United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA 712–C–96–201

June 1996

Health Effects Test

Guidelines

OPPTS 870.3200

Repeated Dose Dermal

Toxicity—21/28 Days

‘‘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 870.3200 Repeated dose dermal toxicity—21/28 days.

(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 OPP 82–2 21–Day Dermal (Pesticide

Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and

Domestic Animals) EPA report 540/09–82–025, 1982; and OECD guideline 410 Repeated Dose Dermal Toxicity: 21–28–Day.

(b) Purpose. A 21/28–day repeated dose dermal study will provide

information on possible health hazards likely to arise from repeated dermal

exposure to a test substance for a period of 21/28 days. This study is

not capable of determining those effects that have a long latency period

for development (e.g., carcinogenicity and life shortening). Extrapolation

from the results of this study to humans is valid only to a limited degree.

It can, however, provide useful information on the degree of percutaneous

absorption, target organs, the possibilities of accumulation, and can be of

use in selecting dose levels for longer-term studies and for establishing

safety criteria for human exposure.

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

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.

Cumulative toxicity is the adverse effect of repeated doses occurring

as a result of prolonged action or increased concentration of the administered test substance or its metabolites in susceptible tissues.

Dose in a 21/28–day repeated dose dermal study is the amount of

test substance applied daily to the skin for 21/28 days. Dose is expressed

as weight of the test substance (grams, milligrams), or as weight of the

test substance per unit body weight of test animal (milligrams per kilogram), or as weight of test substance per unit of surface area (milligrams

per square centimeter)

No-observed-effect level (NOEL) is the maximum dose used in a study

which produces no adverse effects. The NOEL level is expressed in terms

of the weight of a test substance given daily per unit weight of test animal

(milligrams per kilogram per day).

The term repeated dose dermal study is used to embrace the toxic

effects associated with repeated doses of a chemical over part of a life

span of the test animal. Dosing periods lying between a single dose and

10 percent of life span are often referred to as subacute. The term subacute

is semantically incorrect. To distinguish such a dosing period from the
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classical subchronic period, it may be described as short-term repeated

dose study. Study durations have been 14, 21, or 28 days.

Target organ is any organ of a test animal showing evidence of an

effect induced by a test substance.

(d) Limit test. If a test at one dose level of at least 1,000 mg/kg

body weight (expected human exposure may indicate the need for a higher

dose level), using the procedures described for this guideline, produces

no observable toxic effects, or if toxic effects would not be expected based

upon data on structurally related compounds, a full study using three dose

levels might not be necessary.

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

A mammalian species should be used for testing. The rat, rabbit, or guinea

pig may be used. Commonly used laboratory strains should be employed.

If other mammalian species are used, the tester should provide justification/reasoning for their selection. When a subchronic dermal study is conducted as a preliminary to a chronic study, the same species and strain

should be used in both studies.

(ii) Age. (A) Testing should be started with young healthy animals

as soon as possible after weaning and acclimatization.

(B) Dosing should generally begin no later than 8 weeks of age.

(C) At the commencement of the study, the weight variation of animals used should not exceed ± 20 percent of the mean weight for each

sex.

(iii) Sex. Equal numbers of animals of each sex with healthy skin

should be used at each dose level. The females should be nulliparous and

nonpregnant except for specially designed studies.

(iv) Numbers. (A) At least 20 animals (10 animals per sex) should

be used at each dose level.

(B) If interim sacrifices are planned, the number should be increased

by the number of animals scheduled to be sacrificed before completion

of the study.

(C) To avoid bias, the use of adequate randomization procedures for

the proper allocation of animals to test and control groups is required.

(D) Each animal must be assigned a unique identification number.

Dead animals, their preserved organs and tissues, and microscopic slides

should be identified by reference to an animal’s unique number.

(v) Husbandry. (A) Animals should be housed in individual cages.
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(B) The temperature of the experimental animal rooms should be at

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

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

be 30 to 70 percent.

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

12 h dark.

(E) Control and test animals should be fed from the same batch and

lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the

outcome of the test. Animals should be fed and watered ad libitum with

food replaced at least weekly. For nonrodents feeding should be at least

daily and water ad libitum.

(F) The study should not be initiated until animals have been allowed

a period of acclimatization/quarantine to environmental conditions, nor

should animals from outside sources be placed on test without an adequate

period of quarantine.

(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, the vehicle should not elicit toxic effects or substantially

alter the chemical or toxicological properties of the test substance.

