United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–196

June 1996

Health Effects Test

Guidelines

OPPTS 870.2500

Acute Dermal Irritation

‘‘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 870.2500 Acute dermal irritation.

(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 materials used in developing this harmonized OPPTS test guideline are 40 CFR 798.4470 Primary Dermal Irritation; OPP 81–5 Primary Dermal Irritation (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and Domestic Animals)

EPA report 540/09–82–025, 1982; and OECD 404 Acute Dermal Irritation/

Corrosion .

(b) Purpose. Determination of the irritant and/or corrosive effects on

skin of mammals is useful in the assessment and evaluation of the toxic

characteristics of a substance where exposure by the dermal route is likely.

Information derived from this test serves to indicate the existence of possible hazards likely to arise from exposure of the skin to the test substance.

Data on primary dermal irritation are required by 40 CFR part 158 to

support the registration of each manufacturing-use product and end-use

product. See specifically § § 158.50 and 158.340 to determine whether

these data must be submitted.

(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.

Dermal corrosion is the production of irreversible tissue damage in

the skin following the application of the test substance.

Dermal irritation is the production of reversible inflammatory

changes in the skin following the application of a test substance.

Pharmacological effect means any chemically induced physiological

changes in the test animal.

Target organ means any organ of a test animal showing evidence

of an effect of chemical treatment.

(d) Principle of the test methods. (1) The substance to be tested

is applied in a single dose to the skin of several experimental animals,

each animal serving as its own control (except when severe irritation/corrosion is suspected and the stepwise procedure is used (see paragraph

(f)(1)(iii) of this guideline)). The degree of irritation is read and scored

at specified intervals and is further described to provide a complete evaluation of the effects. The duration of the study should be sufficient to permit

a full evaluation of the reversibility or irreversibility of the effects observed but need not exceed 14 days.
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(2) When testing solids (which may be pulverized if considered necessary), the test substance should be moistened sufficiently with water or,

where necessary, a suitable vehicle, to ensure good contact with the skin.

When vehicles are used, the influence of the vehicle on irritation of skin

by the test substance should be taken into account. Liquid test substances

are generally used undiluted.

(e) Initial considerations. (1) Strongly acidic or alkaline substances,

for example with a demonstrated pH of 2 or less, or 11.5 or greater, need

not be tested for primary dermal irritation, owing to their predictable corrosive properties.

(2) It is unnecessary to test materials which have been shown to be

highly toxic (LD50 less than 200 mg/kg) by the dermal route or have

been shown not to produce irritation of the skin at the limit test dose

level of 2000 mg/kg body weight.

(3) It may not be necessary to test in vivo materials for which corrosive properties are predicted on the basis of results from well validated

and accepted in vitro tests. If an in vitro test is performed before the in

vivo test, a description or reference to the test, including details of the

procedure, must be given together with results obtained with the test and

reference substances.

(4) It may not be necessary to test materials for which corrosive potential is predicted from structure-activity relationships.

(f) Test procedures—(1) Animal selection—(i) Species and strain.

The albino rabbit is recommended as the preferred species. If another

mammalian species is used, the tester should provide justification/reasoning for its selection.

(ii) Number of animals. At least six healthy adult animals should

be used unless justification/reasoning for using fewer animals is provided.

It is recommended that a stepwise procedure be used to expose one animal,

followed by additional animals to clarify equivocal responses.

(iii) Stepwise exposure of animals. A single rabbit may be used if

it is suspected that the test material might produce severe corrosion. Three

test patches are applied concurrently or sequentially to the animal. The

first patch is removed after 3 min. If no serious skin reaction is observed,

the second patch is removed after 1 h. If observations indicate that exposure can be continued humanely, the third patch is removed after 4 h and

the responses graded. If a corrosive effect is observed after either 3 min

or 1 h of exposure, the test is immediately terminated by removal of the

remaining patches. If a corrosive effect is observed after an exposure of

up to 4 hours, then further animal testing is not required. If no corrosive

effect is observed in one animal after a 4–h exposure, the test is completed

using two additional animals, each with one patch only, for an exposure
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period of 4 h. If it is expected that the test substance will not produce

severe irritancy or corrosion, the test may be started using three animals,

each receiving one patch for an exposure period of 4 h.

(2) Control animals. Separate animals are not recommended for an

untreated control group. Adjacent areas of untreated skin of each animal

may serve as a control for the test.

(3) Dose level. A dose of 0.5 mL of liquid or 500 mg of solid or

semisolid is applied to the test site.

(4) Preparation of test area. Approximately 24 h before the test,

fur should be removed from the test area by clipping or shaving from

the dorsal area of the trunk of the animals. Care should be taken to avoid

abrading the skin. Only animals with healthy intact skin should be used.

(5) Application of the test substance. (i) The recommended exposure duration is normally 4 h. unless corrosion is observed (see paragraph

(f)(1)(iii) of this guideline). Longer exposure may be indicated under certain conditions (e.g. expected pattern of human use and exposure). At the

end of the exposure period, residual test substance should generally be

removed, where practicable, using water or an appropriate solvent, without

altering the existing response or the integrity of the epidermis.

