United States       Prevention, Pesticides     EPA712-C-98-200
          Environmental Protection    and Toxic Substances     August 1998
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.3150
          90-Day Oral Toxicity in

    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, chap-
ter I, subchapter R of the Code  of Federal Regulations (CFR), the Office
of Pesticide Programs (OPP) which appeared in publications of  the Na-
tional 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. 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.).

    Final  Guideline Release: This guideline is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This guideline is also available electronically
in PDF (portable document format) from EPA's World  Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.3150 90-day oral toxicity in nonrodents.
     (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). Extrapolation from the results  of  this study to humans
is valid only to  a limited  degree. However, it  can be useful in providing
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 select-
ing 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  ad-
ministered test substance or its metabolites in susceptible tissue.

     Dose  in an oral subchronic  study is  the  amount  of test substance
administered daily via the oral route (gavage, capsules, diet or  drinking
water) for 90 days. 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
per day.

     No-observed-effect-level (NOEL) 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).

    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 of the test animal's life span.

    Target organ is any organ of a test animal showing evidence of an
effect induced by the test substance.

    (d) 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.

    (e) Test procedures—(1) Animal  selection—(i) Species and strain.
A mammalian  nonrodent species should be used for testing.  The com-
monly 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/weight. (A) Young adult animals should be used.

    (B) In the case of the dog, dosing should commence after acclimation,
preferably at 4 to 6 months and not later than 9 months of age.

    (C)At the commencement of the study the weight variation of animals
used should be within 20 percent of the  mean weight for each sex.

    (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.

    (C) To  avoid bias, the use of adequate randomization procedures for
the proper allocation  of animals to  test and control groups is required.

    (D) Each animal should be assigned a unique identification number.
Dead  animals, their preserved organs and tissues and microscopic slides
should be identified by reference to the animal's unique number.

    (v) Husbandry. (A) Caging and environmental conditions should be
appropriate to the nonrodent species.  However, it  is  recommended  that
dogs are  housed individually. The  number  of animals per cage must not
interfere with a clear observation of each animal.

     (B) For feeding, conventional laboratory diets may be used with an
unlimited supply of drinking water. The choice  of diet may be influenced
by the  need to  ensure  a suitable admixture  of the test substance when
administered by this method.

     (C) Control and test animals should be fed from the same batch and
lot. The feed should be  analyzed to assure adequacy of nutritional require-
ments of the species tested for impurities that might influence the outcome
of the  test.  For  feeding, conventional laboratory  diets may be used with
an unlimited supply of drinking water.

     (D) The study should not be initiated until animals have been allowed
a period  of acclimatization/quarantine to environmental conditions, nor
should animals from outside sources be placed on test without an adequate
period  of quarantine. An acclimation  period of at least 5 days  is  rec-

     (2) Control and test substances, (i) Where necessary, the test sub-
stance  is dissolved or suspended in a suitable  vehicle. If a vehicle or dilu-
ent is needed, the  vehicle should not elicit toxic effects or substantially
alter the chemical  or toxicological properties of the test substance. It  is
recommended that whenever possible  the usage  of an aqueous  solution
be considered first, followed by consideration of a solution of oil and then
solution in other vehicles.

     (ii) If possible, one lot of the test substance tested should  be used
throughout the duration of the  study and the research sample should be
stored  under conditions that maintain its purity and stability. Prior to the
initiation  of the  study,  there should be characterization of the test sub-
stance,  including the purity of the test compound and, if technically fea-
sible, the names  and quantities of contaminants and impurities.

     (iii) If the  test or  control  substance is to  be incorporated into feed
or another vehicle, the period during which  the test substance is stable
in such a mixture should be determined prior to the initiation of the study.
Its homogeneity and concentration should be determined prior to the initi-
ation of the study and periodically during the study. Statistically random-
ized samples of the mixture should be analyzed  to ensure that proper mix-
ing,  formulation and storage procedures are being followed, and that the
appropriate  concentration of the test or control substance is contained in
the mixture.

