United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–197

June 1996

Health Effects Test

Guidelines

OPPTS 870.2600

Skin Sensitization

‘‘Public Draft’’
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INTRODUCTION

This guideline is one of a series of test guidelines that have been

developed by the Office of Prevention, Pesticides and Toxic Substances,

United States Environmental Protection Agency for use in the testing of

pesticides and toxic substances, and the development of test data that must

be submitted to the Agency for review under Federal regulations.

The Office of Prevention, Pesticides and Toxic Substances (OPPTS)

has developed this guideline through a process of harmonization that

blended the testing guidance and requirements that existed in the Office

of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,

Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the

Office of Pesticide Programs (OPP) which appeared in publications of the

National Technical Information Service (NTIS) and the guidelines published by the Organization for Economic Cooperation and Development

(OECD).

The purpose of harmonizing these guidelines into a single set of

OPPTS guidelines is to minimize variations among the testing procedures

that must be performed to meet the data requirements of the U. S. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

Public Draft Access Information: This draft guideline is part of a

series of related harmonized guidelines that need to be considered as a

unit. For copies: These guidelines are available electronically from the

EPA Public Access Gopher (gopher.epa.gov) under the heading ‘‘Environmental Test Methods and Guidelines’’ or in paper by contacting the OPP

Public Docket at (703) 305–5805 or by e-mail:

guidelines@epamail.epa.gov.

To Submit Comments: Interested persons are invited to submit comments. By mail: Public Docket and Freedom of Information Section, Office

of Pesticide Programs, Field Operations Division (7506C), Environmental

Protection Agency, 401 M St. SW., Washington, DC 20460. In person:

bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Arlington, VA. Comments may also be submitted electronically by sending

electronic mail (e-mail) to: guidelines@epamail.epa.gov.

Final Guideline Release: This guideline is available from the U.S.

Government Printing Office, Washington, DC 20402 on The Federal Bulletin Board. By modem dial 202–512–1387, telnet and ftp:

fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202–512–0132 for disks

or paper copies. This guideline is also available electronically in ASCII

and PDF (portable document format) from the EPA Public Access Gopher

(gopher.epa.gov) under the heading ‘‘Environmental Test Methods and

Guidelines.’’
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OPPTS 870.2600 Skin sensitization.

(a) Scope—(1) Applicability. This guideline is intended to meet testing requirements of both the Federal Insecticide, Fungicide, and

Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances

Control Act (TSCA) (15 U.S.C. 2601).

(2) Background. The source material used in developing this harmonized OPPTS test guideline is the OPPT 40 CFR 798.4100 Dermal Sensitization; OPP 81–6 Dermal Sensitization (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and Domestic Animals)

EPA report 540/09–82–025, 1982; and OECD 406 Skin Sensitization.

(b) Purpose. Determination of the potential to cause or elicit skinsensitization reactions (allergic contact dermatitis) is an important element

in evaluating a substance’s toxicity. Information derived from skin-sensitization tests serves to identify possible hazards to a population exposed

repeatedly to a test substance. The test selected should identify substances

with significant allergenic potential and minimize false negative results.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR

Part 792—Good Laboratory Practice Standards (GLP) apply to this test

guideline. The following definitions also apply to this test guideline.

Challenge exposure is an experimental exposure of a previously treated subject to a test substance following an induction period, to determine

if the subject will react in a hypersensitive manner.

Induction exposure is an experimental exposure of a subject to a test

substance with the intention of inducing a hypersensitive state.

Induction period is a period of a least 1 week following an induction

exposure during which a hypersensitive state may develop.

Skin sensitization (allergic contact dermatitis) is an immunologically

mediated cutaneous reaction to a substance. In the human, the responses

may be characterized by pruritis, erythema, edema, papules, vesicles,

bullae, or a combination of these. In other species, the reactions may differ

and only erythema and edema may be seen.

(d) Principle of the test method. Following initial exposure to a test

substance, the animals are subjected, after a period of not less than

1 week, to a challenge exposure with the test substance to establish whether a hypersensitive state has been induced. Sensitization is determined by

examining the reaction to the challenge exposure and comparing this reaction with that of the initial induction exposure. The test animals are initially exposed to the test substance by intradermal and/or epidermal application (induction exposure). Following a rest period of 10 to 14 days (the

induction period), during which an immune response may develop, the

animals are exposed to a challenge dose. The extent and degree of skin
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reaction to the challenge exposure is compared with that demonstrated by

control animals that undergo sham treatment during induction and then

receive the challenge exposure.

(e) Test procedures. (1) Any of the following test methods is considered to be acceptable.

(i) Buehler test.

(ii) Guinea-pig maximization test (GPMT).

(iii) Other.

(A) Open epicutaneous test.

(B) Maurer optimization test.

(C) Split adjuvant technique.

(D) Freund’s complete adjuvant test.

(E) Draize sensitization test.