(ii) Purity/grade for pesticides. A manufacturing-use product should

be tested to support the registration of a manufacturing-use product and

an end-use product should be tested to support the registration of an enduse product. The lot of the substance tested should be the same throughout

the duration of the study, and the research sample should be stored under

conditions that maintain its purity and stability. The composition of each

lot of the test substance should be determined, including the name and

quantities of known contaminants and impurities, as far as is technically

feasible. The determination should include quantities of unknown materials, if any, so that 100 percent of the test sample is accounted for. The

test substance should be within the limits, if any certified in accordance

with OPPTS 830.1750. When vehicles are used, the influence of the vehicle on irritation of skin by the test substance should by taken into account.

If a vehicle is used, it should not alter the absorption, distribution, metabolism, retention or the chemical properties of the test substance nor should

it enhance, reduce, or alter its toxic characteristics. At the levels used in

the study, the vehicle should not produce physiological effects, and should

resemble the vehicle to be used under expected conditions of use. It should

not interfere with the nutritional status of the animals, nor produce physiological effects. If the test substance is to be incorporated into a vehicle,

the period during which the test substance is stable in such a mixture

should be determined prior to the start of the study. Alternatively, determination of the stability of the test or control substance in statistically

randomized samples of vehicle mixture should be made periodically during
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the study (see 40 CFR Part 160—Good Laboratory Practice Standards

(GLPS)). If the test substance is incorporated into a vehicle, its homogeneity and concentration should be determined prior to the start of the

study and periodically during the study.

(iii) The test substance should be applied to a small area (approximately 6 cm2) of skin and covered with a gauze patch, which is held

in place with nonirritating tape. In the case of liquids or some pastes,

it may be necessary to apply the test substance to the gauze patch and

apply that to the skin. The patch should be loosely held in contact with

the skin by means of a suitable semiocclusive dressing for the duration

of the exposure period. Access by the animal to the patch and resultant

ingestion/inhalation of the test substance should be prevented.

(6) Observation period. The duration of the observation period need

not be rigidly fixed. It should be sufficient to fully evaluate the reversibility or irreversibility of the effects observed. It need not exceed 14 days

after application.

(7) Clinical examination and scoring. (i) After removal of the patch,

animals should be examined for signs of erythema and edema and the

responses scored within 30–60 min, and at 24, 48, and 72 h after patch

removal.

(ii) Dermal irritation should be scored and recorded according to the

grades in the following Table. 1. Further observations may be needed, as

necessary, to establish reversibility. In addition to the observation of irritation, any lesions and other toxic effects should be fully described.

Evaluation of Skin Reaction

Value

Erythema and Eschar Formation:

No erythema ...................................................................................................................... 0

Very slight erythema (barely perceptible) .......................................................................... 1

Well-defined erythema ....................................................................................................... 2

Moderate to severe erythema ............................................................................................ 3

Severe erythema (beet redness) to slight eschar formation (injuries in depth) ................ 4

Maximum possible ............................................................................................................. 4

Edema Formation:

No edema .......................................................................................................................... 0

Very slight edema (barely perceptible) .............................................................................. 1

Slight edema (edges of area well defined by definite raising) .......................................... 2

Moderate edema (raised approximately 1 mm) ................................................................ 3

Severe edema (raised more than 1 mm and extending beyond area of exposure .......... 4

Maximum possible ............................................................................................................. 4

(g) Data and reporting—(1) Data summary. Data should be summarized in tabular form, showing for each individual animal the irritation

scores for erythema and edema at 30 to 60 min, and 24, 48, and 72 h

after patch removal, any lesions, a description of the degree and nature
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of irritation, corrosion and reversibility, and any other toxic effects observed.

(2) Evaluation of results. The dermal irritation scores should be evaluated in conjunction with the nature and reversibility or otherwise of the

responses observed. The individual scores do not represent an absolute

standard for the irritant properties of a material. They should be viewed

as reference values which are only meaningful when supported by a full

description and evaluation of the observations.

(3) Test report. In addition to the reporting recommendations as

specified under 40 CFR part 792, subpart J, the following specific information should be reported:

(i) Species and strain used.

(ii) Rationale for selection of species (if species is other than the species preferred or required by OPP’s toxicology data requirements for pesticide registration).

(iii) Rationale for selections of strain.

(iv) Source of supply of animals.

(v) Description of any pre–test conditioning including diet and quarantine.

(vi) Numbers of animals of each sex in test and control group.

(vii) Age and condition of animals at beginning of study.

(viii) Description of caging conditions including number (and any

change in number) of animals per cage, bedding material, ambient temperature and humidity, photoperiod, and identification of diet of the test

animal.

(ix) Description of treatments used to prevent or control infectious

diseases if such treatment was taken during a test or shortly before a test

was begun.

(x) The report should indicate all ways in which the test procedure

fails to meet applicable standards for acceptable testing contained in OPP’s

toxicology data requirements for pesticide registration and should state the

reasons for such deviations (see 40 CFR part 158).

