.3 Regional Variation in Absorption
Because of functional and structural differences, the regional varia-
tion in the percutaneous absorption of parathion, malathion, and carbaryl was
investigated by Maibach et al. (1971). Using humans, a'test concentration of
4 ug/cm^ was applied to the forearm, palm, abdomen, doraurn of the hand,
scalp, angle of the jaw, postauricular area, forehead, intertriginous axilla,
or the scrotum. Quantitation was based on the 5 day collection of urine with
determination of radioactivity. These results, depicted in the following
table, demonstrate that considerable variation in absorption rates exist for
different chemical structures and regions of the body. Results are shown in
Table 4-2.
Table 4-2. Effect of Anatomic Region on Absorption of Topically
Applied l^C Pesticides in Humans
Percentage Dose Recovered in Urine
Anatomic Region
Forearm
Palm
Foot, ball
Abdomen
Hand, dor sum
Fossa cuubitalis
Scalp
Jaw, angle
Postauricular
Forehead
Ear canal
Axilla
Scrotum
Parathion
8.6
11.8
13.5
18.5
21.0
28.4
32.2
34.0
34.0
36.3
45.6
64.0
101.6
Malathion
6.8
5.8
6.8
9.4
12.5
23.2
28.7
Carbaryl
73.9
69.9
Adapted from Maibach et al (1971)
3.4 ^£ Vitro vs. La Vivo Absorption
Franz (1975) conducted an £n vitro percutaneous absorption study with
12 chemicals and compared these results with those from previously conducted
IP. vivo studies (Feldman and Maibach, 1970). The in. vitro studies used human
abdominal skin taken at autopsy; the in vivo experiments were conducted on the
forearm of volunteers. Although quantitative agreement between the Jjn vivo
and in vitro studies was not good, there was significant (P less than 0.01)
correlation of the relative permeability trends, salicylic acid and caffeine
being the only exceptions. These results are summarized in Table 4-3. Franz
concluded that the study of percutaneous absorption in excised human skin
gives information which is relevant to its living counterpart. It is apparent
however, that these large differences in quantitative rates should be taken
110
-------
into account when comparing interspecies data. The in vitro skin is in a much
different environment, not being perfused by oxygenated blood on the basal
aide, and displaying differences in metabolism as absorbed compound/metabolites
are not swept away into the bloodstream. Questions of effective skin thickness
and epidermal metabolism as well as the temperature at which the test is per-
formed make jin vitro tests for compound absorption difficult to interpret.
Table 4-3. Total Absorption In Human Skin
Compound In Vivo* In Vitrob
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Hippuric acid
Nicotinic acid
Thiourea
Chi orampheni col
Phenol
Urea
Nicotinamide
Acetylsalicylic acid
Salicylic acid
Benzoic acid
Caffeine
Dinitrochlorobenzene
0
0
0
2
4
6
11
21
22
42
47
53
.2
.3
.9
.0
.4
.0
.1
.8
.8
.6
.6
.1
+
7
7
7
7
+
+
+
+
7
+
7
0.1
0.1
0.2
2.5
2.4
1.9
6.2
3.1
13.2
16.5
21.0
12.4
(7)
(3)
(3)
(6)
(3)
(4)
(7)
(3)
(17)
(6)
(12)
(4)
1
3
3
2
10
11
28
40
12
44
9
27
.2
.3
.4
.9
.9
.1
.8
.5
.0
.9
.0
.5
(0.
(0.
(2.
(1.
(7.
(5.
(16
(17
(2.
(29
(5.
(19
8
7
4
0
7
2
>
»
3
»
5
>
, 2.7) (15)
, 8.3) (19)
, 5.5) (52)
, 5.7) (12)
, 26) (7)
, 29) (22)
65) (21)
49) (14)
, 23) (10)
53) (18)
, 20) (17)
33) (18)
Percentage of applied dose, mean +_ standard deviation. The figure in
brackets is the number of subjects studies.
^Percentage of applied dose, median with 95% confidence interval given
in parentheses.
Adapted from Franz (1975) and Feldman and Maibach (1970)
3.5 Use of Abrasion
Because abrasion removes or damages the stratum corneum, it is generally
agreed that this technique will increase the toxicity of a chemical substance.
This has been clearly shown by Frosch and Kligman (1977) to be valid for irri-
tation in their development of the scarification index (the concentration of a
substance necessary to produce irritation on normal versus abraded skin). An
increase in percutaneous absorption of chemical agents after stripping the
stratum corneum has also been demonstrated (Blank et al., 1975; Bettley, 1963).
An increase in absorption would produce a greater concentration of an
agent at target organs and thus, increase its toxicity. If a chemical is
likely to come in contact with damaged skin, the use of abraded skin in animal
studies is justified. However, as this represents an unusual condition of the
skin, the use of abrasion in routine testing should probably not be required.
Ill
-------
3.6 DERMAL LD50 VS ORAL LD50
Gaines (1960) compared the acute oral and dermal LD50 values of a
variety of chlorinated hydrocarbon, organophosphate and miscellaneous pesti-
cides in the rat. He noted that, in general, female rats were more sensitive
than males to organic phosphate compounds. There was a much closer relation-
ship between dermal LDSO's and the occurrence of occupational poisoning than
between oral LDSO's. These comparisons are shown in the following table.
Table 4-4. Relationship Between Toxicity and Safety of Pesticides
Compound
LD50 values
in male rats
(mg/kg)
Oral
Dermal
Kind of exposure
associated with
systemic
poisoning
Greatest
severity of
occupational
poisoning
DDT
Lindane
Dieldrin
Methyl parathion
Guthion
Parathion
Thimet
Phosdrin
11.3
88
46
18
13
13
2
6
2510
1000
90
65
220
21
6
5
Ingestion
Ingest ion
Ingestion and
occupational
Occupational
Occupational
Ingestion and
occupational
Occupational
Occpational
__
Severe
Mild
Mild
Severe
Severe
Severe
Adapted from Gaines (1960)
A comparison of LD50 values by 2 different routes will give an approxi-
mation of the relative penetrability of chemicals through the skin. In the
study by Weil et al. (1971), correlation between dermal LD50 and oral LD50
values of 30 substances was poor, indicating that the substances were absorbed
at different rates.
Dermal and oral LD50 values for a number of chemicals in a variety of
species are reported in the Registry of Toxic Effects of Chemicals (RTECS).
These have been abstracted and are listed in the Appendix. Dermal LD50 values
are greater or equal to the oral LD50 in over 84% of the cases. The greater
toxicity via the oral route is also evident in many other mammals, and birds
as well as rats, rabbits, and humans.
112
-------
4.0 ACUTE DERMAL TOXICITY TESTING
The acute dermal toxicity test is performed with young adult animals.
The use of rabbits, rats and guinea pigs is recommended by OECD while IRLG has
chosen rabbits as the preferred species, because of ease of handling, size and
skin permeability (Table 4-5). Other species may be used in either test but
require justification. The FHSA protocol for acute dermal toxicity testing
specifies that the intact and abraded skin of rabbits be used. Only the OECD
and IRLG protocols allow the use of a limit test; if a dose of 2 g/kg or
2 ml/kg does not lead to mortality, no further testing of acute dermal toxicity
may be necessary. OECD requires intact skin for the acute limit test while
IRLG requires abraded skin. This may significantly increase the sensitivity
of the IRLG protocol. The acute oral limit test is 5 g or 5 ml/kg. Dermal
application is limited by the volume of material that can be applied uniformly
and the available surface area. To apply greater than 2 g/kg body weight would
in many cases involve repeated application or covering much greater than 10%
of the animal's surface with compound. Interpretation and quantitation of the
LD50 would therefore, be quite difficult. If a substance is toxic in the limit
test, acute dermal toxicity testing will still be necessary. However, if toxi-
city is not demonstrated a considerable amount of cost and time will be saved.
4.1 Duration of Exposure
Weil et al. (1971) determined the dermal LD50 values of 30 unspecified
substances in rats and rabbits after 4 and 24 hours of skin contact as well as
for 33 additional substances using only the 4 hour rat and 24 hour rabbit
exposure. The procedure used was a modification of the Draize method (1955).
The LD50 value at the shorter time of exposure correlated very well with the
longer time in both species. Moreover, there was a good correlation of the
logarithmically adjusted LD50 values for the rat 4-hour and rabbit 24-hour
exposure, indicating that either test method will provide adequate information
to assess the toxicity of a substance.
The authors noted that the 4-hour skin test was more realistic in
simulating expected exposures to man. The data obtained from the 4-hour rat
study appears to be as useful as that from the 24-hour exposure of the rabbit
in determining the relative toxicity to animals. This observation should be
verified with various classes of compounds to establish its generality. This
study does not address, for instance, other acute effects besides lethality.
The information provided by these investigators, however, seems to be adequate
justification for the use of a 4-hour exposure, and also indicates the useful-
ness of the rat as an experimental animal.
113
-------
Table 4-5. Comparison of Guidelines for Acute Dermal Toxicity
OECD
IRLG
EPA
Animals
Species
Sex
Age
Weight
Acclimation
Study Design
No./Group 5/sex
Limit Test
Dose Levels
Rabbit, rat or guinea
pig; justify others
Male and female
Females should be
nulliparous and
not-pregnant
Young adult
Rabbit - 2.0-3.0 kg
Rat - 200-300 g
Guinea Pig - 350-450 g
At least 5 days
Maximum Dose
Controls
2 g/kg on intact skin
of 3 males and 3
females
If not mortality,
further testing not
necessary
At least 3 levels
spaced appropriately
to produce test groups
with a range of toxic
effects and mortality
rates. Permits LD50
determination and dose
response curve.
Not mentioned
Not required
Albino rabbits preferred
Justify others
Same
Females should not be
pregnant
Same
Rabbits - Same
Not applicable
Not applicable
Not mentioned
4/sex recommended
2 g/kg on abraded skin
of 3 males and 3
females
Same
Same
Same as IRLG
Same
Same as IRLG
Same
Rabbits - Same
Same as IRLG
Same as IRLG
Same as IRLG
Same as IRLG
5 g/kg on abraded
skin of 4 males'
and 4 females
Same
Same
Maximum dose 2 g/kg
Same
Not mentioned
Same
114
-------
Table 4-5. Comparison of Guidelines for Acute Dermal Toxicity
(Continued)
OECD
IRLG
EPA
Influence of vehicle Vehicle should be non-
on skin penetration
should be considered
Duration of
Exposure
Condition of
test
substance
Condition of
test area
Preparation
24 hours. Then
removal of residual
with water or other
appropriate solvent
Liquids applied
undiluted
Solids pulverized
and moistened with
water or other vehicle
Intact skin
Not less than 10%
of body surface
Apply to dorsal
surface
Clip fur shortly
before test
irritating and of
known low toxicity.
