United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–192

June 1996

Health Effects Test

Guidelines

OPPTS 870.1200

Acute Dermal Toxicity

‘‘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 870.1200 Acute dermal toxicity.

(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 are 40 CFR 798.1100 Acute Dermal Toxicity; OPP 81–2 Acute Dermal Toxicity (Pesticide Assessment Guidelines,

Subdivision F—Hazard Evaluation; Human and Domestic Animals) EPA

report 540/09–82–025, 1982; and OECD 402 Acute Dermal Toxicity.

(b) Purpose. In the assessment and evaluation of the toxic characteristics of a substance, determination of acute dermal toxicity is useful where

exposure by the dermal route is likely. It provides information on health

hazards likely to arise from short-term exposure by the dermal route. Data

from an acute study may serve as a basis for classification and labeling.

It is an initial step in establishing a dosage regimen in subchronic and

other studies and may provide information on dermal absorption and the

mode of toxic action of a substance by this route. An evaluation of acute

toxicity data should include the relationship, if any, between the exposure

of animals to the test substance and the incidence and severity of all abnormalities, including behavioral and clinical abnormalities, the reversibility

of observed abnormalities, gross lesions, body weight changes, effects on

mortality, and any other toxic effects.

(c) Definitions. The definitions in section 3 of the Toxic Substances

Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Laboratory Practice Standards apply to this test guideline. The following definitions also apply to this test guideline.

Acute dermal toxicity is the adverse effects occurring within a short

time of dermal application of a single dose of a substance or multiple

doses given within a 24–h period.

Dosage is a general term comprising the dose, its frequency and the

duration of dosing.

Dose is the amount of test substance applied. Dose is expressed as

weight of test substance (grams, milligrams) per unit weight of test animal

(e.g. milligrams per kilogram).

Dose-effect is the relationship between the dose and the magnitude

of a defined biological effect either in an individual or in a population

sample.

Dose-response is the relationship between the dose and the proportion

of a population sample showing a defined effect.
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LD50 (median lethal dose), dermal, is a statistically derived estimate

of a single dose of a substance that can be expected to cause death in

50 percent of treated animals when applied to the skin. The LD50 value

is expressed in terms of weight of test substance per unit weight of test

animal (milligrams per kilogram).

(d) Approaches to the determination of acute toxicity. (1) EPA

recommends the following means to reduce the number of animals used

to evaluate acute effects of chemical exposure while preserving its ability

to make reasonable judgments about safety:

(i) Using data from substantially similar mixtures. In order to minimize the need for animal testing, the Agency encourages the review of

existing acute toxicity information on mixtures that are substantially similar to the mixture under investigation. In certain cases it may be possible

to glean enough information to make preliminary hazard evaluations that

may reduce the need for further animal testing.

(ii) Limit test. When data on structurally related chemicals are inadequate, a limit test may be considered. If rodents are used, a limit dose

of at least 2,000 mg/kg bodyweight may be administered to a single group

of five males and five females using the procedures described under paragraph (e) of this guideline. If no lethality is demonstrated, no further testing for acute dermal toxicity is needed (Under current policy for pesticide

products, precautionary statements may still be required unless there are

data to indicate the LD50 is greater than 5,000 mg/kg.) If compound-related mortality is produced, further study may need to be considered.

(2) [Reserved]

(e) Conventional acute toxicity test—(1) Principle of the test method. The test substance is applied dermally in graduated doses to several

groups of experimental animals, one dose being used per group. The doses

chosen may be based on the results of a range finding test. Subsequently,

observations of effects and deaths are made. Animals that die during the

test are necropsied, and at the conclusion of the test the surviving animals

are sacrificed and necropsied. This guideline is directed primarily to studies in either rats, rabbits, or guinea pigs but may be adapted for studies

in other species. Animals showing severe and enduring signs of distress

and pain may need to be humanely killed. Dosing test substances in a

way known to cause marked pain and distress due to corrosive or irritating

properties need not be carried out.

(2) Substance to be tested. The composition of each lot of the test

substance must be determined, including the name and quantities of known

contaminants and impurities, as far as is technically feasible. The determinations should include quantities of known materials, if any, so that

100 percent of the test sample is accounted for. Test, control, and reference

substances are discussed at 40 CFR 792.
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(3) Test procedures—(i) Preparations. Healthy young adult animals

are acclimatized to the laboratory conditions for at least 5 days prior to

the test before the test animals are randomized and assigned to the treatment groups.

(ii) Animal selection—(A) Species and strain. The rat, rabbit, or

guinea pig may be used. The albino rabbit is preferred because of its size,

ease of handling, skin permeability, and extensive data base. Commonly

used laboratory strains should be employed. If a species other than rats,

rabbits, or guinea pigs is used, the tester should provide justification and

reasoning for its selection.