(2) The GPMT of Magnusson and Kligman, which uses adjuvant, and

the nonadjuvant Buehler test are given preference over other methods. Although strong preference is given to either the Buehler test or the GPMT,

it is recognized that other tests may give useful results. If other tests are

used, the tester should provide justification/reasoning for their use, methods and protocols must be provided, and each test should include a positive

and a negative control group.

(f) Screening tests. The mouse ear swelling test (MEST) (see paragraphs (i)(9), (i)(10), (i)(11), and (i)(12) of this guideline) or the local

(auricular) lymph node assay (LLNA) (see paragraphs (i)(13), (i)(14),

(i)(15), and (i)(16) of this guideline) in the mouse may be used as screening tests to detect moderate to strong sensitizers. If a positive result is

seen in either assay, the test substance may be designated a potential sensitizer, and it may not be necessary to conduct a further test in guinea

pigs. If the LLNA or MEST does not indicate sensitization, the test substance should not be designated a nonsensitizer without confirmation in

an accepted test using guinea pigs.

(g) Animal selection—(1) Species and strain. The young adult guinea pig is preferred. Commonly used laboratory strains should be employed.

If other species are used, the tester should provide justification/reasoning

for their selection.

(2) Housing and feeding. The temperature of the experimental animal room should be 20 ± 3 °C with the relative humidity 30–70 percent.

Where the lighting is artificial, the sequence should be 12 h light/

12 h dark. Conventional laboratory diets may be used with an unlimited
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supply of drinking water. It is essential that guinea pigs receive an adequate amount of ascorbic acid.

(3) Number and sex. The number and sex will depend on the method

chosen. Either sex may be used in the Buehler test and the GPMT. If

females are used, they should be nulliparous and not pregnant. The Buehler

test recommends using a minimum of 20 animals in the treatment and

at least 10 as controls. At least 10 animals in the treatment group and

5 in the control group should be used with the GPMT, with the stipulation

that if it is not possible to conclude that the test substance is a sensitizer

after using fewer than 20 test and 10 control guinea pigs, the testing of

additional animals to give a total of at least 20 test and 10 control animals

is strongly recommended.

(4) Control animals. (i) The sensitivity and reliability of the experimental technique used should be assessed every 6 months in naive animals

by the use of positive control substances known to have mild-to-moderate

skin-sensitizing properties. In a properly conducted test, a response of at

least 30 percent in an adjuvant test and at least 15 percent in a nonadjuvant

test should be expected for mild-moderate sensitizers. Preferred substances

are hexylcinnamic aldehyde (CAS No. 101–86–0), mercaptobenzothiazole

(CAS No. 149–30–4), and benzocaine (CAS No. 94–09–7). There may

be circumstances where, given adequate justification, other control substances meeting the above criteria may be used.

(ii) Depending upon the test selected, animals may be used as their

own controls, but usually there will be a separate group of sham-treated

animals that are exposed to the test substance only after the induction period, whose reactions are compared to those of the animals that have received both induction and challenge exposures.

(5) Dose levels. The dose level will depend on the test method selected. In the Buehler test, the concentration of the induction dose should

be high enough to cause mild irritation, and the challenge dose should

use the highest non-irritating concentration. In the GPMT, the concentration of the induction dose should be well tolerated systemically, and should

be high enough to cause mild-to-moderate skin irritation; the GPMT challenge dose should use the highest non-irritating concentration.

(6) Observation of animals. (i) Skin reactions should be graded and

recorded after the challenge exposures at the time specified by the methodology selected. This is usually at 24, 48, and 72 h. Additional notations

should be made as necessary to fully describe unusual responses.

(ii) Regardless of the test method selected, initial and terminal body

weights should be taken and recorded.

(7) Procedures. The procedures to be used are those described by

the test method chosen. Brief summaries are given here, but the tester
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should refer to the original literature for more complete guidance on conducting the Buehler test (under paragraphs (i)(1), (i)(2), (i)(3), and (i)(4)

of this guideline) or the GPMT (under paragraphs (i)(5), (i)(6), (i)(7), and

(i)(8) of this guideline).

(i) The Buehler test uses topical administration via a closed patch

on days 0, 6–8, and 13–15 for induction, with topical challenge of the

untreated flank for 6 h on day 27–28. Readings are made approximately

21 and 24 h after removing the challenge patch, and again 24 h after that.

If the results are equivocal, the animals may be rechallenged one week

later, using either the original control group or a new control group for

comparison. See paragraphs (i)(1), (i)(2), (i)(3), and (i)(4) of this guideline.

(ii) The GPMT uses intradermal injection with and without Freund’s

complete adjuvant (FCA) for induction, followed on days 5–8 by topical

irritation/induction, followed by topical challenge for 24 hours on day 20–

22. Readings are made approximately 24 h after removal of the challenge

dose, and again after another 24 h. As with the Buehler test, if the results

are equivocal, the animals may be rechallenged 1 week later. If only 10

animals were used initially and gave equivocal results, the use of an additional 10 experimental and 5 control animals is strongly recommended.

See paragraphs (i)(5), (i)(6), (i)(7), and (i)(8) of this guideline.