Consider effect on
absorption
Same
Liquids - Same
Solids - As paste with
water or saline
Same
Same
Same
Clip fur 24 hours prior
to testing
Acute test of
vehicle first if
toxic potential is
not known. Should
be non-toxic and
not modify toxic
response of test
substance
Same
Liquids - Same
Solids - Same as IRLG
Same
Same
Apply in a band
around the trunk
Same as IRLG
If shaving or chemical No other methods mentioned Same as IRLG
depilation used, must
be 24 hours prior to
test
Retainer
Porous gauze dressing Same
or nonirritating tape
Dressing should be
covered
Cover with impermeable
material in a semi-
occlusive fashion
115
Wrapping material
(type not specified)
See above
-------
Table 4-5. Comparison of Guidelines for Acute Dermal Toxicity
(Continued)
OECD
IRL6
EPA
Study Conduct
Husbandry
Observation
Duration
Evaluation
Necropsy
Histo-
pathology
Conditions of Reference to DHEW (NIH)
temperature, humidity, 74-23
lighting, feeding,
and caging specified
Frequently first day, Same
twice a day there-
after, at least 4 hrs
apart (morning and late
afternoon)
At least 14 days Same
Record onset, Same
severity and
persistence of signs
Irritation not graded Irritation noted and
graded at 24 and 72 hours
Weigh initially,
weekly and at death
Consider for all
animals where
significant signs of
toxicity are observed
Consider for organs
with evidence of gross
pathology (in animals
surviving 24 hr or more)
Same
All animals that die
during test. Terminate
animals if no further
testing planned or if
results are to be used
for labeling purposes
Not mentioned
Similar to IRL6
Same
Same
Same, plus
nature of sign
Irritation noted
and graded - no
time specified
Same
Same
Not mentioned
Reporting of General requirement
Results sufficient to make
independent evaluation
Detailed format specified Same as IRLG
Requirements designated in Same as IRLG
GLP's
116
-------
5.0 LONGER TERM DERMAL TOXICITY TESTING
The requirement to test an agent for longer periods should be based on
the type of exposure expected in man. Substances such as sun screen lotions
or insect repellents that will be applied to a large portion of the body for
extended periods of time should undergo longer term dermal toxicity studies.
Agents that will only be in contact with the skin rarely and for a brief time
probably need not be tested as rigorously.
Pre-chronic studies are often useful in setting doses for chronic study,
but the data generated must also be justified in terms of time and cost. Once
the dermal LD50 of a substance is determined, the long term effects of an agent
might be better established by conducting a subchronic or chronic toxicity
study by another route of exposure because such studies will probably still be
required. Systemic effects after dermal exposure also may not occur due to
insufficient percutaneous absorption. Limit tests on intact skin are therefore
included for subchronic dermal testing allowing the possibility of testing only
one high dose group for the full exposure period if no effects are observed at
1 g/kg.
Three different tests have been included in OECD guidelines: a 21 or 28
day repeated dose, a 90 day subchronic, and a chronic test (Table 4-6). For
subchronic dermal toxicity studies the OECD, IRLG and EPA all recommend that
ideally, exposures should be performed 7 days/week. This may not be practical
because the test animal will require clipping of hair approximately once a week
and therefore there will be no proper period for healing of any associated and
often imperceptible clipping injury before the next week's dosing schedule is
begun. Daily exposures will also require increased expenditures and staffing
by the laboratory for weekend dosing. Testing during the weekend may not pro-
vide enough additional toxicological information to justify the added expense.
For these reasons, OECD's guideline that dosing be performed 5 days/week with
clipping performed on a non-dosing day seems most reasonable. An exposure
schedule of 5 days per week also simulates many industrial exposures. Although
this procedure might allow recovery over the weekend, this effect will be
minimized if maximum tolerated doses are used. The weekend free of exposure
may also, however, permit the loss of tolerance built up during the week.
A comparison of acute and subchronic dermal toxicity methods shows
significant procedural differences. All of the guidelines for acute testing
specify a 24 hour exposure period. For subchronic testing, exposure is for
six hours a day, five to six days a week; the duration of exposure is 21, 28
and/or 90 days depending on which guidelines are followed. For acute studies,
observations are made frequently the first day and twice daily, thereafter,
for a total of at least 14 days. Observations are made daily throughout the
period of exposure for subchronic testing. At evaluation for acute testing,
the IRLG and EPA specify necropsy of all animals that die during the test but
assessment of histopathological changes is not required. According to the
OECD, necropsy should be considered where significant signs' of toxicity are
observed and histopathological studies should be considered for organs with
evidence of gross pathology in animals surviving 24 hours or more. For sub-
chronic toxicity testing, all of the guidelines specify that all animals should
be necropsied, and histological evaluation be performed on all animals in the
117
-------
high dose and control groups. The guidelines for subchronic dermal toxicity
testing differ from those for acute testing by specifying that extensive
clinical tests be performed in some cases. These include hematology, blood
chemistry, and ophthalmological measurements.
118
-------
Table 4-6. Comparison of Guidelines for Subchronic Dermal Toxicity
Subject
OECD
IRLG
EPA
Species
Sex
Age/weight
Rat, Rabbit or
Guinea Pig
Both sexes
Non-pregnant &
nulliparous
Rabbit: 2-3 kg
Rat: 200-300 g
G.P.: 350-450 g
Albino Rabbit or Rat
Same
Same (Rabbit)
Same (Rat)
Albino Rabbit
(preferred); others
accepted if
justified
scientifically
Same as IRLG
Same (Rabbit)
No. of
Animals
10M/10F per dose
Dose
Levels
Control
Limit Test
At least 5M/5F
Rabbits or 10M/10F
Rats
At least 3 plus
extra high dose
group on 90 day
study (sentinnel)
Control or vehicle
control
If toxic effects
produced at 1 g/kg
or higher, no
further dermal
required
At least 3
highest dose non-
irritating but
effective
Same
Maximum quantity of a
test substance plus
vehicle to be applied
should not exceed
2 g/kg. Use abraded
and non abraded sites
At least 5M/5F per
dose when pre-
liminary tests
(i.e. oral LD50,
dermal LD50 or
subchronic oral)
indicate no sex
differences in
response to test
substance. If
sex difference are
noted or such
information is
lacking, at least
8M/8F per dose
At least 3
Same
10M/10F to a single
exaggerated dose
which is maximum
feasible that does
not casue severe
local irritation.
119
-------
Table 4-6. Comparison of Guidelines for Subchronic Dermal Toxicity
(Continued)
Subject
OECD
IRLG
EPA
Preparation
of Animals
Application
of test
substance
Area
Duration
Type of
Covering
Clip fur
Apply as thin and
uniform film.
Uniformly over at
least 10% of the
body surface
Liquid substances
generally not
diluted .
Not stated
Ideally 7 days/
week at least 6
hrs/day for 21/28
days or 90 days,
5 day/week
acceptable
Porous gauze dress-
ing; non-irritating
tape; prevent
animal from ingest-
ing test substances
Same - skin may be
weekly
Not specified
Same, however
Decreased area allowed
Use same volume of
application for all
test doses
Same
Same as OECD, exposure
for 90 days only -
washing permitted
Semi-occlusive
with impermeable
material (i.e.
plastic or rubberized
cloth)
Need not exceed
2 g/kg. If severe
dermal irritant at
level of human or
anticipated human
exposures, it need
not be subjected to
a 90-Day Subchronic
Dermal test.
Same as OECD
Same as OECD
Same as OECD
Dilution allowable
if compound used
in diluted form
Calculate
concentrations per
unit area of skin
Same as IRLG
plus 21 day test
Same as OECD
120
-------
Table 4-6. Comparison of Guidelines for Subchronic Dermal Toxicity
(Continued)
Subject
OECD
IRLG
EPA
Observations Daily
Body weight and
food consumption
weekly
No observation
of irritation
Immobilization No
Hematology
and Blood
Chemistry
Before study starts
and at termination
Hematocrit,
hemoglobin,
total/differential
leucocyte count,
platelet count,
erythrocyte count
All animals
at end of exposure
Calcium,
inorganic
phosphorous,
chloride,
potasssium,
sodium magnesium,
blood pH, BUN,
fasting glucose,
albumin, bili-
rubin. Consider
lipids, hormones,
Same
Body weight weekly
Note Irritation
weekly
No
Housing Individual caging Same
Clinical Laboratory Tests
Rabbits before study
begins; rabbits and
rats at end of study
Same as OECD plus
plus intrinsic and
extrinsic clotting
potential
Same
Rabbits before,
at 6 weeks and at end
of study. Rats at end
of exposure only
Same plus alkaline
phosphatase,
lactic dehydrogenase,
direct and indirect
bilirubin, gamma
glutamyl trans-
peptidase ornithine
decarboxylase. Same
as OECD plus carboxy-
hemoglobin. No
methemoglobin
Same
Body weight
weekly (if
affected, food
consumption
weekly)
Note for 21 days
No
Same
Before and at
end
Sames as OECD
plus sed. rate
and RBC count
High dose and
control (initally)
Same as OECD
Same plus blood
C02, magnesium,
sorbitol dehydro-
genase, glucose 6
phosphatase,
isocitrate dehydro-
genase, ornithine
transcarbamylase,
lactic dehydro-
genase (plus
isozymes) -
121
-------
Table 4-6. Comparison of Guidelines for Subchronic Dermal Toxicity
(Continued)
Subject
OECD
IRLG
EPA
Ophthalmo
logical
and base balance,
methemoglobin,
'cholinesterase
for 21/28 day test
Same as IRLG
for 90 day teat
90 day test only
At least at start and
end of test
cholinesterase if
organophosphate or
carbamate at
beginning, twice
during study and
at end in red
blood cell.
Examine brain
cholinesterase at
termination. Same
as IRLG
Not required
Gross
Necropsy
Organs
weighed
Histo-
pathology
All animals
Liver, kidney,
adrenal, testes
All animals in
high dose and
control.
21/28 days:
Examine treated and
untreated skin
liver, kidney, plus
target organs
Examine lower
doses in target
organ
90 days:
Same
Same as OECD
Same
Same as OECD requires
for 21/28 day study
plus thyroid, spleen,
testes/ovaries, bone
marrow, representative
lymph node, salivary
gland, small intestine,
urinary bladder, thymus
adrenal, lung, gall
bladder plus
Brain (3 levels);
spinal cord (2
levels); sciatic
nerve; pituitary;
thyroid/parathyroid,
trachea, esophagus,
stomach, pancreas,
aorta, prostate,
Same
Same as OECD
Same
Same as OECD
requires for
21/28 days
Routine exam of
brain and spinal
cord only if in-
dicated by
clinical signs or
CNS effects
122
-------
Table 4-6. Comparison of Guidelines for Subchronic Dermal Toxicity
(Continued)
Subject OECD IRLG EPA
uterus, heart,
large intestine,
sternum
Same as IRLG plus
accessary genital
or gan s, du odenum,
jejunum, ileum,
caecum, colon,
rectum, and 3 levels
of the spinal cord
123
-------
6.0 EXAMINATION
6.1 Irritation Assessment
In both the dermal LD50 and subchronic dermal toxicity, IRLG includes
the grading of skin irritation as part of the observation for clinical signs
30 minutes following removal of the test patch and again at 72 hours after
compound application. The grading system is similar to the Draize (1955) scor-
ing systems but also includes noting the occurrence of severe eschar formation
and/or corrosion (see Dermal Irritation). The OECD guidelines do not include
recording of irritation. Many public comments made in response to the FIFRA
and TSCA Test Guidelines recommend that the acute dermal toxicity and irrita-
tion tests be combined. The consideration of this recommendation should be
given high priority, because if the acute test is properly designed, it can
provide information relevant to the irritation potential. This would, of
course, only apply when the rabbit is used to determine acute dermal toxicity.