     (3) 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.

     (4) Satellite group. A satellite group of eight animals (four animals
per sex) may be treated with the high dose level for 90 days and observed
for reversibility, persistence, or delayed occurrence of toxic effects for a
post-treatment period of appropriate length, normally not less than 28 days.
In addition, a control group of 8 animals (4 animals per sex) should be
added to the satellite study.

     (5) Dose levels  and dose selection, (i) In subchronic toxicity tests,
it is desirable to have  a dose response  relationship  as  well as  a NOEL.
Therefore,  at least three dose levels with a control and, where appropriate,
a vehicle control (corresponding to the concentration of vehicle  at the high-
est 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) The highest dose level  should result in toxic effects but not
produce an incidence of fatalities which would prevent a meaningful eval-

     (iii) The intermediate  dose levels should be  spaced to produce a gra-
dation of toxic effects.

     (iv) The lowest  dose  level should not produce any evidence of tox-

     (6) Administration of the test substance, (i) The test substance may
be administered in the diet, drinking water, by gavage or in capsules. Ideal-
ly, if the test substance is administered by gavage or in capsules, the ani-
mals should  be dosed with the test material on a 7-day per  week basis
for a period  of at  least 90 days.  However, based primarily on practical
considerations,  dosing by gavage or with capsules  on a 5-day per week
basis is acceptable. If the  test substance is administered in the drinking
water or mixed in the diet,  then exposure should be  on a 7-day per week

     (ii) All animals should be dosed by the same method during the entire
experimental period.

     (iii) 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 body weight of the animals should
be used; the alternative used should be specified.

     (iv) For a  substance  administered by  gavage or  capsules, 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 body weight.

     (7) Observation period, (i)  Duration of observation should be for
at least 90 days.

     (ii) Animals in the  satellite group (if used) scheduled for follow-up
observations should be kept for at least 28 days further without treatment
to detect recovery from, or persistence of, toxic effects.

     (8) Observation  of animals, (i)  Each animal  should  be observed
twice daily for morbidity and mortality. Appropriate actions should be
taken to minimize loss of animals to the study (e.g., necropsy or refrigera-
tion of  those  animals  found dead  and isolation or sacrifice  of weak or
moribund animals.) General clinical observations should be made at least
once a day, preferably at the same time each day, taking into consideration
the  peak period of  anticipated effects after dosing. The clinical condition
of the animal should be recorded.

     (ii) A careful clinical examination should be made prior to the initi-
ation of treatment and at least once weekly during treatment. Detailed ob-
servations  should be made on all  animals. These observations should be
made, where practical, outside the home cage in a standard arena and pref-
erably at similar times on each occasion. Effort should be made to ensure
that variations in the observation conditions are minimal. Signs of toxicity
should be carefully  recorded, including time of onset,  degree and duration.
Observations should include, but not be limited  to, changes in skin, fur,
eyes, mucous membranes, occurrence of secretions and excretions, and au-
tonomic activity (e.g., lacrimation, piloerection,  pupil size,  unusual res-
piratory pattern).  Changes in level  of  activity,  gait,  posture,  altered
strength, and response to handling as well as the presence of clonic or
tonic movements,  stereotypies (e.g., excessive grooming, repetitive cir-
cling) or bizarre behavior (e.g., self-mutilation) should be recorded.

     (iii) Measurements of feed  consumption  and  water  consumption,
when drinking water is the exposure route, should be made weekly.

     (iv) Animals should be weighed shortly  before the test substance is
administered and weekly during the treatment period.

     (v) Moribund animals should be removed and sacrificed when noticed
and the time of death should be recorded as precisely as possible.

     (vi) At the end of the 90-day period all survivors in the nonsatellite
control and treatment groups should be sacrificed.