(iii) Blind reading of both test and control animals is recommended.

(iv) Removal of the test material should be accomplished with water

or an appropriate solvent, without altering the existing response or the integrity of the epidermis.

(v) Hair is removed from the site of application by clipping, shaving,

or possibly by depilation, depending on the test selected.

(h) Data and reporting. Data should be summarized in tabular form,

showing for each individual animal the skin reaction, results of the induction exposure, and the challenge exposure at times indicated by the method

chosen. As a minimum, the erythema and edema should be graded and

any unusual finding should be recorded.

(1) Evaluation of the results. The evaluation of results will provide

information on the proportion of each group that became sensitized and

the extent (slight, moderate, severe) of the sensitization reaction in each

individual animal.

(2) Test report. In addition to the reporting requirements as specified

under 40 CFR part 158 (for pesticides) and 40 CFR part 792, subpart

J (for toxic substances), the following specific information should be reported:

(i) A description of the method used and the commonly accepted

name.
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(ii) Information on the positive control study, including the positive

control substance used, the method used, and the time conducted.

(iii) The number, species, strain, age, and sex of the test animals.

(iv) Individual weights of the animals at the start of the test and at

the conclusion of the test.

(v) A brief description of the grading system.

(vi) Each reading made on each individual animal.

(vii) The chemical identification and relevant physicochemical properties of the test substance.

(viii) The vehicles used for induction and challenge, and justification

for their use, if other than water or physiological saline. Any material that

might reasonably be expected to react with or enhance or retard absorption

of the test substance should be reported.

(ix) The total amount of test substance applied for induction and challenge, and the technique of application in each case.

(x) The source from which the animals were obtained, housing conditions, diet, evidence for adequate ascorbic acid, and any other information

on animal care that may be relevant.

(xi) Histopathological findings, if any.

(xii) Discussion of results.

(i) References. The following references should be consulted for additional background information on this test guideline.

(1) Buehler, E.V. Delayed contact hypersensitivity in the guinea pig,

Archives of Dermatology 91: 171–177 (1965).

(2) Ritz, H.L. and Buehler, E.V. Planning, conduct and interpretation

of guinea pig sensitization patch tests in Current Concepts in Cutaneous

Toxicity, ed. V. Drill and P. Lazar. Academic, New York, NY. pp. 25–

42 (1980).

(3) Buehler, E.V. Occlusive patch method for skin sensitization in

guinea pigs: the Buehler method. Food and Chemical Toxicology 32:97–

101 (1994).

(4) Buehler, E.V. Prospective testing for delayed contact

hypersensitivity in guinea pigs: the Buehler method; in Methods in

Immunotoxicology, Vol. 2, ed. G. Burleson, A. Munson, and J. Dean.

Wiley, NY, pp. 343–356 (1995).
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(5) Magnusson, B. and Kligman, A.M. The identification of contact

allergens by animal assay. The guinea pig maximization test. Journal of

Investigative Dermatology 52: 268–276 (1969).

(6) Magnusson, B. and Kligman, A.M. Allergic contact dermatitis in

the guinea pig. Charles C. Thomas, Springfield, IL (1970).

(7) Magnusson, B. Identification of contact sensitizers by animal

assay. Contact Dermatology 6:46 (1980).

(8) Magnusson, B. et al. Determination of skin sensitization potential

of chemicals. Predictive testing in guinea pigs. Arbete och Ha ̈lsa: 26(E)

(1979).

(9) Gad, S.C. et al. Development and validation of an alternative dermal sensitization test: the mouse ear swelling test (MEST). Toxicology

and Applied Pharmacology 84: 93–114 (1986).

(10) Maisey, J. and Miller, K., Assessment of the ability of mice fed

on Vitamin-A supplemented diet to respond to a variety of potential contact sensitizers. Contact Dermatitis 15: 17–23 (1986).

(11) Thorne, P.S. et al., The noninvasive mouse ear swelling assay.

I. Refinements for detecting weak contact sensitizers. Fundamental and

Applied Toxicology 17:790–806 (1991).

(12) Thorne, P.S. et al. The noninvasive mouse ear swelling assay.

II. Testing the contact sensitizing potency of fragrances. Fundamental and

Applied Toxicology 17:807–820 (1991).

(13) Kimber, I. et al. The murine local lymph node assay for identification of contact allergens: a preliminary evaluation of in situ measurement of lymphocyte proliferation. Contact Dermatitis 21:215–220 (1989).

(14) Kimber, I. et al. Identification of contact allergens using the murine local lymph node assay: comparisons with the Buehler Occluded Patch

Test in guinea pigs. Journal of Applied Toxicology 10: 173–180 (1990).

(15) Kimber, I. et al. The murine local lymph node assay: results

of an interlaboratory trial. Toxicology Letters 55:203–213 (1991).

(16) Basketter, D.A. et al. Interlaboratory evaluation of the local

lymph node assay with 25 chemicals and comparison with guinea pig test

data. Toxicology Methods 1:30–43 (1991).