Inclusion of irritation assessment in the dermal LD50 protocol would
require further description of the patch test unit. Currently, no information
on the patch size, concentration of test substances or conditions of the skin
is given in any of the proposed guidelines. Without this information, some
caution must be exercised in drawing conclusions on the relative toxicity and
irritation of the various test agents.
6.2 Histopathology and Clinical Measurements
Both the OECD and IRLG suggest a "battery" of clinical chemical para-
meters and tissues for histopathological evaluations. Extensive monitoring of
clinical chemistry and hematological parameters has, historically, been
associated with the, need to assess human clinical health status. Recently,
the trend has been to include a battery of clinical biochemistry tests (appli-
cable for humans) in subchronic and chronic animal studies. These tests are
being proposed regardless of the purpose of the study, test chemical, economic
impact, or significance and usefulness of the data obtained. Although these
tests are an indirect assessment of cellular status, proponents assert that
they do reflect the function of living cells. Histopathology is seen as an
evaluation of the structure of dead and artifically preserved cells. However,
most of the available literature indicates that histopathology is more useful
than clinical chemistry in evaluating the toxic effects of chemical substances.
6.2.1 Clinical Chemistry (derived in part from Public Comments on EPA
Guidelines)
Numerous studies have been published illustrating the variability of
clinical chemistry determinations and the ultimate reliance on histopatho-
logical examination to identify treatment-related responses (Buttar et al.,
1076; Chow et al., 1977; Verschuuren et al., 1976; and Oser et al., 1975). The
existence of a correlation between serum enzyme levels and a histopathological
effect has long been a subject of discussion. A summary of relevant statements
received by EPA are presented below.
124
-------
Korsrud et al. (1972) found that microscopic examination was more sensi-
tive than enzyme analysis. According to the latter authors: "The sensitivity
and the degree of quantitation of responses varied between methods. Minimal
histologic changes were seen at a low CCl^ dose (0.0125 ml/kg). Alterations
in the other criteria were not observed at this dose" (emphasis added). The
sensitivity of several serum enzymes for the detection of induced liver damage
in rats was subsequently reported by Korsrud et al. (1973). The authors con-
cluded that histological examination of the liver was useful for assessing the
sensitivity of serum parameters since histological evidence of damage was
detectable at lower doses of each toxicant than were serum changes.
An inhalation study on peroxyacetyl nitrate in rats (Kruysse et al.,
1977) revealed that the 4.1 ppm level produced histopathological changes in
the respiratory tract without any significant adverse effects on the hemato-
logical parameters monitored.
Dose-dependent effects of coumarin or butylated hydroxytoluene were
examined by Nievel et al. (1976) with the following conclusion: "It appears
that studies on any individual, randomly selected enzymes and biochemical
parameters are insensitive and possibly unsuitable indicators of toxicity
unless an overall change in the pattern of blood chemical pathology is cor-
related with the early molecular changes underlying nucleic acid and protein
synthesis that are detectable within hours of the administration of many
compounds of relatively low toxic potential." They continued, "Our evaluation
of the biochemical parameters widely used in the assessment of and monitoring
toxic damage of the liver in the course of many safety assessment programes
therefore indicated no reliable correlation between these analytical chemical
changes and toxicity."
However, although the use of isoenzyme determination might be useful in
noninvasive measurement of organ damage in a more specific fashion, histopath-
ology once again was more sensitive. Cornish et al. (1971) reported that
"although the use of isozyme patterns,...have become relatively common as an
aid in clinical diagnosis, the technique has not as yet been widely used in
toxicology". These investigators reported the markedly different pattern seen
in response to liver and kidney damage in rats, but cautioned that preparation
of isozyme assays required that "conditions of blood collection and treatment
of samples be uniform and carefully controlled". Ttie sensitivity of isozyme
patterns compared with the degree of morphological damage to the liver or
kidney of rats was reported by Grice et al. (1971). Generally, advanced
degenerative changes, including necrosis, had occurred in both these tissues
before enzyme alterations were seen.
By the time that function tests show a significant variation from the
control range, a very large amount of histologically visible damage has usually
occurred. Our experience on the Carcinogenicity Testing Program within the
National Toxicology Program also suggests that extensive clinical chemistry
measurements do not add to the information provided by histopathological deter-
minations. Several special protocols for halogenated and aromatic compounds
failed to demonstrate sufficient sensivity. Histopathology, on the other hand,
detected lesions at 1/2 to 1/4 the dosage necessary to demonstrate liver and
kidney tissue damage by clinical chemistry measurements.
125
-------
Clinical chemistry and histopathology studies should be considered com-
plementary. At times one is more sensitive and in other instances the other is
more sensitive. Data from one may also give clues as to what to look for in
the other type of investigation. Histopathology documents lasting or irrever-
sible changes quite well, but transiet effects may be shown better by clinical
chemistry if the right sample is taken at the right time.
In summary it seems evident that an extensive battery of clinical
chemistry measurements should not be required for all substances. However,
some agents that exert their toxicity by inhibition of an enzyme, cholines-
terase inhibitors for example, should be monitored by appropriate techniques.
The evidence suggests that histopathology can serve as the principal method
for the detection of tissue damage with clinical chemistry measurements being
utilized when appropriate.
6.2.2 Hematology
The effectiveness of the normal routine hematological evaluations in
detecting slight toxicological cellular alterations apparently is also limited
in comparison to histopathological examination. Van Logten et al. (1974)
reported that no striking effects on hematological or biochemical parameters
were seen, except for a doubling of the percentage of neutrophil granulocytes
in the highest dosage group in rats fed sodium bromide for 90 days. However,
histopathologically, a dose-related disturbance of the endocrine system and
some of its target organs was found. The results of a chronic study on
tetrasul in rats was published by Verschuuren et al. (1973) in which the
hematological parameters were generally comparable to control, whereas micro-
scopic alterations were observed in the liver and thyroid. Furthermore, in
short-term comparative studies on tetrasul in six animal species, Verschuuren
et al. (1973) found that histopathological changes in the liver and the induc-
tion of microsomal liver enzyme activity were the most sensitive parameters.
No statistically significant dose-related changes in the hematological para-
meters were observed.
Histological examination was also shown to be a better indicator of
toxicity than hematological determinations when hamsters, rats and rabbits
were exposed to acrolein (Feron et al. 1978). The investigators found that:
"Hematological values in rats and rabbits were not affected by acrolein, but
in Hamsters, females of the top dose group showed statistically significant
increases in the number of erythrocytes, packed cell volume, hemoglobin
content, and lymphocytes accompanied by a decrease in the number of neutro-
philic leucocytes. All enzyme activities were within normal ranges and no
statistically significant differences occurred between tne various test groups
and tne control group" (emphasis added). It was further reported that marked
histopathological changes were seen in different portions of the respiratory
tract of all animals at the top dose. Hematological measurements, however,
may detect changes occuring in the bone marrow and spleen.
6.2.3 Recommendations
In humans and veterinary clinical cases, histopathology is not always
possible and clinical chemistry and hematology studies may be effective
126
-------
diagnostic tools. However, in animal toxicity studies, there is little support
for requiring extensive clinical determinations as a screening procedure for
possible toxic manifestations. The bulk of the data indicates that histo-
pathology is the best method for the detection of adverse effects for most
chemicals. In certain specific instances, however, clinical chemistry measure-
ments and hematology studies will be the only means of monitoring toxicity.
127
-------
7.0 ADDITIONAL PUBLIC COMMENTS
The following additional topics appeared in the public comments on
proposed EPA guidelines for dermal toxicity studies and deserve consideration.
o The requirement for measurement of food consumption is generally
more applicable for oral toxicity tests where the diet contains
the test substance. In a dermal study, if weekly weighings
indicate normal growth, there should be no need to measure food
consumption.
o Many comments emphasized that preliminary studies might provide
information that should lessen the requirements for acute and
subchronic dermal studies. No demonstrable toxicity in a limit
test (maximum dose) should eliminate the need for extensive test-
ing at lower doses. This can either be due to a lack of absorption
or a lack of systemic toxicity. Detoxification by the skin is also
a possibility but is less likely. Likewise if no significant his-
topathologic changes occur at the highest dose, the requirements
for further analysis at lower doses should be dropped.
o Several comments were made that rigorous requirements for dermal
studies were unnecessary, costly, and provided little new informa-
tion if studies by more conventional routes of administration had
been previously conducted. Specific reference was made to target
organ damage and information derived from clinical chemistry and
histopathology.
o Other commenters felt that the "shot-gun" approach for clinical
laboratory tests was not supported by the scientific literature
and experience. They felt that there was no scientific basis for
the specification of 14-16 clinical measurements.
o Pre-test bleeding was criticized as providing no useful information
and being traumatic. Untreated or vehicle treated groups with
large numbers of animals satisfy the requirement for control data.
128
-------
8.0 CONCLUSION
Because of differences in permeability, and other variables such as the
area of the patch and the amount and concentration of the test substance, it is
extremely difficult to quantitatively predict the dermal effects of a chemical
in man even after extensive animal studies. The same quality of information
could be obtained by determining the relative rate of percutaneous absorption
of a substance and integrating this information with toxicity studies by other
routes of administration.
Sufficient scientific evidence has been assessed that indicates that no
single species will accurately predict the dermal effects of all classes of
chemicals in humans. The guinea pig and rat have been evaluated as possible
alternatives to the rabbit with favorable results. A definite advantage in
using the rat in dermal toxicity study is that much more information by other
routes of exposure is available and costs are lower. A drawback is the diffi-
culty in restraining rats and maintaining the patch for the desired length of
the test. Skin permeability for both species is usually greater than in man,
thus allowing a conservative extrapolation. A comparison of L050 values indi-
cates that for most chemical substances there is good agreement between the 2
species.
All of the variables that influence the reproducibility and accuracy of
irritation studies are involved and further complicated in dermal toxicity
studies. An area of critical concern is the patch test unit: The patch size,
material, type of tape, and degree of occlusion can influence the results of a
toxicity study. Standardization of the method of applying the patch, degree
of occlusion, and the amount and concentration of test substance is essential
if meaningful interpretations are to made.
The concentration of the test substance and the area of application in
relation to the total surface area or body weight of the animal can also
influence the outcome of a study. If the animals are all within a close range
of body weights, a standard size patch applied to the same surface area will
eliminate some problems. If the animals vary in size this must be taken into
account when deciding on the test area and the size of the patch to be used.
Whenever possible, the same concentration of test substance for the various
dose levels should be used.
The use of abraded skin in dermal toxicity studies does not seem to be
justified unless the agent is expected to come in contact with damaged skin.
Non-uniformity of abrasive techniques and difficulty in interpreting results
should rule out this procedure for routine testing.
Since some test substances are not removed simply by washing the skin
with water, various suggestions on how to remove the agent without causing
injury to the skin have been made. If exposure in humans is expected to be
continuous because of the difficulty in removing materials such as glues or
dyes, animal experiments should be designed to simulate this condition (i.e.,
the excess compound should be removed if possible by washing, but residues
left on skin should serve as a model for actual exposure).