     (9) Clinical pathology. Hematology  and  clinical chemistry examina-
tions  should be made  on all animals, including  controls, of  each sex in
each group. The hematology and clinical  chemistry parameters should be
examined prior to treatment, either at monthly intervals or midway through
the  treatment period, and at the end of the treatment period on all groups
of animals, including concurrent controls.  Overnight fasting of the animals

prior to blood sampling is recommended.  Overall, there  is a need for a
flexible approach in the measures examined, depending on the observed
or expected effects from a chemical, and  in the frequency of measures,
depending on the duration of potential chemical exposures.

    (i) Hematology. The  recommended parameters are red blood  cell
count,  hemoglobin  concentration,  hematocrit, mean corpuscular volume,
mean corpuscular  hemoglobin,  and mean corpuscular hemoglobin con-
centration,  white blood  cell  count,  differential  leukocyte count, platelet
count,  and  a measure of clotting potential, such as prothrombin time and
activated partial thromboplastin time.

    (ii) Clinical chemistry. (A) 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
and signs of clinical toxicity.

    (B)  The recommended clinical chemistry determinations  are potas-
sium, sodium, calcium,  phosphorus,  chloride, glucose, total cholesterol,
urea nitrogen, creatinine, total protein, total bilirubin, and albumin.  Sug-
gested hepatic  enzymes  include  alanine  aminotransferase,  aspartate
aminotransferase,  alkaline  phosphatase,  sorbitol  dehydrogenase,   and
gamma glutamyl transpeptidase. Measurements of addtional enzymes (of
hepatic or other origin) and bile acids may also be useful.

    (C)  If a test  chemical has an  effect  on the hematopoietic system,
reticulocyte counts and bone marrow cytology may be indicated.

    (D) Other determinations that should be carried out if the test chemi-
cal is known or suspected of affecting  related  measures include fasting
triglycerides, hormones, methemoglobin, and cholinesterases.

    (iii) Urinalysis should  be  performed prior  to treatment,  midway
through treatment and at the end of the study using timed urine  collection.
Urinalysis determinations include:  appearance, volume, osmolality or  spe-
cific gravity, pH, protein, glucose, and blood/blood cells.

    (10) Ophthalmological examination. Ophthalmological examination,
using an ophthalmoscope  or equivalent suitable equipment,  should be
made on all animals prior to the administration  of the test substance and
at termination of the study, preferably in all animals but at least the high
dose and control groups. If changes in the eyes are detected,  all animals
in the other dose groups should be examined.

    (11) 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 (with gall bladder), kidneys, adrenals, testes,
epididymides, ovaries, uterus, thyroid (with parathyroid), thymus,  spleen,
brain, and heart 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:

     (A)  Digestive  system—salivary glands, esophagus, stomach,  duode-
num, jejunum, ileum, cecum, colon, rectum,  liver, pancreas, gallbladder.

     (B)  Nervous  system—brain (multiple  sections,  including  cerebrum,
cerebellum and medulla/pons), pituitary, peripheral nerve (sciatic or tibia,
preferably in close proximity to the muscle), spinal cord (three levels, cer-
vical, mid-thoracic and lumbar), eyes (retina, optic nerve).

     (C) Glandular system—adrenals, parathyroid, thyroid.

     (D) Respiratory system—trachea, lungs, pharynx, larynx, nose.

     (E) Cardiovascular/hematopoietic system—aorta, heart, bone marrow
(and/or a fresh aspirate), lymph nodes (preferably one lymph node cover-
ing the route of administration and  another one  distant  from the route of
administration to cover systemic effects), spleen, thymus.

     (F)  Urogenital system—kidneys,  urinary  bladder, prostate,  testes,
epididymides, seminal vesicle(s), uterus, ovaries, female mammary gland.

     (G) Other—all gross lesions and masses, skin.

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

     (i) Full histopathology on the organs and tissues, listed in paragraph
(e)(ll)(iii)  of this  guideline,  in at  least all animals in the control- and
high-dose groups.  The examination  should be extended to all animals in
all dosage  groups  if treatment-related changes are observed in  the high-
dose group.

