United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–259

June 1996

Health Effects Test

Guidelines

OPPTS 870.8700

Subchronic Oral Toxicity

Test

‘‘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 870.8700 Subchronic oral toxicity test.

(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 is OPPT 40 CFR 795.260 Subchronic Oral

Toxicity Test.

(b) Purpose. In the assessment and evaluation of the toxic characteristics of a test substance, the determination of subchronic oral toxicity may

be carried out after initial information on toxicity has been obtained by

acute testing. The subchronic oral study has been designed to permit the

determination of the no-observed-effect level and toxic effects associated

with continuous or repeated exposure to a test substance for a period of

90 days. The test is not capable of determining those effects that have

a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time. It will

provide information on target organs, the possibilities of accumulation, and

can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

(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.

Cumulative toxicity is the adverse effects of repeated doses occurring

as a result of prolonged action on, or increased concentration of, the administered substance or its metabolites in susceptible tissue.

Dose is the amount of test substance administered. Dose is expressed

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

animal (e.g. expressed as milligrams per kilogram), or as weight of test

substance per unit weight of food or drinking water.

No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level (NOEL) is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed

in terms of the weight of a substance given daily per unit weight of test

animal (expressed as milligrams per kilogram). When administered to animals in food or drinking water, the NOEL is expressed as milligrams per

kilogram of food or milligrams per milliliter of water.

Subchronic oral toxicity is the adverse effects occurring as a result

of the repeated daily exposure of experimental animals to a chemical for

a part (approximately 10 percent for rats) of a life span.
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(d) Principle of the test method. The test substance is administered

orally in graduated daily doses to several groups of experimental animals,

one dose level per group, for a period of 90 days. During the period of

administration the animals are observed daily to detect signs of toxicity.

Animals which die during the period of administration are necropsied. At

the conclusion of the test all animals are necropsied and histopathological

examinations carried out.

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

mice should be used.

(ii) Age. (A) Young adult animals should be employed. At the commencement of the study the weight variation of animals used should not

exceed ± 20 percent of the mean weight for each sex.

(B) Dosing should begin as soon as possible after weaning, before

the animals are 6 weeks old, and in any case not more than 8 weeks old.

(iii) Sex. (A) Equal numbers of animals of each sex should be used

at each dose level.

(B) The females should be nulliparous and nonpregnant.

(iv) Numbers. (A) At least 20 rats and 20 mice (10 females and

10 males of each species) should be used at each dose level.

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

by the number of animals scheduled to be sacrificed before the completion

of the study.

(2) 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 administering the test substance, a vehicle control group. If the

toxic properties of the vehicle are not known or cannot be made available,

both untreated and vehicle control groups are required.

(3) Satellite group. A satellite group of 20 rats and 20 mice (10

females and 10 males of each species) should be treated with the high

dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of not less

than 28 days.

(4) Dose levels and dose selection. (i) In subchronic toxicity tests,

it is desirable to have a dose response relationship as well as no-observedtoxic-effect level. Therefore, at least three dose levels with a control and,

where appropriate, a vehicle control (corresponding to the concentration

of vehicle at the highest exposure level) 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.
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(ii) The highest dose level should result in toxic effects but not

produce an incidence of fatalities which would prevent a meaningful evaluation.

(iii) The lowest dose level should not produce any evidence of toxicity. Where there is a usable estimation of human exposure the lowest

dose level should exceed this.

(iv) The intermediate dose levels should produce minimal observable

toxic effects. If more than one intermediate dose is used, the dose levels

should be spaced to produce a gradation of toxic effects.

(v) The incidence of fatalities in low and intermediate dose groups

and in the controls should be low to permit a meaningful evaluation of

the results.

(5) Exposure conditions. The animals should be dosed with the test

substance on a 7–day per week basis over a period of 90 days. However,

based primarily on practical considerations, dosing by gavage or capsule

studies on a 5–day per week basis should be acceptable.

(6) Observation period. (i) Duration of observation should be for

at least 90 days.

(ii) Animals in the satellite group scheduled for followup observations

should be kept for not less than 28 days without treatment to detect recovery from, or persistence of, toxic effects.

(7) Administration of the test substance. (i) The test substance

should be administered in the diet or in capsules. Alternatively, it may

be administered by gavage or in the drinking water.

(ii) All animals should be dosed by the same method during the entire

experimental period.

(iii) Where necessary, the test substance is dissolved or suspended

in a suitable vehicle. If a vehicle or diluent is needed, it should not elicit

important toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever

possible the usage of an aqueous solution be considered first, followed

by consideration of a solution of oil, and then by possible solution in other

vehicles.

(iv) For substances of low toxicity, it is important to ensure that when

administered in the diet the quantities of the test substance involved do

not interfere with normal nutrition. When the test substance is administered

in the diet, either a constant dietary concentration (given in parts per million) or a constant dose level in terms of animal body weight should be

used; the alternative used should be specified.
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(v) For a substance administered by gavage or capsule, the dose

should be given at similar times each day, and adjusted at intervals (weekly or biweekly) to maintain a constant dose level in terms of animal body

weight.

(8) Observation of animals. (i) Each animal should be handled and

its physical condition appraised at least once each day.