129
-------
The frequency of blood sampling and the extent of hematological and
clinical chemistry testing should be selective rather than requiring that
every blood parameter be determined. Unnecessary bleeding of animals can be
detrimental and could theoretically alter the outcome of the test. Cardy and
Warner (1979) reported a severe weight gain depression when rats were regular-
ly bled. Also, the value and sensitivity of hematological examination has been
questioned. Evidence has been presented that suggests that histopathological
examination will demonstrate tissue damage at lower doses before it is detect-
able by clinical studies. This is not to say that hematological testing has
no value, but rather that flexibility in the guidelines is necessary.
130
-------
9.0 REFERENCES
Bartek, M.J.; J.A. LaBudde, and H.I. Maibach (1972). Skin Permeability In
Vivo: Comparison in rat, rabbit, pig and man. J. Invest. Dermatol. 58,
114-123.
Bettley, F.R. (1963). The irritant affect of soap in relation to epidermal
permeability. Brit. J. Dermatol. 75, 113.
Blank, I.H., R.D. Griesemer, and E. Gould (1957). The penetration of an
anticholinesterase agent (SARIN) into skin. J. Invest. Dermatol. 29, 299.
Buttar, H.S., E.A. Nera, and R.H. Downie (1966). Serum enzyme activities
and hepatic triglyceride levels in acute and subacute acetaminophen
treated rats. Toxicology 6, 9-20.
Cardy, R.H. and J.W. Warner (1979). Effect of sequential bleeding on body
weight gain in rats. Laboratory Animal Science 29:179.
Casarett and Doull's. Toxicology - The Basic Science of Poisons (1980) 2nd
Ed. Macmillan Publishing Co, Inc.
Chow, A.Y.K., G.H. Hirsh, and H.S. Buttar (1977). Nephrotoxic and hepa-
totoxic effects of triclosaw and chlorhexidine in rats. Toxicol. Appl.
Pharmacol. 42, 1-10
Cornish, H.H. (1971) Problems posed by observations of serum enzyme dangers
in toxicology. Crit. Rev. Toxicol. 1, 1-32
Deichmann, W.B., U.E. MacDonald, M. Coplan, F. Woods, and E. Blum (1978)
Di-(4-Aminophenyl)-Methane (MDA) - 4.7 year dog feeding study.
Toxicology 11:185-188.
Draize, J.H. (1955) Dermal Toxicity. Food Drug Cosmetic Law Journal. 10,
722-732.
Durbridge, T.C., F. Edwards, R.G. Edwards, and M. Atkinson (1976).
Evaluation of benefits of screening -tests done immediately on admission
to hospital. Clinical Chemistry 22, 968-971.
Feldman, R.J. and H.I. Maibach (1970) Absorption of some organic com-
pounds through the skin in man. J. Investigative Dermatology 54, 399-404.
Feron, V.J., A. Kruysse, H.P. Til; et al. (1978). Repeated exposure to
acsolain vapor-subacute studies in hamsters, rats, and rabbits.
Toxicology 9, 47-57.
Franz, T.J. (1975) Percutaneous absorption. On the relevance of in vitro
data. J. Invest. Dermatol. 64, 190-195.
Freundt, K.J., G.P. Liebaldt, and E. Lieberwirth (1977). Toxicity studies
on trans 1,2-dichloroethylene. Toxicology 7, 141-153.
131
-------
Frosch, P.J., and A.M. Kligman (1977) The chamber scarification test for
assessing irritancy of topically applied substances. In: Cutaneous
Toxicity, ed. Drill, V.A. New York: Academic Press p. 127-154.
Gaines, T.B. (1960) The acute toxicity of pesticides to rats. Toxicol.
Appl. Pharmacol. 2, 88-99.
Galambos, J.T., and C.E. Willis (1978) Relationship between 505 paired liver
tests and biopsies in 242 obese patients. Gastroenterology
74(6)til91-1195.
Grice, H.C., M.L. Berth, H.H. Cornish, et al. (1971) Correlation between
serum enzymes, isoenzyme patterns and histologically detectable organ
damage. Food Cosmet. Toxicol. 9, 847-855.
Interagency Regulatory Liason Group (IRLG; 1981) Testing Standards and
Guidelines Work Group. Recommended Guidelines for Acute Dermal Toxicity,
Draft Guideline for Subchronic Dermal Toxicity Tests.
Korsrud, G.O., H.C. Grice, and J.M. McLauglan (1972). Sensitivity of
several serum enzymes in detecting carbon tetrachloride-induced liver
damage. Toxicol. Appl. Pharmacol. 22, 474-483.
Korsrud, G.O., H.C. Grice, T. Kuiper-Goodman, J.E. Knipfel, and J.M.
MeLaughIan, (1973). Sensitivity of several serum enzymes for the
detection of thioacetamide-, dimethylnitrosamine-, and
diethanolalmine-induced liver damage in rats. Toxicol. Appl. Pharmacol.
26, 299-313.
Kruysse, A., V.J. Feron, H.K. Immel; et al. (1977). Short-term inhalation
toxicity studies with peroxyacetyl nitrate in rats. Toxicology 8,
231-249.
Maibach, H.I., Feldman, R.J., Milby, T.H. and W.F. Serat (1971) Regional
variation in percutaneous penetration in man, pesticides. Arch. Environ.
Health 23, 208-211.
Nievel, J.G., J. Anderson, and P.J. Bray (1976). Biochemical changes in
liver and blood underlying parenclyrnal damage of rat liver induced by a
dose-dependent effect of drugs before and after development of liver
enlargement and modular hyperplasia. Biochemical Society Transactions 4,
932-933.
NIOSH Criteria for a recommended standard .... Occupational Exposure to
Nitroglycerin and Ethylene Glycol Dinitrate, HEW No. 78-167.
OECD 1981. Organization for Economic Cooperation and Development. OECD
Guidelines for Testing of Chemicals. Acute Dermal Toxicity, Repeated
Dose Dermal Toxicity - 21/28-day, Subchronic Dermal Toxicity - 90-day.
OECD Publications and Information Center, Suite 1207, 1750 Pennsylvania
Ave., N.W., Washington, D.C. 20006.
132
-------
Oser, B.L., K. Morgmreidge, G.E. Cox, and S. Carson (1975) Short-term
toxicity of ethyrlene chlorohydrin in rats, dogs, and monkeys. Food and
Cosmetic Toxicology 13, 313-315.
Roudabush, R.L., C.J. Terhaar, D.W. Fassett and S.P. Dziuba (1965)
Comparative acute effects of some chemicals on the skin of rabbits and
guinea pigs. Tbxicol. Appl. Pharmacol. 7, 559-565.
Van Logten, M.J., M. Wolthuis, Rauws; et al. (1974). Semichronic toxicity
study of sodium bromide in rats. Toxicology 2, 257-267.
Verschuuren, H.G., R. Kroes, and G.J. Van Esch (1973). Toxicity studies on
tetrasul. I. Acute, long-term, and reproduction studies. Toxicology 1,
63-78
Verschuuren, H.G., R. Kroes, and E.M. Den Tonkelaar, (1973). Toxicity
studies on tetrasul. II. Short-term comparative studies in 6 animal
species. Toxicology 1, 103-12.
Verschuuren, H.G., R. Kroes, Den Tonkelaar, M. Engelina, (1973). Toxicity
studies on tetrasul. III. Short-term comparative studies in rats with
tetrasul and structurally related acaricides. Toxicology 1, 113-23.
Wester, R.C., and ft. I. Maibach (1975) Rhesus monkey as an animal model for
percutaneous absorption. In: Maibach, H.I. ed. Animal Models in
Dermatology: Relevance to Human Dermatopharmacology and Dermatotoxico-
logy. Edinburgh, Churchill, Livingstone, pp. 133-137.
Weil, C.S., N.I. Condra, and C.P. Carpenter (1971). Correlation of 4-hour
vs 24-hour contact skin penetration toxicity in the rat and rabbit and
use of the former for predictions of relative hazard of pesticide
formulations. Toxicol. Appl. Pharmaol. 18:734-42.
133
-------
Chemical
APPENDIX
Comparison of Oral and Dermal Lethality8
Oral LD5g Dermal LD^g
Species (mg/kg) mg/kg
Dermal/Oral Rabbit/Rat
Acet amide, 2-chloro-N,N-diallyl-
Acet amide, 2-fluoro-
Acetamide, 2-fluoro-N-methyl-N-
(1-naphthyl)-
Acetanilide, 2-chloro-N-isopropyl-
Acetic acid, 2-(tert-butyl)-4,6-
dinitro-m-tolyl ester
Acetic acid, (2,4-dichlorophenoxy)-
Acetic acid, diphenyl-, 3-fluoroethyl
eater
Acetic acid, fluoro-, methyl ester
Acetic acid, mercaptophenyl-, ethyl
ester, S-ester with 0,0-dimethyl
phosphorodithioate
Acetic acid, thiocyanato-, isobornyl
ester
Acetone
.
Acrolein
Acrylanilide , 3',4'-dichloro-2-methyl
Acrylonitrile
Rat
Rat
Rat
House
Guinea pig
Rabbit
Rat
Rat
Rabbit
Rat
Rabbit
Rabbit
Mouse
Rabbit
Rabbit
Rat
Rabbit
Rat
Rat
Rabbit
Guinea Pig
700
5.8
115
200
2
710
42
370
6
0.6
0.5
150
630
5,300
1,250
7
1,800
82
93
50
360
80
213
372
5
380
1,300
1,500
1,400
4
7
2
2,620
6,000
20,000
1,800
52
1,780
148
250
460
0.51
13.7
1.85
1.86
2.50
0.35
30.9
4.05
0.67
11.7
4.00
17.5
9.52
3.77
0.14
: 7.43 .
0.99
1.81
2.69
9.20
Dermal 0.93
Oral 0.1C
Dermal 1.75
Oral 4.24
Dermal 11.11
Oral 1.1
Dermal 1.6'.
obtained from Registry of Toxic Effects of Chemical Substances (RTECS), U.S. Department of Health
and Human Services, Public Health Service, Center for Disease Control, National Institute for Occupa-
tional Safety and Health. Cinci-mati Ohio.
