United States       Prevention, Pesticides     EPA712-C-96-200
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.3150
          Subchronic Nonrodent
          Oral Toxicity—90-Day
                'Public Draft"

     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

     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:

     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,  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

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-

     (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.

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.

    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.

     (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

     (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

     (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  (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

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-

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

     (10)  Histopathology. The following histopathology  should be per-

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

     (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:

     (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
     (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).