United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–200

June 1996

Health Effects Test

Guidelines

OPPTS 870.3150

Subchronic Nonrodent

Oral Toxicity—90–Day

‘‘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 870.3150 Subchronic nonrodent oral toxicity—90–day.

(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 is OPP 82–1 90–Day Oral—Two Species,

Rodent and Nonrodent (Pesticide Assessment Guidelines, Subdivision F—

Hazard Evaluation; Human and Domestic Animals) EPA report 540/09–

82–025, 1982; and OECD 409 Subchronic Oral Toxicity—Nonrodent: 90–

Day.

(b) Purpose. The determination of subchronic oral toxicity in the assessment and evaluation of the toxic characteristics of a chemical 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 (NOEL) 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 provides 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 test 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. 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 is the maximum dose used in a test which produces

no observed adverse effects. A NOEL is expressed in terms of the weight

of a substance given daily per unit weight of test animal (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.
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Subchronic oral toxicity is the adverse effects occurring as a result

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

the oral route for a part (approximately 10 percent) of a life span.

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

body weight (BW) (expected human exposure may indicate the need for

a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected

based upon data of structurally related compounds, a full study using three

dose levels might not be necessary.

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

A mammalian species should be used for testing. The commonly used

nonrodent species is the dog, preferably of a defined breed; the beagle

is frequently used. If other mammalian species are used, the tester should

provide justification/reasoning for his or her selection.

(ii) Age—(A) Young adult animals should be used. 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) In the case of the dog, dosing should commence after acclimation,

preferably at 4 to 6 mon and not later than 9 mon of age.

(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 eight animals (four females and four

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

it is desirable to have a dose response relationship as well as a no-observed-toxic-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.

(ii) There should be no fatalities at the highest dose level.

(iii) The lowest dose level should not produce any evidence of toxicity. The lowest dose level should exceed a usable estimation of human

exposure if available.

(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) There should be no fatalities in the low and intermediate dose

groups.

(4) Exposure conditions. The animals are 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 in gavage or capsule

studies on a 5–day per week basis is considered to be acceptable.

(5) 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 at least 28 days further without treatment to detect

recovery from, or persistence of, toxic effects.

(6) Administration of the test substance. (i) The test substance may

be administered in the diet or in capsules.

(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 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
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in the diet either a constant dietary concentration (parts per million) or

a constant dose level in terms of the BW of the animals should be used;

the alternative used should be specified.

(v) For a substance administered by gavage or capsule, the dose

should be given at approximately the same time each day, and adjusted

at intervals (weekly or biweekly) to maintain a constant dose level in terms

of animal BW.

(7) Observation of animals. (i) Each animal should be observed

daily and, if necessary, handled to appraise its physical condition.

(ii) Additional observations 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 feed consumption or

water consumption when the test substance is administered in the feed

or drinking water.

(vi) Animals should be weighed weekly.

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

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

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

made on all animals of each sex in each group.

(A) Certain hematology determinations should be carried out at least

two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice

at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte

count, total and differential leukocyte count, and a measure of clotting

potential such as clotting time, prothrombin time, thromboplastin time, or

platelet count.

(B) Certain clinical biochemistry determinations on blood should be

carried out at least two times during the test period on all groups of animals including concurrent controls—after 30 days of test and just prior
5

to terminal sacrifice at the end of the test period. Clinical biochemistry

test areas 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. Suggested determinations include calcium,

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

fasting appropriate to the species), serum alanine aminotransferase, as

serum aspartate aminotransferase, ornithine decarboxylase, gamma

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 high dose and

control groups. If changes in the eyes are detected, all animals should

be examined.

(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.

(B) Urinalysis is not recommended on a routine basis, and should

be carried out only when there is an indication based on expected and

or observed toxicity.

(9) 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, and gonads should be

weighed wet as soon as possible after dissection to avoid drying. In addition, for the nonrodent, the thyroid with parathyroids also should be

weighed wet.

(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; lungs—which should be

removed intact, weighed, and treated with a suitable fixative to ensure

that lung structure is maintained (perfusion with the fixative is considered

to be an effective procedure); nasopharyngeal tissues; brain—including

sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary;

thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow;

salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus;

accessory genital organs (epididymis, prostate, and, if present, seminal

vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representa-
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tive lymph node; (mammary gland); (thigh musculature); peripheral nerve;

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

cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal

glands).

(10) Histopathology. The following histopathology should be performed:

(i) Full histopathology on the organs and tissues, listed above, of all

nonrodents.

(ii) All gross lesions in all animals.

(iii) Target organs in all animals.

(iv) The tissues listed above if indicated by signs of toxicity of target

organ involvement.

(v) Lungs, liver, and kidneys of all animals.

(g) 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 types 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 accepted 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 will 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, BW

changes, effects on mortality and any other general or specific toxic effects. A properly conducted subchronic test should provide a satisfactory

estimation of a no-effect-level.

(ii) Further investigation to establish absorption and bioavailability

of the test substance should be considered in any study which demonstrates

an absence of toxic effects.

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

under 40 CFR part 792, subpart J (Good Laboratory Practice Standards)

the following specific information should be reported:
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(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.

(ii) Individual animal data. (A) Date of death during the study or

whether animals survived to termination.

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

course.

(C) BW data.

(D) Feed 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.

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

(1) Boyd, E.M., Chapter 14—Pilot Studies, 15—Uniposal Clinical Parameters, 16—Uniposal Autopsy Parameters, in Predictive Toxicometrics.

Williams and Wilkins, Baltimore, MD (1972).

(2) Fitzhugh, O.G., Subacute Toxicity, in Appraisal of the Safety of

Chemicals in Foods, Drugs and Cosmetics. The Association of Food and

Drug Officials of the United States (1959, 3rd Printing 1975) pp. 26–35.

(3) Food Safety Council. Subchronic Toxicity Studies, in Proposed

System for Food Safety Assessment. Food Safety Council, Columbia (1978)

pp. 83–96.

(4) 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).

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

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

World Health Organization, Geneva (1978).