134
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral L05g Dermal LD5g
Species (ing/kg) mg/kg
Dermal/Oral Rabbit/Rat
Aeetophenone, 2' ,3' ,4' ,5' ,6'-
pentachloro-
Rabbit
Guinea pig
93
50
250
460
2.69
9.20
Allodan
Ammonium, alky 1 dime thy Ibenzyl-,
chloride
Ammonium, alkyldimethyl(ethylbenzyl)-,
chloride
Ammonium, (2-chloroethyl)trimethyl-,
chloride
Aniline
Aniline, p-chloro
Aniline, 4,4'-(imidocarbonyl)bis(N,N-
dimethyl-, hydrochloride
p-Aniaidine
Arsine, Dichloro (2chloro vinyl)-
Calcium arsenate
Sodium arsenite
Potassium arsenite
Benzaldehyde, p-nitro-
Benzaldehyde, p-nitro-, oxime
Benzamide, 2,6-dichlorothio-
Benzene nitro-
Benzene thiol-
Benzhydrol, 4,4'-Dichloro-alpha
(Trichloromethyl)
Rat
Rat
Rat
940
280
300
1,000
1,560
1,420
1.06
5.57
4.73
Rabbit
Rat
Rat
Mouse
Rat
Rat
Rabbit
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rat
Rabbit
150
250
370
480
1,400
50
20
70
14
4,700
180
757
640
46
575
1810
232
1,400
3,200
300
3,200
24
6
2,400
150
150
16,000
7,100
1,000
2,100
f
300
100
1870
1.55
5.60
8.65
0.63
2.29
0.48
120
2.14
10.7
3.40
39.4
1.32
3.28
6.52
0.17 Oral 3.15
1.03 Dermal 18.70
135
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LD50 Dermal LD50
Species (mg/kg) nig/kg
Dermal/Oral Rabbit/Rat
2-Benzifflidazolecarbamic acid, methyl
ester
1-Benziraidazolecarbbxylic acid,
5,6-dichlorc-2-(Trifluoranethyl)-
Benzimidazole, 2-(2-furyl)-
phenyl ester
Benzoic acid, benzyl ester
Benzoic acid, p-hydroxy-, isopropyl
ester, ester with isopropyl ethyl-
phosphoramidothioate
1H-2 , 1 , 3-Benzoth iadiazin-4( 3H) -one-
2,2-dioxide, 3-isopropyl-
Benzyl alcohol
4,4'-8ipyridinium, 1 , 1 ' -dimethyl- ,
di chloride
Rat
Rat
Rat
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
Rabbit
Rat
Duck
6,400
283
1,100
1,700
28
1,100
1,040
57
1,200
2,000 0.31
700 2.47
1,000 0.91
4,000 2.35 Dermal 1.00
4,000
188 6.71 Dermal 0.86
162
2,500 2.27
2,000 1.92
236 -- Dermal 2.9
80 1.40
1,260 1.05
1-Butanol, 2-ethyl Rabbit 1,200
t-Butyl hydroperoxide Rat 406
Butyric acid, 4-(4-chloro-o-
tolyl)oxy)-, sodium salt Rat 700
Butyric acid, 4-(2,4-dichlorophenoxy)- Rat 700
Butyric acid, ester with dimethyl-
(2,2,2-trichloro-l-hydroxyethyl)
phosphonate Rat 1,100
Carbamic acid, diethylthio-, S-
(p-chlorobenzyl)ester Rat 1,903
Carbamic acid, (3-(dimethylamino)
propyl)- propyl ester, hydrochloride Rat 10,900
1,260
790
1,000
800
7,000
2,900
5,000
1.05
1.95
1.43
1.14
6.36
1.52
.50
136
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
Species (mq/kg)
Oral LD50 Dermal LD50
mg/kg Dermal/Oral Rabbit/Rat
Carbamic acid, dimethyl-, 1- ((dime thy 1-
amino)carbonyl)-5-methyl-lH-pyrazol-
3-yl ester
Carbamic acid, dimethyl-, 1-isopropyl-
-3-methylpyrazol-5-yl ester
Carbamic acid, (mercaptoacetyl)methyl-,
ethyl ester, S-ester with 0.0-
diethylphosphorodithioate
Carbamic acid, methyl-, o-sec-butyl-
phenyl ester
Carbamic acid, methyl-, o-cumenyl ester
Carbamic acid, methyl-, m-cym-5-yl
ester
Carbamic acid, methyl-, 2,3-dihydro-
2,2-dimethyl-7-benzofuranyl ester
Carbamic acid, methyl-, 4-dimethyl-
amino-m-tolyl ester
Carbamic acid, methyl-, 4-dimethyl-
amino-3,5-xylyl ester
Carbamic acid, methyl-, 0-(((2,4-
dimethyl-l,3-dithiolan-2-yl)-
methylene)amino) deriv.
Carbamic acid, methyl-, 2,3-
(dimethylmethylenedioxy)phenyl ester
Carbamic acid, methyl-, o-(l,3-
dioxolan-2-yl)phenyl ester
Rat
Rat
Rat
Mouse
Mouse
Rat
Human
Rat
Bird
Rabbit
Rat
Rat
Rat
Rat
Mouse
Rat
25
11
36
173
150
74
11
5.3
0.42
30
14
1
179
61
60
600
5.6
380
340
7,600
450
10,000
120
100
885
275
1,500
300
1,000
1,660
32.000
24.0
0.51
10.6
1.97
50.7
6.08
909 Dermal 7.38
22.6
238
~
9.17
107
300
5.59
27.2
533
Carbamic acid, N-methyldithio-,
sodium salt
Rabbit
320
800
2.50
Carbamic acid, methyl-, 3-(ethyl-
thiomethyDphenyl ester
Rat
411
1,000
2.43
137
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
Species
Oral LDjg Dermal LDjg
(mg/kg) mg/kg
Dermal/Oral Rabbit/Rat
Carbanic acid, methyl-, o-isopropoxy-
phenyl ester
Carbanic acid, methyl-, m-( 1-methyl-
butyDphenyl ester mixed with
carbamic acid, methyl-, m-(l-
ethylpropyl)phenyl eater (4:1)
Carbamic acid, N-methyl-, 4-(methyl-
thio)-3,5-xylyl ester
Carbamic acid, methyl-, 1-naphthyl
ester
Carbamic acid, methyl-, m-tolyl ester
Carbanilic acid, m-chloro-, 4-ehloro-
2-butynyl ester
Carbonic acid, dithio-, cyclic-5,5-
(6-methyl-2,3-quinoxalinediyl) eater
Carbonoch lor idle acid, methyl ester
Carbon tetrachloride
Coumarin, 3-(alpha-acetonylbenzyl)-
4-hydroxy-
Coumarin, 3-chloro-7-hydroxy-4-
methyl-, 0-ester with 0,0-diethyl
phosphorothioate
m-Cresol
o-Cresol
p-Cresol
Rat
Rabbit
Rat
Rabbit
Rat
Bird (Wild)
Rabbit
Rat
Rat
Rabbit
Rat
Mouse
Rat
Rat
Rat
Bird (Wild)
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
83
87
60
5
710
250
268
600
1,100
67
2,800
1,550
16
3
242
~
121
~
207
__
800 9.64
1,450
242 2.78
400
350 5.83
100 20.0
2.000 2.82
4.000 16.0
6,000 22.4
23,000 38.3
500 0.46
1,750 26.2
5,070 1.81
1,400 0.90
_
860 53.8
7.5 2.50
620 2 . 56
2050
1,100
890
750
301
Dermal 1.81
Dermal 1.65
Oral 2.84
Dermal 0.50
Dermal 3.31
Dermal 0.81
Dermal 0..40
138
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
o-Creaol, 4,6-dinitro-
o-Cresol, 4,6-ditnitro-, sodium salt
Crotonic Acid, 3-Hydroxy-, alpha-
Methylbenzyl Ester, Dimethyl Phosphate
(E)-
Crotonic Acid, 3-Hydroxy-, Methyl
Eater, Dimethyl Phosphate, (E)-
Crontonic Acid, 3-Methyl ,2-sec-Butyl-
4,6-Dinitrophenol Ester
Crontonic Acid, 2-(l-Methylheptyl)-
4,6-Dinitrophenyl Ester
Cyanamide, Calcium Salt (1:1)
1,3,5-Cycloheptatriene
Cyclohexane, 1,2,3,4,5,6-Hexachloro-
Cyclohexane, 1,2,3,4,5,6-Hexachloro-,
gamma- isomer
Cyclopropanecarboxanilides, 3',4'-
dichloro-
Decaborane (14)
1,4:5, 8-Dimethanonphthalene , 1,2,3,4,
10,10-Hexachloro-6,7-Epoxy-l 4,4a,5,6,
7,8,8a-0ctahydro-, endo, endo-
1,4:5, 8-D imeth anonph thalene , 1,2,3,4,
10 , 10-Hexachloro-6 ,7-Epoxy-l ,4 ,4a ,5,6,
7,8,8a-0ctahydro-, endo, endo-
Species
Rat
Rat
Rat
Rat
Mouse
Duck
Rat
Rabbit
Rabbit
Rabbit
Rat
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
Oral LD5Q
(mg/kg)
10
26
74
3.7
4
4.63
58
2,000
1,400
57
100
76
76
3,028
64
3
7
46
45
Dermal LD5Q
mg/kg
200
200
202
4.7
40
11
720
750
9,400
590
442
900
500
500
3,038
740
71
12
60
10
250
Dermal/Oral
20.0
7.69
2.73
1.27
10.0
2.38
12.4
4.70
0.42
7.75
9.00
6.58
6.58
1.00
11.6
4.00
8.57
0.22
5.56
Rabbit/Rat
Dermal 1.04
Oral 0.76
Dermal 0 56
Dermal 0 . 10
Oral 2.33
Dermal 5.00
Oral 0.98
Dermal 25.00
139
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LDjg Dermal LD5g
Species (mg/kg) mg/kg
Dermal/Oral Rabbit/Rat
l,4:5,8-Dimethanonphthalene, 1,2,3,4,
10,10-Hexachloro-l,4,4a,5,8,8a-
Hexahydro-, endo, endo
l,4:5,8-Dimethanonphthalene, 1,2,3,4,
10,10-Hexachloro-l,4,4a,5,8,8a-
Hexahydro-, endo, endo
p-Dioxane
m-Dioxane , 5-ethyl-5-nitro-2-propyl-
Diatannoxane, Hexabutyl
Distamoxance, Hexakia(beta, beta-
Dime thy Iphenethyl) -
Diaulfide, BisCDimethylphosphinothioyl)
mixed with Diaulfide, Bia
(Diisopropylphospinothioyl) (75S:25S)
Ethane, 2,2,-Bia(p-methoxyphenyol)-l,
1-1-Trichloro-mixed with 0,0-Diethyl
0-(2-Iaopropyl-4-Methyl-6-Pyrimidinyl)
Phosphorothioate
Ethane, 1,2,-dibromo-
Ethane, l,l,l-Trichloro-2,2-Bis
( p-Chloropheny 1 ) -
Ethanol , 2-amino-
Ethanol, 2-butoxy-
Ethanol, 2-(2-Butoxyethoxy) -, Acetate
Ethanol, 2-Chloro-
Rat
Rat
Rabbit
Rat
Rat
Rabbit
Rat
Rat
Rabbit
Rat
Rat
Rabbit
Rat
Rabbit
Guinea Pig
Rabbit
Rat
Rabbit
Guinea Pig
Rabbit
Rat
Rabbit
7
39
2,000
2,000
87
2,630
265
2,000
108
55
113
250
150
320
2,100
1,000
1,200
2,600
91
23
'
98
7,600
9,400
11,700
900
1,000
480
8,000
8,000
300
300
1,931
300
1,000
490
1,500
1,000
230
1,500
84
56
3.29
2.51
3.80
4.70
134
0.38
1.81
.