(ii) One lot of the test substance should be used if possible, throughout the duration of the study, and the research sample should be stored

under conditions that maintain its purity and stability. Prior to the initiation

of the study, there should be a characterization of the test substance, including the purity of the test compound, the amount of contaminants and

impurities, and their identity, if possible.

(iii) If the test or control substance is to be incorporated into feed

or another vehicle, the period during which the test substance is stable

in such a mixture should be determined prior to the initiation of the study.

Its homogeneity and concentration should be determined prior to the initiation of the study and periodically during the study. Statistically randomized samples of the mixture should be analyzed to ensure that proper mixing, formulation, and storage procedures are being followed, and that the

appropriate concentration of the test or control substance is contained in

the mixture.

(3) Control groups. A concurrent control group is required. This

group should be an untreated or sham-treated control group or, if a vehicle

is used in the application of the test substance, a vehicle control group.

If the toxic properties of the vehicle are not known or not available, both

untreated/sham-treated and vehicle control groups are required.
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(4) Satellite group. A satellite group of 20 animals (10 animals per

sex) may be treated with the high dose level for 21/28 days and observed

for reversibility, persistence, or delayed occurrence of toxic effects for a

post-treatment period of 14 days. In addition a control group of 20 animals

(10 animals of each sex) should be added to the satellite study.

(5) Dose levels and dose selection. (i) For the repeated dose study,

it is desirable to determine a dose-response relationship as well as a

NOEL. Therefore, at least three dose levels plus a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle

at the highest dose level) group should be used. Doses should be spaced

appropriately to produce test groups with a range of toxic effects. The

data should be sufficient to produce a dose-response curve.

(ii) The highest dose level should result in toxic effects but not

produce severe skin irritation or an incidence of fatality which would prevent a meaningful evaluation. If application of the test substance produces

severe skin irritation, the concentration may be reduced, although this may

result in a reduction in, or absence of, other toxic effects at the high dose

level. If the skin has been badly damaged early in the study, it may be

necessary to terminate the study and undertake a new one at lower concentrations.

(iii) The intermediate dose levels should be spaced to produce a gradation of toxic effects.

(iv) The lowest dose level should not produce any evidence of toxic

effects.

(6) Preparation of animal skin. Fur should be clipped from not less

than 10 percent of the body surface area shortly before testing for application of the test substance. In order to dose approximately 10 percent of

the body surface, the area starting at the scapulae (shoulders) to the wing

of the ileum (hipbone) and half way down the flank on each side of the

animal should be shaved. Shaving should be carried out approximately

24 h before the test. Repeated clipping or shaving is usually needed at

approximately weekly intervals. When clipping or shaving the fur, care

should be taken to avoid abrading the skin (which could alter its permeability).

(7) Preparation of test substance. (i) Liquid test substances are generally used undiluted, except as indicated in paragraph (e)(5)(ii) of this

guideline.

(ii) Solids should be pulverized when possible. The substance should

be moistened sufficiently with water or, when necessary, a suitable vehicle

to ensure good contact with the skin. When a vehicle is used, the influence

of the vehicle on toxicity of, and penetration of the skin by, the test substance should be taken into account.
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(iii) The volume of application should be kept constant, e.g. less than

300 μL for the rat; different concentrations of test solution should be prepared for different dose levels.

(8) Administration of test substance. (i) The duration of exposure

should be at least for 21/28 days.

(ii) The animals should be treated with test substance for at least 6

h/day on a 7–day per week basis. However, based on practical considerations, application on a 5–day per week basis is acceptable. Dosing should

be conducted at approximately the same time each day.

(iii) The test substance should be applied uniformly over the treatment

site.

(iv) The surface area covered may be less for highly toxic substances.

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

as possible.

(v) During the exposure period, the test substance should be held in

contact with the skin with a porous gauze dressing. The test site should

be further covered with nonirritating tape to retain the gauze dressing and

the test substance and to ensure that the animals cannot ingest the test

substance. Restrainers may be used to prevent the ingestion of the test

substance, but complete immobilization is not recommended.

(9) Observation period. The animals should be observed for a period

of 21/28 days. Animals in the satellite group, (if one is used) scheduled

for follow-up observations should be kept for at least 14 days further without treatment to assess reversibility.

(10) Observation of animals. (i) Observations should be made at

least once each day for morbidity and mortality. 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 or sacrifice of

weak or moribund animals).