     (A) All gross lesions in all animals.

     (B) Target organs in all animals.

     (C)  When a satellite  group is  used, histopathology  should be per-
formed on  tissues  and organs identified as showing effects in the  treated

     (ii) If  excessive early deaths or  other problems occur in the high dose
group  compromising the significance  of the data, the next dose level
should be examined for complete histopathology.

     (iii) An attempt should be made to correlate gross observations with
microscopic findings.

     (iv) Tissues  and  organs  designated for microscopic  examination
should be fixed in 10  percent buffered formalin  or a recognized suitable
fixative as  soon as necropsy is performed and no less than 48 hours prior
to trimming.

     (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 types of lesions and the  percentage of animals displaying each type
of lesion.

     (ii)  When applicable, all numerical results  should be evaluated by
an appropriate and generally acceptable 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,  body
weight changes, effects on mortality and any other general or specific toxic
effects. A properly conducted subchronic test should provide a satisfactory
estimation  of a NOEL.  It can  also  indicate  the  need for an additional
longer-term study and provide information on selection of dose levels.

     (3) Test report. In addition to the reporting requirements as specified
under 40 CFR part 792, subpart J (Good Laboratory Practice Standards),
40 CFR  part 160 and the OECD Principles of GLP (ISBN 92-64-12367-
9) the following specific information should be reported:

     (i) Test substance  characterization should include:

     (A) Chemical identification.

     (B)  Lot or batch numbers

     (C)  Physical properties.

     (D) Purity/impurities.

     (ii) Identification and composition of any vehicle used.

     (iii) Test system should contain data on:


     (A)  Species  and strain of animals used and rationale for selection
if other than that recommended.
     (B) Age, including body weight data and sex.
     (C) Test environment including cage conditions, ambient temperature,
humidity, and light/dark periods.
     (D) Identification of animal diet.
     (E) Acclimation period
     (iv) Test procedure should include the following data:
     (A) Method of randomization used.
     (B) Full description of experimental design and procedure.
     (C) Dose regime including levels, method, and volume.
     (v) Test results should include:
     (A) Group animal data. Tabulation of toxic response data by species,
strain, sex, and exposure level for:
     (7) Number of animals exposed.
     (2) Number of animals showing signs of toxicity.
       Number of animals dying.
     (B)  Individual animal data. Data should be presented  as  summary
(group mean) as well as for individual animals.
     (7) Date of death during the  study  or whether  animals survived to
     (2) Date of observation of each abnormal  sign and its subsequent
     (3) Body weight data.
     (4) Feed and water (when collected) consumption data.
     (5) Achieved dose (mg/kg/day) as a time-weighted average if the test
substance is administered in the diet or drinking water.
     (6) Ophthalmological examination data
     (7) Hematological tests employed and all results.
     (8) Clinical biochemistry tests employed and all results.
     (9) Urinalysis  tests employed and all results.

     (10)  Necropsy  findings,  including  absolute and relative (to  body
weight) organ weight data.

     (11} Detailed description of all histopathological findings.

     (72) Statistical treatment of results, where appropriate.

     (h) Quality  control. A system should be developed and  maintained
to assure and  document adequate  performance of laboratory staff and
equipment. The study must be conducted in compliance with GLP regula-

     (i) 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 aus-
pices of the Committee on  Toxicology,  National Research Council, Na-
tional Academy of Sciences, Washington, DC (1977).

     (5) Weingand K., Brown G., Hall R. et al. Harmonization of Animal
Clinical Pathology  Testing in  Toxicity and Safety Studies. Fundam. &
Appl. Toxicol.  29:198-201 (1996).

     (6) World Health Organization. Part I. Environmental Health Criteria
6, in Principles and Methods for Evaluating the  Toxicity of  Chemicals.
World Health Organization, Geneva (1978).