4.00
2.78
5.45
17.1
1 20
6.67
1.53
0.71
1.53
0.192
0.58
0.92
__
Dermal 0.08
Dermal 1.00
Oral 0.51
Dermal 1.00
Oral 2.21
Dermal 0 . 16
Oral 0.48
Dermal 0.67
Dermal Q-. 67
140
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LD50 Dermal LD5Q
Chemical
Ethanol, 2-ethoxy-
Ethanol, 2-(2-ethoxyethoxy)
Ethanol, 2-methoxy-
Ether, 2,4-Dichlorophenyl
p-Nitrophenyl
Ether, 2'-Hydrooxy-2,4,4'-
Trichlorodiphenyl
Formamide, N,N-dimethyl
Formamidine, N -(4-Chloro-o-Tolyl)-
N,N-Oimethyl-
Formamidine, N ' -(4-chloro-o-tolyl)-
N,N-dimethyl-, hydrochloride
Formic acid, chloro , ethylene ester
Formic acid, chloro-, isopropyl ester
Formic acid, chloro-, oxydiethylene
ester
Formic acid, chloro-, propyl ester
Glyoxylonitrile, phenyl-, oxime,
0,0-diethyl phosphorothioate
Heptanethiol, methyl
2,4-Hexadien-l-ol
1,3-Hexanediol, 2-ethyl-
Hydracrylic acid, phenethyl ester
Species
Rabbit
Rat
Rabbit
Rabbit
Rat
Rat
Rat
Rabbit
Rat
Mouse
Rabbit
Rat
Mouse
Mouse
Rabbit
Mouse
Mouse
Rat
Rat
Rat
Rabbit
Rat
(mg/kg)
3,100
6,500
3,620
890
740
3,700
2,800
170
160
625
225
1,100
178
813
650
1,845
85
2,140
2,600
7,800
mg/kg
3,500
6,000
16,400
1,280
5,000
9,300
5,000
5,000
4,000
225
640
4,000
2.000
12
11,300
2,000
10
1,000
1,954
1,010
2,000
10,000
Dermal/Oral
1.13
0.92
4.53
1.44
6.76
2.51
1.79
23.5
1.41
1.02
17.8
1.82
0.07
2.46
0.02
0.54
23.0
0.47
0.77
1.28
Rabbit/Rat
Oral 0.50
Dermal 2 . 73
Dermal 1 . 00
Oral 3.68
Dermal 0 . 16
141
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LDjg Dermal LD5Q
Species (mq/kq) mg/kg
Dermal/Oral Rabbit/Rat
Imidocarbonic acid, ( diethoxyphos-
phinothioyl)dithio-f cyclic
ethylene ester
1,3-Indandione, 2-((p-chlorophenyl)-
phenylacetyl)-
Iron, carbonyl
Isopropylamine
Lactonitrile, 2-methyl-
Me thane, isothiocyanato-
4,7-Methanoindan, 1,2,4,5,6,7,8,8-
octachloro-3a,4,7,7a-tetrahydro-
4,7-Methanoindene, 1,4,5,6,7,8,8-
heptachloro-3a,4 ,7 ,7a-tetrahydro-
4,7-Methanoisobenzofuran, 1,3,4,5,6,7,
8,8-octachloro-l,3,3a,4,7,7a-
hexahydro
1,3, 4-M«theno-2H-cyclobuta( cd)
pentalen-2-one, l,la,3,3a,4,5,5,5a,
5b,6-decachloroctahydro-
Naphthalimide, N-hydroxy-, diethyl
phosphate
Nicotine
Bird (Wild)
Rabbit
Rabbit
Rabbit
Guinea pig
Rabbit
Rat
Mouse
Rabbit
Rabbit
Rat
Rat
Rabbit
Guinea pig
Rabbit
Rat
Rat
Rabbit
1.8
50
12
3,200
9
13.5
97
97
100
40
6.2
4
2
65
70
50
__
-
10
200
240
550
150
17
2,780
1,020
33
780
119
.
5
12
2
345
140
140
50
5.56
4.00
20.0
0.17
16.7
1.26
28.7 Dermal 0.01
18.7
- ~
7.8 7.8
2.98
0.81 Oral 0.65
3.00 Dermal 2.4
1.00
5.31
2.00
2.80 Dermal Q.36
Nicotine, sulfate (2:1) Rat
2-Norbornanone, endo-3-chloro-endo-
6-cyano-, 0-(methylcarbamoyl)oxime Rat
55
19
285
303
5.18
15.9
142
-------
- APPENDIX (Continued)
Comparison of Oral and Oernal Lethality
Oral LD50 Dermal
Chemical
5-Norbornene-2,3-dicarboximide, N-(2
ethylhexyl)-
5-Norbornene-2,3-dijnethanol, 1,4,5,
6,7,7-hexachloro-, cyclic sulfite
Optunal
7-Oxabicyclo( 2 , 2 , l)heptane-2 , 3-
dicarboxylic acid, disodiuro salt
l,2,4-Oxadiazolidine-3,5-dione,
2-(3,4-dichlorophenyl)-4-methyl-
l,4-Oxathiin-3-carboxamide, 5,6-
dihydro-2-methyl-N-phenyl-
2,4-Pentanediol, 2-methyl-
3-Penten-2-one, 4-methyl-
Phenethyl alcohol
Phenol
Phenol, 2-sec-butyl-4,6-dinitro-
Phenol, 4,4'-Isopropylidenedi-
Phenol, pentachlorb-
Phenol, m-phenoxy-
Phenol, 2,4,6-tri9
(dimethylaminomethyl)-
Phosphine oxide, tris(l-aziridinyl)-
Speciea
Rat
Rat
Rabbit
Bird (Wild)
Rat
Rabbit
Rat
Rat
Rabbit
Rabbit
Guinea pig
Rat
Rabbit
Rat
Rabbit
Rat
Rat
Rat
Rat
Mouse
(rag/kg)
2,800
18
0.75
51
10,200
430
3,200
1,000
400
414
25
2,230
50
1,211
1,200
37
292
mg/kg
470
74
167
1.3
750
100
1,350
1,050
13,200
5,150
5,000
669
850
80
3,000
105
2,750
1,280
87
375
Dermal/Oral Rabbit/Rat
0.17
4.11 Dermal 2.26
1 73
14.7 Dermal 0.13
0.13
2.44
4 13
5.15
12.5
1.62 Dermal 1.27
3.20
1.35
2.10
2.27
1.07
2.35
1.29
Phosphine oxide, tris(l-d-methyl)
aziridinyl)-
Rat
136
183
1.35
143
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LDcn Dermal LD.Q
Species (mg/kq)
mg/kg Dermal/Oral Rabbit/Rat
Phosphonamidothioic Acid, Ethyl-,
MMethylthio)-m-Tolyl Cater
Phosphonic Acid, l-(BUtylamino)
Cyclohexyl-, Dibutyl Eater
Phosphonic Acid, (2,2,2-Trichloro-
1-Hydroxyethyl) - , Dimethyl Ester
Bird (Wild)
Rat
Rabbit
Rat
Rabbit
3.2
3,000
450
1,450
75
1,200
500
2,000
5,000
23.5
0.40
'-
4.44
3.45
Dermal 0.42
Oral 3.22
Dermal 2.50
Phosphonium, (5-Chloro-2-Thienyl)
Methyltributyl-, Chloride
Rat
Phosphonodithioic Acid, Chloromethyl ,
S,S-Diethyl Ester Rat
455
35
1,000
79
2.20
2.26
Phosphonodithioic Acid, Ethyl ,
0-Ethyl S-Phenyl Ester
Rat
147
49.0
Phosphonodithioic Acid, Methyl-,
S_((N-Methoxycarbony1)-N-
MethylcarbamoyDMethyl 0-Methyl Ester Rat
57
720
12.6
Phosphonothioic Acid, Ethyl , 0-Ethyl
0-(2,4,5-Trichlorophenyl) Ester
Phosphonothioic Acid, Phenyl-,
0-(4 Bromo-2,5-Dichlorophenyl)
0-Methyl Ester
Phosphonothioic Acid, Phenyl-,
0-Ethyl O-(p-Nitrophenyl) Ester
Rat
Rabbit
Rat
Duck
15
124
8
3
64
800
25
400
4.27
6 45
3.13
333
Phosphoramidic Acid, Isopropyl ,
4-(Methylthio)-m-Tolyl Ethyl Ester
Duck
Rat
1.68
8
24
72
14.3
9 00
Phosphoramidic Acid, Methyl-,
4-tert-Butyl-2-Chlorophenyl Methyl
Ester Rabbit 400
Phosphoramidocyanidic Acid,
Dimethyl-, Ethyl Ester Rabbit 16
2,000
5.00
2.19
144
-------
APPENDIX (Continued)
Comparison, of Oral and Dermal Lethality
Chemical
Oral LD5Q
Species (nig/ kg)
Dermal
'50
Dermal/Oral Rabbit/Rat
Phosphoramidothioic Acid, Acetimidoyl-,
0,0-Bia(p-Chlorophenyl) Eater
Phoaphoramidothioic Acid,
0,5-0imethyl Eater
Rat
Rat
Rabbit
Phosphoramidothioic Acid, N-Ethyl-,
0-(1-1sopropoxycarbony1-l-Propen-2-YL)
0-Methyl Eater
Phoaphoric Acid, (7-Chloro-Bicyclo
(3.2.0)Hepta-2,6-Dien-6-Yl)Dimethyl
Eater
Phosphoric Acid, 2-Chloro-l-
(2,4-Dichlorophenyl)Vinyl Diethyl
Ester
Phosphoric Aicd, 2-Chlorovinyl
Diethyl Ester
Phosphoric Acid, l,2-Dibromo-2,
2-Dichloroethyl Dimethyl Eater
Phosphoric Acid, 3,2-Dichlorovinyl
Dimethyl Ester
Rat
Rat
Rat
Rabbit
Rabbit
Rat
Rat
Rabbit
3.70
7.5
10
100
96
20
500
250
32
1.25
25
50
118
1,500
2,925
30
400
18
800
75
107
6.76
6.67
11.B
15.0
30.5
1 50
0.80
6.00
3.20
2.34
85.6
Oral 1.33
Dermal 2.36
Oral 25.0
Dermal 13.3
Oral 0.04
Dermal 1.43
Phosphoric Acid, 2,2-Dichlorovinyl
Methyl Ester, Calcium Salt mixed
with 2,2-Dichlorovinyl Phosphoric
Acid Calcium Salt
Mouse
250
3,040
12.2
Phosphoric Acid, Diethyl Ester,
Ester with 6-Chloro-3-(Hydroxymethyl)-
2-Benzoxazolinone
Rat
35
400
11.4
Phosphoric Acid, Dimethyl Ester,
ester with 2-Chloro-N,N-Diethyl-3-
Hydroxycrotonamide
Rat
Duck
Rabbit
17
3.81
125
26
267
7.35 Dermal 2.14
6.82
145
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
Species
Oral LD5()
(mg/kg)
Dermal LD-g
mg/kq
Dermal/Oral Rabbit/Rat
Phosphoric Acid, Dimethy Ester,
eater with (E)-3-Hydroxy-
N,N-Dimethylcrotonamide
Rat 16
Bird (Wild) 2
Rabbit
42
1.3
168
2.63
0.65
Dermal 4.00
Phosphoric Acid, Dimethyl Eater,
ester with (E)-3-Hydroxy-N-
Methoxy-N-Methylcrotonamide Rabbit
Phosphoric Acid, Dimethyl Ester,
ester with (E)-3-Hydroxy-N-
Methy1cr otonamide
Phosphorodithioic Acid,
S(((p-Chlorophenyl)Thio)Methyl)
0,0-Diethyl Eater
11
Rat 21
Duck 3.36
Bird (Wild) 1.6
Phosphorodithioic Acid, S-(2-Chloro-
l-(l,3-Dioxo-2H-Isoindol-2H-Isoindol-
2-YL)Ethyl) Rabbit 35
Phosphorodithioic Acid, 5-((6-Chloro-
2-Oxo-3(2H)-Benzoxazolyl)Methyl)0,0-
Diethyl Ester Rabbit 120
Rabbit 1,250
Phosphorodithioic Acid,
S(((p-Chlorophenyl)Thio)Methyl)
0,0-Dimethyl Ester Rat 98
107
112
30
4.2
145
390
1,270
190
9.73
5.33
8.93
2.63
4.14
3.25
1.02
1.94
Phosphorodithioic Acid,
S(((p-Dichlorophenyl)Thio)Methyl)
0,0-Diethyl Ester
Rat
61
652
10.7
Phosphorodithioic Acid, 0,0-Diethyl
Ester, S-Ester with N-Isopropyl-2-
Mercaptoacetamide
Rat 8
Rabbit 85
100
14
12.5
1.65
Oral 1.06
Dermal 0 14
Phosphorodithioic Acid, 0,0-Diethyl
Ester, S-Ester with 3-(Mercaptomethyl)- Rat
Phosphorodithioic Acid, 0,0-Diethyl,
S-((Ethylsulfinyl)Ethyl) Ester
Rat
Mouse
3.5
12
250
192
263
2.78
54.5
21.9
146
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LD5Q Dermal LD5Q
Species (mg/kg) mg/kg
Dermal/Oral Rabbit/Rat
Phosphorodithioic Acid, 0,0-Diethyl
S-(2-Ethylthio)Ethyl) Ester
Phosphorodithioic Acid, 0.,0-Diethyl,
S-(Ethylthio)Methyl Ester
Rat
Rat
Guinea Pig
Duck
2
1.10
20
2.55
6 .
