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:

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    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
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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.

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                                           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

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                               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

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                         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

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                         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

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                         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

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                         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

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                                    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

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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

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                               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

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       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.

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       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

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       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

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                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

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                               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.

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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

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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

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                 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

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       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

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     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

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             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

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       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

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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

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                        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

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                        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

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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

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         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

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       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

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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

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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

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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

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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

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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

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Davies, R.E., K.H. Harper; S.R. Kynoch.  1972.  Inter-species Variation in
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Draize, J.H., G. Woodard and H.O. Calvery.  1944.  Methods for the Study of
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Draize, J.H.  1959.  Dermal Toxicity.  In:  Appraisal of the Safety of Chemi-
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Frosch, P.J. and A.M. Kligman.  1979.  The Duhring Chamber:  An Improved
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   Contact Dermatitis. 5:73-81
                                      42

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                                      43

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                                      44

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                                      45

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                                      46

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                             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

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       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

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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

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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

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       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

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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

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                                                     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

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                                               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

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                                               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

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       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

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       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

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       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

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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

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       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

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       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

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"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

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       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

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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

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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

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                     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

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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

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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

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                                               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

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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

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                                    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

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                                    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

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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

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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

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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

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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

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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

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                                 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

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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

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        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

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             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

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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

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                                           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)

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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

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                                             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)

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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

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                                          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

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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

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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

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                           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

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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

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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

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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

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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

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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

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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

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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

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           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

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            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

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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

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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

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      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

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      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

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      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

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      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

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      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

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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

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       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

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       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

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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

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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

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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

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       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

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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,
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Bettley, F.R. (1963).  The irritant affect of soap in  relation  to epidermal
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Blank, I.H., R.D. Griesemer, and E. Gould (1957).  The penetration of an
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Buttar, H.S., E.A. Nera, and R.H. Downie (1966).  Serum enzyme  activities
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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
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Chow, A.Y.K., G.H. Hirsh, and H.S. Buttar (1977).  Nephrotoxic  and hepa-
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Cornish, H.H. (1971) Problems posed by observations of serum enzyme dangers
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Deichmann, W.B., U.E. MacDonald, M. Coplan, F. Woods,  and E. Blum (1978)
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Draize, J.H. (1955) Dermal Toxicity.  Food Drug Cosmetic Law Journal.  10,
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Durbridge, T.C., F. Edwards, R.G. Edwards, and M. Atkinson (1976).
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Feldman, R.J. and H.I. Maibach (1970)  Absorption of some organic com-
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Feron, V.J., A. Kruysse, H.P. Til; et al. (1978).  Repeated exposure to
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Franz, T.J. (1975) Percutaneous absorption.  On the relevance of in vitro
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Freundt, K.J.,  G.P. Liebaldt, and E. Lieberwirth (1977).  Toxicity studies
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Frosch, P.J., and A.M. Kligman (1977) The chamber scarification test  for
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Gaines, T.B. (1960) The acute toxicity of pesticides to rats.  Toxicol.
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Galambos, J.T., and C.E. Willis (1978) Relationship between 505 paired  liver
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Grice, H.C., M.L. Berth, H.H. Cornish, et al. (1971)  Correlation between
   serum enzymes, isoenzyme patterns and histologically detectable organ
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Interagency Regulatory Liason Group (IRLG; 1981) Testing Standards and
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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.
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   detection of thioacetamide-, dimethylnitrosamine-, and
   diethanolalmine-induced liver damage in rats.  Toxicol. Appl. Pharmacol.
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Kruysse, A., V.J. Feron, H.K. Immel; et al. (1977).  Short-term inhalation
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Maibach, H.I.,  Feldman, R.J., Milby, T.H. and W.F. Serat (1971) Regional
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Nievel, J.G., J. Anderson, and P.J. Bray (1976).  Biochemical changes  in
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   dose-dependent effect of drugs before and after development  of liver
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NIOSH Criteria for a recommended standard .... Occupational Exposure  to
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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.
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   Ave., N.W.,  Washington, D.C. 20006.
                                    132

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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

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                                          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

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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)

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