(ii) A careful clinical examination should be made at least once weekly. 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).

(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree, and duration.
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(iv) Individual weights of animals should be determined shortly before the test substance is administered, weekly thereafter, and at death.

(v) Food consumption should also be determined weekly if abnormal

body weight changes are observed.

(vi) Moribund animals should be removed and sacrificed when noticed. The time of death should be recorded as precisely as possible.

(vii) At termination, all survivors in the treatment groups should be

sacrificed.

(11) Clinical pathology. Hematology and clinical chemistry examinations should be made on all animals, including controls, of each sex in

each group at the end of the study.

(i) Hematology. The recommended parameters are: Hemoglobin and

hematocrit concentrations, red blood cell count, white blood cell count,

differential leukocyte count, platelet count, and a measure of clotting potential such as prothrombin time or thromboplastin time.

(ii) Clinical chemistry. Parameters which are considered appropriate

to all studies are electrolyte balance, carbohydrate metabolism, and liver

and kidney function. The selection of specific tests will be influenced by

observations on the mode of action of the substance and signs of clinical

toxicity. Suggested blood clinical chemistry determinations:

(A) Electrolytes.

(1) Calcium.

(2) Chloride.

(3) Magnesium.

(4) Phosphorus.

(5) Potassium.

(6) Sodium.

(B) Enzymes.

(1) Alkaline phosphatase.

(2) Alanine aminotransferase.

(3) Aspartate aminotransferase.

(4) Gamma glutamyl transferase.

(5) Sorbitol dehydrogenase.
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(C) Other.

(1) Albumin.

(2) Blood creatinine.

(3) Blood urea nitrogen.

(4) Globulin.

(5) Glucose (fasting) .

(6) Total bilirubin.

(7) Total cholesterol.

(8) Total serum protein.

Other determinations which may be necessary for an adequate toxicological evaluation include analyses of lipids, hormones, acid/base balance, methemoglobin and cholinesterase activity. Additional clinical biochemistry may be employed where necessary to extend the investigation

of observed effects.

(iii) Urinalysis is not recommended on a routine basis, but only when

there is an indication based on expected OR observed toxicity.

(12) Optional immunotoxicity screen. In partial fulfillment of requirements for an immunotoxicity screen, subpopulations of splenic or peripheral blood lymphocytes in the rodents should be enumerated and quantified. Total T-, Total B-, Total T-helper, T-suppressor/cytotoxic and Natural Killer (NK) cell populations should be determined on at least 10 rodents of each sex in each group at the end of 21/28 days.

(13) Ophthalmological examination. Ophthalmological examinations using an ophthalmoscope or an equivalent device should be made

on all animals prior to the administration of the test substance and on

all high dose and control groups at termination. If changes in the eyes

are detected, all animals in the other dose groups should be examined.

(14) Gross necropsy. (i) All animals should be subjected to a full

gross necropsy which includes examination of the external surface of the

body, all orifices, and the cranial, thoracic and abdominal cavities and their

contents.

(ii) The liver, lungs, brain, kidneys, spleen, thymus, and gonads

should be trimmed and weighed wet, as soon as possible after dissection.

(iii) The following organs and tissues, or representative samples thereof, should be preserved in a suitable medium for possible future

histopathological examination:
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(A) Digestive system.

(1) Salivary glands.

(2) Esophagus.

(3) Stomach.

(4) Duodenum.

(5) Jejunum.

(6) Ileum.

(7) Cecum.

(8) Colon.

(9) Rectum.

(10) Liver.

(11) Pancreas.

(12) Gall bladder (dogs).

(B) Nervous system.

(1) Brain (multiple sections).

(2) Pituitary.

(3) Peripheral nerve(s).

(4) Spinal cord (three levels).

(5) Eyes (retina, optic nerve).

(C) Glandular system.

(1) Adrenals.

(2) Parathyroids.

(3) Thyroids.

(D) Respiratory system.

(1) Trachea.

(2) Lung.

(3) Pharynx.

(4) Larynx.
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(5) Nose.

(E) Cardiovascular/Hematopoieitic system.

(1) Aorta (thoracic).

(2) Heart.

(3) Bone marrow.

(4) Lymph nodes.

(5) Spleen.

(6) Thymus.

(F) Urogenital system.

(1) Kidneys.

(2) Urinary bladder.

(3) Prostate.

(4) Testes.

(5) Epididymides.

(6) Seminal vesicles.

(7) Uterus.