(ii) Additional observation should be made daily with appropriate actions 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).

(iii) Signs of toxicity should be recorded as they are observed including the time of onset, degree, and duration.

(iv) Cage-side observations should include, but not be limited to,

changes in skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity,

and behavior pattern.

(v) Measurements should be made weekly of food consumption or

water consumption when the test substance is administered in the food

or drinking water, respectively.

(vi) Animals should be weighed weekly.

(vii) At the end of the 90–day period all survivors in the nonsatellite

treatment group should be sacrificed. Moribund animals should be removed and sacrificed when noticed.

(9) Clinical examinations. (i) The following examinations should be

made on at least five animals of each sex in each group of rats.

(A) Certain hematology determinations should be carried out just

prior to terminal sacrifice at the end of the test period. The following hematology determinations should be carried out: hematocrit, hemoglobin

concentration, erythrocyte count, total and differential leucocyte count, and

a measure of clotting potential such as clotting time, prothrombin time,

thromboplastin time, or platelet count.

(B) Certain clinical biochemistry determinations should be carried out

just prior to terminal sacrifice at the end of the test period. The following

clinical biochemical test areas should be carried out: Electrolyte balance,

carbohydrate metabolism, and liver and kidney function. The selection of

additional tests should be influenced by observations on the mode of action

of the substance. Suggested additional determinations include calcium,

phosphorus, chloride, sodium, potassium, fasting glucose (with period of

fasting appropriate to the species/breed), serum alanine aminotransferase,

serum aspartate aminotransferase, ornithine decarboxylase, gamma
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glutamyl transpeptidase, urea nitrogen, albumen, blood creatinine, total bilirubin, and total serum protein measurements. 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.

(ii) The following examinations should be made on at least five animals of each sex in each group.

(A) Ophthalmological examination, using an ophthalmoscope or

equivalent suitable equipment, should be made prior to the administration

of the test substance and at the termination of the study. If changes in

the eyes are detected, all animals should be examined.

(B) Urinalysis is required only when there is an indication based on

expected or observed toxicity.

(10) 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) At least the liver, kidneys, adrenals, gonads, and brain should

be weighed wet, as soon as possible after dissection to avoid drying.

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

histopathological examination—all gross lesions, brain—including sections

of medulla/pons, cerebellar cortex and cerebral cortex, pituitary, thyroid/

parathyroid, thymus, lungs, trachea, heart, sternum with bone marrow, salivary glands, liver, spleen, kidneys/adrenals, pancreas, gonads, uterus, accessory genital organs (epididymis, prostrate, and, if present, seminal vesicles), aorta, (skin), (nonrodent gall bladder), esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, urinary bladder, representative

lymph node, (mammary gland), (thigh musculature), peripheral nerve,

(eyes), (femur including articular surface), (spinal cord at three levels—

cervical, midthoracic and lumbar), and, (rodent-exorbital lachrymal

glands).

(11) Histopathology. (i) Full histopathology should be performed on

the organs and tissues, listed under paragraphs (e)(10)(ii) and (e)(10)(iii)

of this guideline of all animals in the control and high-dose groups, and

all animals that died or were killed during the study.

(ii) Histopathology should be performed on all gross lesions in all

animals.

(iii) Histopathology should be performed on target organs in all animals.
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(iv) Histopathology should be performed on the tissues mentioned in

parentheses under paragraph (e)(10)(iii) of this guideline if indicated by

signs of toxicity or target organ involvement.

(v) Histopathology should be performed on lungs, liver, and kidneys

of all animals. Special attention to examination of the lungs should be

made for evidence of infection since this provides a convenient assessment

of the state of health of the animals.

(vi) For the satellite group, histopathology should be performed on

tissues and organs identified as showing 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 the number

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

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

of lesion.

(ii) All observed results, quantitative and incidental, should be evaluated by an appropriate statistical method. Any generally acceptable statistical methods may be used; the statistical methods should be selected during the design of the study.

(2) Evaluation of the study results. (i) The findings of a subchronic

oral toxicity study should be evaluated in conjunction with the findings

of preceding studies and considered in terms of the toxic effects and the

necropsy and histopathological findings. The evaluation should include the

relationship between the dose of the test substance and the presence or

absence, the incidence and severity, of abnormalities, including behavioral

and clinical abnormalities, gross lesions, identified target organs, body

weight changes, effects on mortality and any other general or specific toxic

effects. The test should provide a satisfactory estimation of a no-effect

level.

(ii) In any study which demonstrates an absence of toxic effects, further investigation to establish absorption and bioavailability of the test substance should be considered.

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

in the TSCA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information should be reported:

(i) Group animal data. Tabulation of toxic response data by species,

strain, sex, and exposure level for:

(A) Number of animals dying.

(B) Number of animals showing signs of toxicity.

(C) Number of animals exposed.
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(ii) Individual animal data. (A) Time of death during the study or

whether animals survived to termination.

(B) Time of observation of each abnormal sign and its subsequent

course.

(C) Body weight data.

(D) Food consumption data when collected.

(E) Hematological tests employed and all results.

(F) Clinical biochemistry tests employed and all results.

(G) Necropsy findings.

(H) Detailed description of all histopathological findings.

(I) Statistical treatment of results where appropriate.