United States       Prevention, Pesticides     EPA712-C-96-259
          Environmental Protection    and Toxic Substances     June 1996
          Agency         (7101)
&EPA    Health Effects Test
           OPPTS 870.8700
           Subchronic Oral Toxicity
                 '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 870.8700   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.

     (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.

     (ii)  The highest dose level  should result in toxic effects  but not
produce an incidence of fatalities which would prevent a meaningful  eval-

     (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

     (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.

     (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

     (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

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

     (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

     (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

     (iii) Histopathology should be performed  on target  organs in all  ani-

     (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

     (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.

     (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
     (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.