(8) Ovaries.

(G) Others.

(1) All gross lesions and masses.

(2) Skin.

(3) Sternum and/or femur

(15) Histopathology. (i) The following histopathology should be performed:

(A) Full histopathology on the organs and tissues, listed under paragraph (e)(14)(iii) of this guideline, of all animals in the control and high

dose groups.

(B) All gross lesions in all animals.

(C) Target organs in all animals.

(D) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents should be made for evidence of infection
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as this provides a convenient assessment of the state of health of the animals.

(ii) If excessive early deaths or other problems occur in the high dose

group, compromising the significance of the data, the next dose level

should be examined for complete histopathology.

(iii) An attempt should be made to correlate gross observations with

microscopic findings.

(iv) Tissues and organs designed for microscopic examination should

be fixed in 10 percent buffered formalin or a recognized suitable fixative

as soon as necropsy is performed and no less than 48 hours prior to trimming. Tissues should be trimmed to a maximum thickness of 0.4 cm for

processing.

(v) When a satellite group is used, histopathology should be performed on tissues and organs identified as showing toxic effects in the

treated groups.

(f) Data and reporting—(1) Treatment of results. (i) Data should

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

animals at the start of the test, the number of animals showing lesions,

the types of lesions and the percentage of animals displaying each type

of lesion.

(ii) All observed results, (quantitative and qualitative) should be evaluated by an appropriate statistical method. Any generally accepted statistical method should be used; the statistical methods including significance

criteria should be selected during the design of the study.

(2) Evaluation of study results. The findings including observed

toxic effects, necropsy, and histopathological changes should be evaluated.

The evaluation should include the relationship between the dose of the

test substance, the incidence and severity of abnormalities including behavioral and clinical abnormalities, gross lesions, identified target organs,

body weight changes, effect on mortality and any other general or specific

toxic effects. A properly conducted 21/28–day repeated dose dermal study

will provide information on the effects of repeated application of a substance and a satisfactory estimation of a NOEL. It also can indicate the

need for an additional longer-term study and provide information on the

selection of dose levels.

(3) Test report. In addition to reporting requirements specified under

EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart

J and 40 CFR part 160, and the OECD principles of GLP (ISBN 92–

64–12367–9), the following specific information should be reported:

(i) Test substance characterization should include:.
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(A) Chemical identification.

(B) Lot or batch numbers

(C) Physical properties.

(D) Purity/impurities.

(E) Identification and composition of any vehicle if used.

(ii) Test system should contain data on:

(A) Species and strain of animals used and rationale for selection

if other than that recommended.

(B) Age including body weight data and sex.

(C) Test environment including cage conditions, ambient temperature,

humidity, and light/dark periods.

(D) Identification of animal diet.

(iii) Test procedure should include the following data:

(A) Method of randomization used.

(B) Full description of experimental design and procedure.

(C) Dose regime including levels, method, and volume.

(iv) Test results should include:

(A) Group animal data: Tabulation of toxic response data by species,

strain, sex and exposure level for:

(1) Number of animals exposed.

(2) Number of animals showing signs of toxicity.

(3) Number of animals dying.

(B) Individual animal data. Data should be presented as summary

(group mean) as well as for individual animals.

(1) Date of death during the study or whether animals survived to

termination.

(2) Date of observation of each abnormal sign and its subsequent

course.

(3) Body weight data.

(4) Feed consumption data, when collected.

(5) Results of ophthalmological examination, when performed.
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(6) Results of the hematology tests performed.

(7) Results of the clinical chemistry tests performed.

(8) Results of the immunochemistry screen, when performed.

(9) Necropsy findings, including absolute and relative organ weight

data.

(10) Detailed description of all histopathological findings.

(11) Statistical treatment of results, where appropriate.

(g) Quality control. A system should be developed and maintained

to assure and document adequate performance of laboratory staff and

equipment. The study must be conducted in compliance with GLP regulations.

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

(1) Draize, J.H. Dermal toxicity, Appraisal of Chemicals in Food,

Drugs and Cosmetics. The Association of Food and Drug Officials of the

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

(2) 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 (1977).

(3) Organization for Economic Co-Operation and Development.

Guidelines for Testing of Chemicals, Section 4—Health Effects, Part 410,

Repeated Dose Toxicity Study, Paris (1981).

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

6, Principles and Methods for Evaluating the Toxicity of Chemicals.

(World Health Organization, Geneva) (1978).