United States Prevention, Pesticides EPA712-C-96-259
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA 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 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 870.8700 Subchronic oral toxicity test.
(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 material used in developing this har-
monized OPPTS test guideline is OPPT 40 CFR 795.260 Subchronic Oral
Toxicity Test.
(b) Purpose. In the assessment and evaluation of the toxic character-
istics 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 short-
ening). It provides information on health hazards likely to arise from re-
peated 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 establish-
ing 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 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-ob-
served-effect level (NOEL) is the maximum dose used in a test which pro-
duces 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 ani-
mals 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 com-
mencement 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 de-
layed 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-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 ef-
fects. 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 eval-
uation.
(iii) The lowest dose level should not produce any evidence of tox-
icity. 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 recov-
ery 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 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 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 mil-
lion) 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 (week-
ly 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 ac-
tions 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 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 re-
moved 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 he-
matology 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 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 at least five ani-
mals 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 there-
of, 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, sali-
vary glands, liver, spleen, kidneys/adrenals, pancreas, gonads, uterus, ac-
cessory genital organs (epididymis, prostrate, and, if present, seminal vesi-
cles), aorta, (skin), (nonrodent gall bladder), esophagus, stomach, duode-
num, 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 ani-
mals.
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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 evalu-
ated by an appropriate statistical method. Any generally acceptable statis-
tical methods may be used; the statistical methods should be selected dur-
ing 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, fur-
ther investigation to establish absorption and bioavailability of the test sub-
stance 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, sub-
part 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.
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