(B) Age. Young adult animals, rats 6– to 10–weeks–old on receipt

and between 8 and 12 weeks old at the beginning of dosing; rabbits and

guinea pigs at least 12 weeks old at the beginning of dosing should be

used. The weight variation of animals used in a test should not be greater

or less than 20 percent of the mean weight for each sex.

(C) Number and sex of animals. (1) At least five experimentally

naive animals with healthy intact skin are used at each dose level. They

should all be of the same sex. After completion of the study in one sex,

at least one group of five animals of the other sex is dosed to establish

that animals of this sex are not markedly more sensitive to the test substance. The use of fewer animals may be justified in individual circumstances. Where adequate information is available to demonstrate that

animals of the sex tested are markedly more sensitive, testing in animals

of the other sex may be dispensed with.

(2) The females should be nulliparous and nonpregnant.

(3) In acute toxicity tests with animals of a higher order than those

mentioned above, the use of smaller numbers should be considered.

(D) Assignment of animals. Each animal must be assigned a unique

identification number. A system to randomly assign animals to test groups

and control groups is required.

(E) Housing. Animals should be housed in individual cages.

(1) The temperature of the experimental animal rooms should be at

22 ± 3 °C for rodents, 20 ± 3 °C for rabbits.

(2) The relative humidity of the experimental animal rooms should

be 30 to 70 percent.

(3) Where lighting is artificial, the sequence should be 12–h light/

12–h dark.

(4) For feeding, conventional laboratory diets may be used with an

unlimited supply of drinking water.
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(iii) Dose levels and dose selection. (A) Three dose levels should

be used and spaced appropriately to produce test groups with a range of

toxic effects and mortality rates. The data should be sufficient to produce

a dose-response curve and permit an acceptable estimation of the median

lethal dose. Range finding studies using single animals may help to estimate the positioning of the dose groups so that no more than three dose

levels will be necessary.

(B) Limit test. This test is described under paragraph (d)(2)(ii) of this

guideline.

(C) Vehicle. Solids should be pulverized when possible, the test substance should be moistened sufficiently with water or, where necessary,

a suitable vehicle to ensure good contact with skin. If a vehicle or diluent

is needed, it should not elicit toxic effects itself nor substantially alter

the chemical or toxicological properties of the test substance. In addition,

the influence of the vehicle on penetration of skin by the test substance

should be taken into account. It is recommended that wherever possible

the use of an aqueous solution be considered first, followed by consideration of a solution in oil (e.g. corn oil), and then by consideration of possible solution in other vehicles. For nonaqueous vehicles the toxic characteristics of the vehicle should be known, and if not known should be determined before the test.

(iv) Exposure and exposure duration. The test substance should be

administered over a period of 24 h.

(v) Preparation of animal skin. Fur should be clipped from the dorsal area of the trunk of the test animals. Shaving may be employed, but

it should be carried out at least 24 h before dosing. Care must be taken

to avoid abrading the skin, which would alter its permeability.

(vi) Application of test substance. (A) The test substance should

be applied uniformly over a shaved area which is approximately 10 percent

of the body surface area. The area starting at the scapulae (shoulders) to

the wing of the ileum (hip bone) and half way down the flank on each

side of the animal should be shaved. The volume of application should

be kept constant, e.g. less than 300 μL for the rat; different concentrations

of the test solution should be prepared for different dose levels. With highly toxic substances, the surface area covered may be less, but as much

of the area as possible should be covered with as thin and uniform a film

as practical. The test material is not removed after application. In the case

where less than 10 percent of the surface area is covered an approximation

of the exposed areas should be determined.

(B) The test substance should be held in contact with the skin with

a porous gauze dressing (<8 ply) and nonirritating tape throughout a

24–h exposure period. The test site should be further covered in a suitable

manner to retain the gauze dressing and test substance and ensure that
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the animals cannot ingest the test substance. Restrainers may be used to

prevent the ingestion of the test substance, but complete immobilization

is not a recommended method. Although a semiocclusive dressing is preferred, an occlusive dressing will also be acceptable.

(C) At the end of the exposure period, residual test substance should

be removed where practicable using water or an appropriate solvent.

(vii) Observation period. Although 14 days is recommended as a

minimum observation period, the duration of observation should not be

fixed rigidly. It should be determined by the toxic reactions, rate of onset,

and length of recovery period, and may thus be extended when considered

necessary. The time at which signs of toxicity appear, their duration, and

the time to death are important, especially if there is a tendency for deaths

to be delayed.

(viii) Observation of animals. (A) A careful clinical examination

should be made at least once each day.

(B) Additional observations should be made daily, especially in the

early days of the study. Appropriate actions should be taken to minimize

loss of animals to the study (e.g. necropsy or refrigeration of those animals

found dead and isolation of weak or moribund animals).