2.50
20
203
3.00
2.27
1.00
79.6
Phosphorodithioic Acid,
0,0-Diisopropyl Ester, S-Ester with
N-(2-Mercaptoethyl) Rat 770
Phosphorodithioic Acid, 0,0-Dimethyl
S-(2-Acetamidoethyl) Ester Mouse 342
Phosphorodithioic Acid, 0,0-Dimethyl
Ester, S-Ester with N-Ethyl-2-
Mercaptoacetamide Rat 125
3,950
472
2,000
5.13
1.38
16.0
Phosphorodithioic Acid, 0,0-Dimethyl
Ester, S-Ester with N-Formyl-2-
Mercapto-N-Methyl-
Phosphorodithioic Acid, 0,0-Dimethyl
Ester, S-Ester with 4-(Mercaptoacetyl)
Morpholine
Phosphorodithioic Acid, 0,0-Dimethyl
Ester, S-Ester with 2-Mercapto-N-
(2-Methoxyethyl)
Phosphorodithioic Acid, 0,0-Dimethyl
Ester, S-Ester with 2-Mercapto-N-
Methylacetamide
Rat
Rat
Rat
Rat
Guinea Pig
Phosphorodithioic Acid, 0,0-Dimethyl
Ester, S-Ester with 3-(Mercaptomethyl)-
l,2,3-Benzotriazin-4(3H)-One Rat
250
190
600
152
350
13
353
283
1,600
353
1,000
220
1.41
1.49
2.67
2.32
2.86
16.9
Phosphorodithioic Acid, 0,0 Dimethyl
Ester, S-Ester with 4-(Mercaptomethyl)-
2-Methoxy-delta(sup 2)-1.3,4-
Thiadiazolin-5-One Rat
Rabbit
20
63
25
375
1.25
5.95
Oral 3.15
Dermal 15.0
147
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LD-g Dermal LD5Q
Species (mg/kg) mg/kg
Dermal/Oral Rabbit/Rat
Phoaphorodithioic Acid, 0,0-Dimethyl
Eater, S-Ester with N-(Mercaptomethyl)
Phthalimide
Phosphor odithioic Acid, S,S'-p-Dioxane-
2,3-Diyl 0,0,0',0'-Tetraethyl Ester
Phosphor odithioic Acid, 0-Ethyl,
S,S-Oipropyl Ester
Phoaphorodithioic Acid, S,S-Methylene
O.O.O'O'-Tetraethyl Ester
Phosphorofluoridic Acid,
Bis(l-Methylethyl) Ester
Phosphorothioic Acid, S-Benzyl
0,0-Oiisopropyl Ester
Phosphorothioic Acid 0-14-Bromo-
2-Chlorophenyl)-0-Ethyl-S-Propyl Ester
Phosphorothioic Acid, 0-(4-Bromo-
2,5-Dichlorophenyl) 0,0-Diethyl Ester
Rat
Rabbit
Rat
Rabbit
Rat
Duck
Rabbit
Rat
Guinea Pig
Rabbit
Mouse
Mouse
Rat
Rabbit
Rat
Rabbit
147
63
-
34
1.26
13
40
~
37
1,760
400
700
52
-
1,550
3,160
20
85
60
11
26
62
915
890
72
5,000
300
472
1,000
1,366
10.5
0.32
1.77
8.73
-
4.77
22.9
1.95
2.84
0.75
0.67
19.2
Dermal 2.04
Dermal 4.25
Dermal 0.43
Dermal 14 . 35
Oral 1.75
Dermal 1.57
Dermal 1.37
Phosphorothioic Acid, 0-(4-Bromo-
2,5-Dichlorophenyl) 0,0-Dimethyl Ester Rabbit 720
Phosphorothioic Acid, 0-(2-Chloro-
l-Isopropylimidazol-4-YL) 0,0-Diethyl
Ester Rat 40
2,181
290
3.03
7.25
Phosphoroth.ioic Acid, c. (2-Chloro-
4-Nitrophenyl) 0,0-Dimethyl Ester Rat
Phosphorothioic Acid, 0 (3-Chloro-
4-Nitrophenyl) 0,0-Dimethyl Ester Rat
330
880
790
1,500
2.39
1.71
148
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
Phosphorothioic Acid, S-(((Cyano-l
-Methy 1-Ethy 1) Carbamoy 1) Methyl )
0,0-Oiethyl Eater
Phoaphorothioic Acid, 0-(2,5-
Dichloro-4-Iodophenyl) 0,0-Oimethyl
Ester
Phosphorothioic Acid, 0,0-Oiethyl
0-((2,5-Dichloro-4-Methylthio)Phenyl)
Eater
Phosphorothioic Acid, 0,0-Diethyl
0-(2-(Oiethylamino)-6-Methyl-4-
Pyrimidinyl) Ester
Phosphorothioic Acid, 0,0-Diethyl
Ester, 0,0-Diethyl ESter, 0-Ester with
6-Hydroxy-2-Phenyl-3(2H)Pyridazinone
Phosphorothioic Acid, 0,0-Diethyl
0-(2-(Ethylthio)Ethyl) Eater, mixed
with 0,0-Diethyl S (2-
(Ethylthio)Ethyl) Ester (7:3)
Phosphorothioic Acid, 0,0-Diethyl
0-(2-Isopropyl-6-Methyl-4-Pyrimidinyl)
Ester
Phosphorothioic Acid, 0,0-Diethyl
0-(p-(Methylsulfinyl.)Phenyl) Ester
Phosphorothioic Acid, 0,0-Diethyl
O-(p-Nitrophenyl) Ester
Oral LD5Q Dermal LD5Q
Speciea (mg/kg) mg/kg
Rat
Rat
Rabbit
Rat
Rabbit
Rat
Rat
Rat
Bird (Wild)
Rabbit
Rat
Rabbit
Duck
Bird (Wild)
Rat
Rat
Mouse
Rabbit
Guinea Pig
Duck
Bird (Wild)
3.5
2,000
2,000
7.8
20
140
850
1.7
7
76
-
7.47
2.4
2
2
6
10
8
2.34
2
.
105
1,800
500
58
48
1,000
2,100
8.2
1.8
24
455
400
3
4.2
3
6.8
32.4
40
600
28
1.80
Dermal/Oral
30.0
0.90
0.25
7.44
2.40
7.14
2.47
4.82
0.26
~
5.99
0.40
1.75
1.50
3.40
5.40
4.00
75.0
12.0
0.90
Rabbit/Rat
Oral 1.00
Dermal 0.28
Oral 2.56
Dermal 0.83
Dermal 2.93
Dermal 0.88
Oral 5.PO
Dermal 5.88
149
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
Phosphorothioic Acid, 0,0-Diethyl
0-(5-Phenyl-3-Isoxazolyl) Ester
Phosphorothioic Acid, 0,0-Diethyl
0-(l-Phenyl-l,2,4-Triazolyl) Ester
Phosphorothioic Acid, 0,0-Diethyl
0-Pyrazinyl Ester
Phosphorothioic Acid, 0,0-Oiethyl
0-(2-Quinoxalinyl) Ester
Phosphorothioic Acid, 0,0-Diethyl
0-(3,5,6-Trichloro-2-Pyridyl) Eater
Phosphorothioic Acid, 0,0-Dimethyl
Ester, 0,0-Diester with 4,4'-
Thiodiphenol
Phosphorothioic Acid, 0,0-Oimethyl
Ester, 0-Ester, 0-Ester with p-
Hydroxybenzonitrile
Phosphorothioic Acid, 0,0-Dimethyl
Ester S-Ester with 2-((2-Mercapto-
ethyl)Thio)-N-Methylpropionamide
Species
Rat
Mouse
Rat
Rat
Duck
Rat
Rat
Rabbit
Rat
Rabbit
Rat
Mouse
Rabbit
Oral LDcQ
(mg/kqr
112
98
64
3.50
1.68
26
97
1,000
1,000
18
40
_
Dermal LD.g
may kg Dermal/Oral Rabbit/Rat
450 4.02
193 1.97
1,100 . 17.2
8 2.29
74.17
300 11.5
202 2.08 Oral 10.31
2,000 Dermal 9.90
1,370 1.37 Dermal 0.71
1,000
800 44.4
1,500 37.5
160
Phosphorothioic Acid, 0,0-Dimethyl
Ester, S-Ester with 2-(Mercaptomethyl)
-5-Methoxy-4H-Pyran-4-One
Rat
23
130
5.65
Phosphorothioic Acid, 0,0-Dimethyl
S-(2-(Ethylsulfonyl)Ethyl) Ester
Rat
40
500
12.5
Phosphorothioic Acid, 0,0-Dimethyl
S-(2-(Methylthio)Ethyl) Ester
Rat
68
0.81
Phosphorothioic Acid, 0,0-Dimethyl-,
0-(4-Methylthio)-m-Tolyl) Ester
Rat
Duck
215
6
330
44
1.54
7.33
150
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Chemical
Oral LDcg
Species (mg/kg)
Dermal LD-g
mg/kg
Dermal/Oral Rabbit/Rat
Phosphorothioic Acid, 0,0-Difflethyl
O-(p-Nitrophenyl) Ester
Phosphorothioic Acid, 0,0-Dimethyl
0-(4-Nitro-m-Tolyl) Ester
Phosphorothioic Acid, 0,0-Dimethyl
0(2,4,5-Trichlorophenyl) Ester
Phosphorothioic Acid, 0-Ethyl
S-Propyl 0-(2,4,6-Trichlorophenyl)
Ester
Rat
Rabbit
Duck
Mouse
Duck
6
420
61
715
1,190
Rat 906
Rabbit 420
Guinea Pig 1,4000
Rat
200
63 10.5 Oral 70.00
300 0.71 Dermal 4.76
54 0.88
2,500 3.50
504 0.42
2,000 2.21 Oral 0.46
1,000 2.38 Dermal 0.50
2,000 1.43
250 1.25
Phosphorothioic Acid, S-(2-
(Ethylsulfinyl)Ethyl) 0,0-Dimethyl
Ester
Rat
Phosphorothioic Acid, S-(2-
(Ethylsulfinyl)-l-Methylethyl)
0,0-Dimethyl Ester Rat
Phoaphorothioic Acid, 0-(2-
(Ethylthio)Ethyl) 0,0-Oimethyl Ester,
mixed with Phosphorothioic Acid, S-(2-
(Ethylthio)Ethyl) 0,0-Dimethyl Ester
(7:3) Rat
Phosphorotrithioic Acid, S,S,S-
Tributyl Ester Rat
Phosphorotrithious Acid, Tributyl
Ester Rat
30
103
65
150
910
100
1,000
300
168
615
3.33
9.71
4.62
1.12
0.68
Phosphorotrithious Acid, Trimethyl
Ester Rat 105
Phthalic Acid, Bis(2-Ethylhexyl)
Ester Rabbit 3400
Phthalimide, N-(2,6-Dioxo-3-
Piperidyl)- Rat 113
1,030
2,500
1,550
9.81
0.74
13.7
151
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LDeQ Dermal LD5Q
Speciea (mq/kq)
Dermal/Oral Rabbit/Rat
Piperidinium, l-Allyl-l-(3,7-
Dimethyloctyl)-, Bromide
Pivalic acid
Poly(Oxy(Methyl-l,2-Ethanediyl)),
alpha-Butyl-omega-Hydroxy-
Polypropylene Glycol Monobutylether
Propane, l-Chloro-2-Nitro-
Propane , 1 , 2-Dibromo-3-Chloro-
Propane, Dichloro- mixed with