(4) Format. (i) The test report should include all information necessary to provide a complete and accurate description and evaluation of

the test procedures and results. A test report should contain at least three

parts: A summary and evaluation of the test results, a description of the

test procedures, and the data and information required by each applicable

section of OPP’s toxicology data requirements for pesticide registration.
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Particular information, data, or analysis may be required more than once

in the test report, and it should be referenced each time that it is required.

(ii) Test chemical. The test report should include:

(A) Physical nature and, where appropriate, concentration and pH

value for the test substance.

(B) Chemical name, molecular structure, and a qualitative and quantitative determination of its chemical composition (including names and

quantities of known contaminants and impurities, so far as technically feasible; the determinations should also include quantities of unknown materials, if any, so that 100 percent of the sample tested is accounted for.

(C) Manufacturer and lot number of the test substance.

(D) Identification and composition of any vehicles (e.g. diluents, suspending agents, and emulsifiers) or other materials used in administering

the test substance.

(iii) Test procedures. The test report should include:

(A) Specification of test methods, including a full description of the

experimental design and procedures, the length of the study, and the dates

on which the study began and ended.

(B) All dose levels administered, method and frequency of administration (including hour of dosing in relation to photoperiod), total volume

of material (i.e., test substance plus vehicle), individual dosings, and duration of treatment.

(C) The method of randomization used in selecting samples to assay,

the assay method used to determine the stability and homogeneity of the

test substance being administered, and results of the assay if the test substance is administered by a vehicle.

(D) Include references to statistical and any other methods employed

for analyzing the raw data, and references to any published literature used

in developing the test protocol, performing the testing, making and interpreting the observations, and compiling and evaluating the results.

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

(1) Bermes, E.W. et al. Chapter 2. Statistics, normal value and quality

control. Fundamentals of Clinical Chemistry. Tietz, N., ed. W.B. Saunders,

Philadelphia (1976).

(2) Dharan, M. Total Quality Control in the Chemical Laboratory.

C.V. Mosby: St. Louis (1977).
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(3) Dixon, W.J. ed. Biomedical Computer Programs (BMD). 2nd edition, University of California Press: Los Angeles (1970).

(4) Draize, J.H. Dermal Toxicity. Appraisal of the Safety of Chemicals

in Foods, Drugs and Cosmetics. Association of Food and Drug Officials

of the United States (1959, 3rd printing 1975) pp. 46–59.

(5) Draize, J.H. et al. Methods for the Study of Irritation and Toxicity

of Substances Applied Topically to the Skin and Mucous Membranes,

Journal of Pharmacology and Experimental Therapeutics. 83:377–390

(1944).

(6) Feigenbaum, A.V. Total Quality Control Engineering and Management. McGraw-Hill, New York (1961).

(7) Galen, R.S. and S.R. Gambino. Beyond Normality (The Predictive

Value and Efficiency of Medical Diagnosis). Wiley, New York (1975).

(8) Inhorn, S.L., ed., Quality Assurance Practices for Health Laboratories. American Public Health Association: Washington, D.C. 20036

(1978).

(9) Marzulli, F.N. and Maibach, H.I. Dermatotoxicology and Pharmacology, Advances in Modern Toxicology. Vol. 4. (New York: Hemisphere Publishing Corp., 1977).

(10) National Academy of Sciences. Principles and Procedures for

Evaluating the Toxicity of Household Substances. A report prepared by

the Committee for the Revision of NAS Publication 1138, Under the auspices of the Committee on Toxicology, National Research Council, National Academy of Sciences, Washington, DC, (1978).

(11) Nie, N.H. et al. Statistical Package for the Social Sciences

(SPSS). 2nd Ed. McGraw-Hill, New York (1975).

(12) Oranization for Economic Coperation and Development. OECD

Guideline 402, Paris (1987).

(13) Reed, A.H. and R.J. Henry. Chapter 12. Accuracy, precision and

control charts. In Clinical Chemistry: Principles and Technics. 2nd Ed.

Henry, R.J., ed. Harper and Row, New York (1974).

(14) Remington, R.D. and M.A. Schork. Statistics with Applications

to the Biological and Health Sciences. Prentice-Hall, New York. (Includes

a chapter on non–parametric methods (Chapter 12. Distribution Free and

Nonparametric Methods) which are useful for non–normally distributed

data.) (1970)

(15) Rohlf, F.J. and R.R. Sokal. Statistical Tables. W.H. Freeman and

Company: San Francisco (1969).
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(16) Sokal, R.R. and F.J. Rohlf. Biometry. Freeman, San Francisco

(1969).

(17) Von Fraunhofer, J.A. and J.J. Murray. Statistics in Medical, Dental and Biological Studies. Tri-Med Books, London (1976).

(18) World Health Organization. Part I. Environmental Health Criteria

6, Principles and Methods for Evaluating the Toxicity of Chemicals. Geneva, World Health Organization (1978).

(19) Young, J.R. et al. Classification as corrosive or irritant to skin

of preparations containing acidic or alkaline substances without testing on

animals. Toxicology In Vitro, 2,19 (1988).