United States Prevention, Pesticides EPA712-C-96-200
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 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 870.3150 Subchronic nonrodent oral toxicity—90-day.
(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 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 as-
sessment 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 estab-
lishing 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 ad-
ministered 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-ob-
served-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, pro-
duces 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 commence-
ment 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-ob-
served-toxic-effect level. Therefore, at least three dose levels with a control
and, where appropriate, a vehicle control (corresponding to the concentra-
tion of vehicle at the highest exposure level) should be used. Doses should
be spaced appropriately to produce test groups with a range of toxic ef-
fects. 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 tox-
icity. 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 sub-
stance 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 toxi-
cological 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 includ-
ing 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, cir-
culatory, 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 re-
moved 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 concur-
rent controls: After 30 days of test and just prior to terminal sacrifice
at the end of the test period. Hematology determinations which are appro-
priate 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 ani-
mals including concurrent controls—after 30 days of test and just prior
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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 se-
lection 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 bil-
irubin, 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 nec-
essary, 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 addi-
tion, for the nonrodent, the thyroid with parathyroids also should be
weighed wet.
(iii) The following organs and tissues, or representative samples there-
of, 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; duo-
denum; 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 per-
formed:
(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 evalu-
ated 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 ef-
fects. 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 ad-
ditional background information on this test guideline.
(1) Boyd, E.M., Chapter 14—Pilot Studies, 15—Uniposal Clinical Pa-
rameters, 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).
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