Propane, Dichloro-
l ,3-Propanediol, 2-Ethyl 2-
(Hydroxymethyl)-, Cyclic Phosphate
1,3-Propanediol, 2-Ethyl-2-
(Hydroxymethyl)-, Cyclic
Phosphite (1:1)
2-Propanol, 1-methoxy-
2-Propanone, Chloropentafluoro-,
Hydrate
2-Propanone, 1,3-Dichloro-l, 1,3,3-
Tetrafluoro-
2-Propanone , 1,1,1,3,3,3 Hexach loro-
2-Propanone, Hexafluoro , Hydrate
2-Propanone , 1 , 1 ,3-Trichloro-l ,3 , 3-
Trifluoro-
Rat
Rat
Rabbit
Rabbit
Rat
Rabbit
Rabbit
Rat
Rabbit
Rat
Rat
Mouse
Rabbit
Rat
Rat
Rat
Rabbit
Rat
Rat
Rabbit
360
900
23,900
23,900
197
ISO
140
3.08
a. 39
7
8,000
85
61
1,290
190
277
-_
115
1,900
2,100
2,100
362
362
1,400
2,100
2,100
50
929
4
13,000
81
91
2,980
2,980
113
770
770
0.32
2.11
0.09
0.09
1.84
7.78
15.0
16.2
110
0.57
1.63
0.95
1.49
2.31
0.60
2.78
._
Dermal 1.00
Dermal 1.00
Dermal 1.00
Dermal 1.00
152
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LD5Q Dermal U>50
Species (mg/kg) mq/kg
Dermal/Oral Rabbit/Rat
Propionaldehyde, 2-Methyl-2-
(Methylsulfonyl)-, 0-Methylcarbamoyl)-
Oxime
Propionaldehyde, 2-Methyl-2-
(Methylthio)-, O-(Methylcarbamoyl)-
Oxime
Propionic Acid, 2-Chloro-3-(4-
Chlorophenyl)-, Methyl Ester
Propionic Acid, 2-(2,4-
Dich lo r ophenoxy ) -
Propionitrile, 2-((4-Chloro-6-
Ethylamino)-3-Triazin-2-Ylamino)-
2-Methyl-
Pyridine, 2-Chloro-6-(Trichloro-
methyl)-
2,5-Pyridinedicarboxylic Acid,
Dipropyl Ester
Pyrophosphoric Acid, Tetraethyl
Ester
Pyrophosphoric Acid, Tetraethyl
Ester (liquid mixture)
Pyrrolidine, 1-Butyl-
Ryania
Serine, p-nitrophenyl-
Succinic Acid, Mercapto-, Diethyl
Ester, S-ester with 0,0-Dimethyl
Phosphorodithioate
Rat
Rabbit
Rat
Rabbit
Mouse
Rat
Rat
Rabbit
Rat
Rabbit
Rat
Duck
Rabbit
Rat
Mouse
Rat
Rat
Rabbit
Rat
26.8
900
500
400
800
149
500
5,230
-
0.5
3.56
~
1.05
51
750
24,000
250
885
1,000
1,000
2.5
756
1,400
1,400
1,200
850
9,400
9,500
2.4
64
5
2. -40
1,000
750
16,000
4,100
4,444
37.3
.._
0.003
1.51
3.50
1.75
0.05
1.70
1.80
-
4.80
18.0
2.29
19.6
i.nu
0.67
16.4
5.02
Dermal 1.00
Dermal 1.01
Dermal 2.08
Oral 0.28
Dermal 0.92
153
-------
Chemical
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LD.Q Dermal LD5Q
Species (mg/kq)
mg/kg Dermal/Oral Rabbit/Rat
Sulf amide, N-((Oichlorofluoromethyl)-
Thio)-N',N'-Dimethyl-N-(p-Tolyl)-
1,2,4-Thiadiazole, S-EtHoxy-3-
(Trichloromethyl)-
Thiocyanic Acid, 2-(2-Butoxyetnoxy)
Ethyl Eater
Thiopyrophosphoric acid,
Tetrapropyl ester
as-Triazir>-5(4H)-ONE, 4-Amino-6-
tert-8utyl-3-(Methylthio)-
Tributylamine
T riethylenetetramine
s-Trioxane, 2,4,6-trimethyl-
1,3,5, 2,4, 6-Triphosphatriborin,
l,2,3,4,5,6-Hexahydro-l,2,3,4,5,6-
Hexamthyl-
m-Toluamide, N,N-diethyl-
Toluene, alpha-(2-(2-Butoxyethoxy)
Ethoxy)-4,5-(Methylenedioxy)-2-
Propyl-
Toxaphene
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
Rat
Rabbit
Rabbit
Rabbit
Rat
Rat
Rabbit
Rabbit
Rat
Rabbit
1,000
779
90
35
450
2,200
615
5,500
3,304
13.5
1,950
1,584
7,500
40
500
1,700
250
125
1800
3830
2,000
250
820
14,000
1,800
5,000
3,180
200
600
1,025
0.50
2.18
2.78
3.57
4.00
0.91
0.41
0.15
4.24
133
2.56
2.01
0.03
15.0
Oral 0.39
Dermal 0.50
Dermal 2.13
Oral 0.81
Dermal 0.64
Dermal 1.71
x Triazine, 2-Chloro-4-Ethylamino-
6-Isopropylamino- Rabbit 750
v-Trithiane, 5-(Dimethylamino)-,
Oxalate Rat 310
Urea, 3-(Hexahydro-4,7-Methanoindan-
5-YL)-l,l-Dimethyl- Rat 2,000
7,500
1,000
23,000
10.0
3.23
11.5
154
-------
APPENDIX (Continued)
Comparison of Oral and Dermal Lethality
Oral LOeg Dermal LD,-
Chemical Species (mg/kg) mg/kg Dermal/Oral Rabbit/Rat
2,4-Xylenol Rat 3,200 1,040 0.33
2,6-Xylenol Rabbit 700 1,000 1.43
Mouse 980 920 0.94
155
-------
2g^Rr DOCUMENTATION .-u"«^ "» " *
«,,.-'-... PASfc EPA 560/11-82-002
s?^!^^ -: '
Selected issueaFiri Testing for Dermal Toxicity, Including
7. Autnorts)
9. Pertormifti Organization Nama and Addran
Tracor Jitco, Inc.
1776 East Jefferson Street
Rockville, MD 20852-4081
12. Sponsoring Organization Nama and Address
Office of Toxic Substances
U.S. Environmental Protection Agency
Washington, D.C. 20460
*i 1)0^*4 da^-Ma^^ai ^^^^itfMft fltteW"
January, 1982
>
8. Parfbrmtnc Organization Rapt.
No.
10. Pro|eet/Taa«/Worti Untt No.
Work Assignmpnf 02
11. ContracttO or OranttG) No.
(0 68-01-6176
(Q
U. Typa of Report & Parted Covered
Final Technical Report
14.
IS. Supplementary Natea
is. Abatract (Limit: 200 «ord»> Four categories of Dermatotoxicity testing are examined: Dermal Irritatior,!
Sensitization, Systemic Toxicity, and Phototoxicity. The rabbit is most widely used for ir-
ritation; the guinea pig is also acceptable, as its sensitivity is comparable. Factors affect-
ing dermal irritation include: the degree of occlusion, use of abrasion, the application site,
and duration of exposure and observation. This review suggests a tier-like strategy utilizing
pH limits and preliminary screening in the hairless mouse may be useful in evaluating ir-
ritation potential. Eight guinea pig methods are considered acceptable for determining
sensitization; they utilize intradermal and/or epicutaneous routes of administration. The
Maximization and Closed Patch tests are most widely used, but no one method has been
sufficiently validated to support its selection as the method of choice. Ultraviolet light
can alter a non-toxic chemical to one causing direct phototoxicity or allergenicity.
Following topical or intraperitoneal application of a compound a test site is irradiated
with UV light and reactions are scored. Dermal toxicity testing determines whether substances
can be absorbed sufficiently to produce systemic effects. Factors influencing irritation also
influence systemic toxicity. Relative rates of percutaneous absorption of a series of compounds
can be estimated from LD50 values via different routes. Comparison of dermal LDSO's for rabbit
and rats shows that more than 75% of the values varied by less than a factor of four. Neither
spacies clearly showed greater sensitivity- Dermal LD50 values were similar for a 24-hour
study in rabbits and a 4-hour study in rats.
17. Document Analysis a. Oescnptors
Toxic Tolerances; Skin; Absorption; Laboratory Animals; Guinea Pigs; Skin Effect; Toxicology;
Photosensitivity; Lethal Dosage; Contact Dermatitis; Allergic Skin Diseases; Skin Test Agents;
Tests; Experimental Design; Standards
o. ldentifi*rs/Ooen*Cnded Terms
Dermal Toxicity Testing Methods; Dermatotoxicity; Skin Permeability; Patch Tests; Guidelines;
Organization for Economic Cooperation and Development; OECD; Environmental Protection Agency;
EPA; Interagency Regulatory Liaison Group; IRLG; Sensitization
c. COSATt Held/Group
is. Availability statement Document is available to the public
through the National Technical Information Service,
Springfield, VA 22151.
19. Security Class (This Report)
20. Security Class (This
21. No. of Pitts
155
22. Price
($ ANSJ-Z39.1S)
Sea Instructions on Reverse
OPTIONAL FORM Z72 (4-7T
(Formerly NTIS-3S)
------- |