United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–98–196

August 1998

Health Effects Test

Guidelines

OPPTS 870.2500

Acute Dermal Irritation
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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.).

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

Government Printing Office, Washington, DC 20402 on disks or paper

copies: call (202) 512–0132. This guideline is also available electronically

in PDF (portable document format) from EPA’s World Wide Web site

(http://www.epa.gov/epahome/research.htm) under the heading ‘‘Researchers and Scientists/Test Methods and Guidelines/OPPTS Harmonized Test

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 three healthy adult animals (either

sex) 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 irritation/ 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 hour. If observations indicate that exposure can be continued humanely, the third patch is removed

after 4 hours and the responses graded. If a corrosive effect is observed

after either 3 min or 1 hour 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–hour exposure,

the test is completed using two additional animals, each with one patch
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only, for an exposure period of 4 hours. 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 hours.

(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 hours 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) 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. Although water or saline is the

preferred agent to be used for moistening dry test materials, other agents

may be used providing the use is justified. Acceptable alternatives include:

gum arabic, ethanol and water, carboxymethyl cellulose, polyethylene glycol, glycerol, vegetable oil, and mineral oil.

(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.
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(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 hours 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.

Table 1.—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 hours

after patch removal, any other dermal lesions, a description of the degree

and nature 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, strain, sex, age and source of test animal.

(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).
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(iii) Tabulation of erythema and edema data and any other dermal

lesions/responses for each individual animal at each observation time point

(e.g. 30–60 minutes and 24, 48, and 72 hours until end of test/reversibility).

(iv) Description of any lesions observed.

(v) Narrative description of the degree and nature of irritation or corrosion observed.

(vi) Description of any systemic effects observed.

(vii) Description of any pre-test conditioning, including diet, quarantine and treatment of disease.

(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 test animal.

(ix) Manufacturer, source, purity, and lot number of test substance.

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

value for the test substance.

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

the test substance.

(xii) A list of references cited in the body of the report, i.e., references

to any published literature used in developing the test protocol, performing

the testing, making and interpreting observations, and compiling and evaluating the results.

(h) 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).

(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.
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(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).

(12) U.S. EPA, Atlas of Dermal Lesions, Office of Pesticides and

Toxic Substances, Report 20T–2004, August 1990.

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

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

(14) 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).