(C) Observations should be detailed and carefully recorded, preferably

using explicitly defined scales. Observations should include, but not be

limited to, evaluation of skin and fur, eyes and mucous membranes, respiratory and circulatory effects, autonomic effects such as salivation,

central nervous system effects, including tremors and convulsions, changes

in the level of motor activity, gait and posture, reactivity to handling or

sensory stimuli, grip strength, and stereotypies or bizarre behavior (e.g.

self-mutilation, walking backwards).

(D) Individual weights of animals should be determined shortly before

the test substance is administered, weekly thereafter, and at death. Changes

in weights should be calculated and recorded when survival exceeds one

day.

(E) The time of death should be recorded as precisely as possible.

(ix) Gross pathology. (A) At the end of the test, surviving animals

should be weighed and sacrificed.

(B) A gross necropsy should be performed on all animals under test.

All gross pathology changes should be recorded.

(C) If necropsy cannot be performed immediately after a dead animal

is discovered, the animal should be refrigerated (not frozen) at temperatures low enough to minimize autolysis. Necropsies shall be performed

no later than 16 hours after death.
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(x) Additional evaluations. Microscopic examination of organs

showing evidence of gross pathology in animals surviving 24 h or more

should also be considered because it may yield useful information.

(xi) Data and reporting—(A) Treatment of results. Data should

be summarized in tabular form, showing for each test group the number

of animals at the start of the test, body weights, time of death of individual

animals at different dose levels, number of animals displaying other signs

of toxicity, description of toxic effects and necropsy findings. Any methods used for calculation of the LD50 or any other parameters should be

specified and referenced. Methods for parameter estimation are described

under paragraphs (f)(2), (f)(3), and (f)(4) of this guideline.

(B) Evaluation of results. An evaluation should include the relationship, if any, between exposure of the animals to the test substance and

the incidence and severity of all abnormalities, including behavioral and

clinical abnormalities, gross lesions, body weight changes, effects on mortality, and any other toxic effects. The LD50 value should always be considered in conjunction with the observed toxic effects and any necropsy

findings. The LD50 value is a relatively coarse measurement, useful only

as a reference value for classification and labelling purposes, and for an

expression of the lethal potential of the test substance by the ingestion

route. Reference should always be made to the experimental animal species in which the LD50 value was obtained.

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

under 40 CFR part 792, subpart J and 40 CFR part 160, subpart J, the

following specific information should be reported. The test report should

include:

(1) Species, strain, sex,and source of test animals.

(2) Method of randomization in assigning animals to test and control

groups.

(3) Rationale for selection of species, if other than that recommended.

(4) Tabulation of individual and test group data by sex and dose level

(e.g. number of animals exposed, number of animals showing signs of

toxicity and number of animals that died or were killed during the test).

(i) Description of toxic effects, including their time of onset, duration,

reversibility, and relationship to dose.

(ii) Body weights.

(iii) Time of dosing and time of death after dosing.

(iv) Dose-response curves for mortality and other toxic effects (when

permitted by the method of determination).
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(v) Gross pathology findings.

(vi) Histopathology findings and any additional clinical chemistry

evaluations, if performed.

(5) Description of any pre-test conditioning, including diet, quarantine

and treatment for disease.

(6) Description of caging conditions including: Number (or change

in number) of animals per cage, bedding material, ambient temperature

and humidity, photoperiod, and identification of diet of test animals.

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

(8) Relevant properties of substance tested including physical state

and pH (if applicable).

(9) A list of references cited in the body of the report. References

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

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

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

(1) American Society for Testing and Materials. Standard Test Method for Estimating Acute Oral Toxicity in Rats, a standard issued under

the fixed designation E 1163–87, under the jurisdiction of ASTM Committee E–35 on Pesticides and is the direct responsibility of Subcommittee

E3526 on Safety to Man, Philadelphia, PA (1987).

(2) Chanter, D.O. and Heywood, R., The LD50 Test: Some Considerations of Precision, Toxicology Letters 10:303–307 (1982).

(3) Finney, D.J. Chapter 3—Estimation of the median effective dose

and Chapter 4-Maximum likelihood estimation, Probit Analysis, 3rd ed.

Cambridge, London (1971).

(4) Finney, D.J. The Median Lethal Dose and Its Estimation. Archives

of Toxicology 56:215–218 (1985).

(5) Organization for Economic Cooperation and Development. OECD

Guidelines for Testing of Chemicals. Guideline 420: Acute Oral Toxicity—Fixed Dose Method. Endorsed by the Joint Meeting of the Chemicals Group and Management Committee, 20th–22nd November, 1991,

ENV/EPOC(92)15.

(6) Organization for Economic Cooperation and Development. OECD

Guidelines for Testing of Chemicals. Guideline 402: Acute Dermal Toxicity. Adopted: February 24, 1987.