United States Prevention, Pesticides EPA712-C-96-196
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 pub-
lished 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. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
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 "Environ-
mental 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 com-
ments. 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, Ar-
lington, 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 Bul-
letin 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 test-
ing 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 har-
monized OPPTS test guideline are 40 CFR 798.4470 Primary Dermal Irri-
tation; OPP 81-5 Primary Dermal Irritation (Pesticide Assessment Guide-
lines, 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 pos-
sible 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/corro-
sion is suspected and the stepwise procedure is used (see paragraph
(f)(l)(iii) of this guideline)). The degree of irritation is read and scored
at specified intervals and is further described to provide a complete evalua-
tion of the effects. The duration of the study should be sufficient to permit
a full evaluation of the reversibility or irreversibility of the effects ob-
served but need not exceed 14 days.
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(2) When testing solids (which may be pulverized if considered nec-
essary), 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 corro-
sive 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 corro-
sive 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 po-
tential 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/reason-
ing 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 expo-
sure 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 expo-
sure duration is normally 4 h. unless corrosion is observed (see paragraph
(f)(l)(iii) of this guideline). Longer exposure may be indicated under cer-
tain 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 end-
use 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 mate-
rials, 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 vehi-
cle 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, metabo-
lism, 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 physio-
logical 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, deter-
mination 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 homo-
geneity 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 (approxi-
mately 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 reversibil-
ity 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 irrita-
tion, any lesions and other toxic effects should be fully described.
Evaluation of Skin Reaction
Value
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)
Maximum possible
Edema Formation:
No edema
Very slight edema (barely perceptible)
Slight edema (edges of area well defined by definite raising)
Moderate edema (raised approximately 1 mm)
Severe edema (raised more than 1 mm and extending beyond area of exposure
Maximum possible
0
1
2
3
4
4
0
1
2
3
4
4
(g) Data and reporting—(1) Data summary. Data should be sum-
marized 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 ob-
served.
(2) Evaluation of results. The dermal irritation scores should be eval-
uated 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 informa-
tion should be reported:
(i) Species and strain used.
(ii) Rationale for selection of species (if species is other than the spe-
cies preferred or required by OPP's toxicology data requirements for pes-
ticide registration).
(iii) Rationale for selections of strain.
(iv) Source of supply of animals.
(v) Description of any pre-test conditioning including diet and quar-
antine.
(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 tem-
perature 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 nec-
essary 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 quan-
titative determination of its chemical composition (including names and
quantities of known contaminants and impurities, so far as technically fea-
sible; the determinations should also include quantities of unknown mate-
rials, 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, sus-
pending 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 adminis-
tration (including hour of dosing in relation to photoperiod), total volume
of material (i.e., test substance plus vehicle), individual dosings, and dura-
tion 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 sub-
stance 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 inter-
preting the observations, and compiling and evaluating the results.
(g) References. The following references should be consulted for ad-
ditional 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 edi-
tion, 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 Man-
agement. 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 Labora-
tories. American Public Health Association: Washington, D.C. 20036
(1978).
(9) Marzulli, F.N. and Maibach, H.I. Dermatotoxicology and Phar-
macology, Advances in Modern Toxicology. Vol. 4. (New York: Hemi-
sphere 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 aus-
pices of the Committee on Toxicology, National Research Council, Na-
tional 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, Den-
tal 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. Gene-
va, 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).
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