[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2006]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR799.9310]
[Page 349-354]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 799_IDENTIFICATION OF SPECIFIC CHEMICAL SUBSTANCE AND MIXTURE
TESTING REQUIREMENTS--Table of Contents
Subpart H_Health Effects Test Guidelines
Sec. 799.9310 TSCA 90-day oral toxicity in rodents.
(a) Scope. This section is intended to meet the testing requirements
under section 4 of the Toxic Substances Control Act (TSCA). In the
assessment and evaluation of the toxic characteristics of a chemical,
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-effects level (NOEL) and toxic effects associated with
continuous or repeated exposure to a test substance for a period of 90
days. This study 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 useful in providing
information on health hazards likely to arise from repeated exposure by
the oral route over a limited period of time, such as target organs, the
possibilities of accumulation, and can be of use in selecting dose
levels for chronic studies and for establishing safety criteria for
human exposure.
(b) Source. The source material used in developing this TSCA test
guideline is the Office of Prevention, Pesticides, and Toxic Substances
(OPPTS) harmonized test guideline 870.3100 (August 1998, final
guideline). This source is available at the address in paragraph (h) of
this section.
(c) Definitions. The following definitions apply to this section.
Cumulative toxicity is the adverse effects of repeated doses
occurring as a result of prolonged action on, or increased concentration
of, the administered test substance or its metabolites in susceptible
tissue.
Dose in a subchronic oral study is the amount of test substance
administered daily via the oral route (gavage, drinking water or diet)
for a period of 90 days. Dose is expressed as weight of the test
substance (grams, milligrams) per unit body weight of test animal
(milligram per kilogram) or as weight of the test substance in parts per
million in food or drinking water per day.
No-observed-effects level (NOEL) is the maximum dose used in a study
which produces no adverse effects. The NOEL is usually expressed in
terms of the weight of a test substance given daily per unit weight of
test animal (milligrams per kilogram per day).
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%) of the test
animal's life span.
Target organ is any organ of a test animal showing evidence of an
effect induced by a test substance.
(d) Limit test. If a test at one dose level of at least 1,000 mg/kg
body weight (expected human exposure may indicate the need for a higher
dose level), using the procedures described for this study, produces no
observable toxic effects or if toxic effects would not be expected based
upon data of structurally related compounds, then a full study using
three dose levels might not be necessary.
(e) Test procedures--(1) Animal selection--(i) Species and strain. A
variety of rodent species may be used, although the rat is the preferred
species. Commonly used laboratory strains must be employed.
(ii) Age/weight. (A) Testing should be started with young healthy
animals as soon as possible after weaning and acclimatization.
(B) Dosing of rodents should generally begin no later than 8-9 weeks
of age.
(C) At the commencement of the study the weight variation of animals
used must be within 20% of the mean weight for each sex.
(iii) Sex. Equal numbers of animals of each sex must be used at each
dose level, and the females shall be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 20 rodents (10 males and 10 females) at
each dose level.
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(B) If interim sacrifices are planned, the number must 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 must be assigned a unique identification number.
Dead animals, their preserved organs and tissues, and microscopic slides
must be identified by reference to the animal's unique number.
(v) Husbandry. (A) Animals may be group-caged by sex, but the number
of animals per cage must not interfere with clear observation of each
animal. The biological properties of the test substance or toxic effects
(e.g., morbidity, excitability) may indicate a need for individual
caging.
(B) The temperature of the experimental animal rooms should be at 22
3 [deg]C.
(C) The relative humidity of the experimental animal rooms should be
50 20%.
(D) Where lighting is artificial, the sequence should be 12 hours
light/12 hours dark.
(E) Control and test animals must be fed from the same batch and
lot. The feed should be analyzed to assure adequacy of nutritional
requirements of the species tested and 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.
(F) 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 five days is recommended.
(2) Control and test substances. (i) Where necessary, the test
substance is dissolved or suspended in a suitable vehicle. If a vehicle
or diluent 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 wherever possible the usage of an
aqueous solution be considered first, followed by consideration of a
solution in 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 a characterization of
the test substance, including the purity of the test compound and, if
technically feasible, 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 initiation of the study and periodically during the study.
Statistically randomized samples of the mixture should be analyzed to
ensure that proper mixing, 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 must 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 20 animals (10 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 20 animals (10 animals of
each sex) should be added to the satellite study.
(5) Dose levels and dose selection. (i) In subchronic toxicity
tests, it is desirable to determine a dose-response relationship as well
as a NOEL. Therefore, at least three dose levels plus a control and,
where appropriate, a vehicle control (corresponding to the concentration
of vehicle at the highest dose
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level) must 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
evaluation.
(iii) The intermediate dose levels should be spaced to produce a
gradation of toxic effects.
(iv) The lowest dose level should produce no evidence of toxicity.
(6) Administration of the test substance. (i) If the test substance
is administered by gavage, the animals are dosed with the test substance
on a 7-day per week basis for a period of at least 90 days. However,
based primarily on practical considerations, dosing by gavage 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 basis.
(ii) All animals must 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 body weight
should be used; the alternative used should be specified.
(iv) For a substance administered by gavage, 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 body
weight.
(7) Observation period. (i) The animals must be observed for a
period of 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) Observations must be made at least
twice each day for morbidity and mortality. Appropriate actions should
be 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). 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 must be made at least once
weekly. Observations should be detailed and carefully recorded,
preferably using explicity defined scales. Observations should include,
but not be limited to, evaluation of skin and fur, eyes and mucous
membranes, respiratory and circulatory effects, autonomic effects such
as salivation, central nervous system effects, including tremors and
convulsions, changes in the level of activity, gait and posture,
reactivity to handling or sensory stimuli, altered strength, and
stereotypes or bizarre behavior (e.g., self-mutilation, walking
backwards).
(iii) Signs of toxicity should be recorded as they are observed
including the time of onset, degree and duration.
(iv) Measurements of food consumption and water consumption, if
drinking water is the exposure route, must be made weekly.
(v) Individual weights of animals must be determined shortly before
the test substance is administered, weekly thereafter, and at death.
(vi) Moribund animals should be removed and sacrificed when noticed
and the time of death should be recorded as precisely as possible.
(vii) At termination, all survivors in the treatment and control
groups must be sacrificed.
(9) Clinical pathology. Hematology and clinical chemistry
examinations must be made on all animals, including controls, of each
sex in each group. The hematology and clinical chemistry parameters
should be examined at terminal sacrifice at the end of the study.
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
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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 concentration,
white blood cell count, differential leukocyte count, platelet count,
and a measure of clotting potential, such as prothrombin time or
activated partial thromboplastin time.
(ii) Clinical chemistry. (A) Parameters 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 potassium,
sodium, glucose, total cholesterol, urea nitrogen, creatinine, total
protein and albumin. More than 2 hepatic enzymes, (such as alanine
aminotransferase, aspartate aminotransferase, alkaline phosphatase,
sorbitol dehydrogenase, or gamma glutamyl transpeptidase) should also be
measured. 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
chemical is known or suspected of affecting related measures include
calcium, phosphorus, fasting triglycerides, hormones, methemoglobin, and
cholinesterases.
(iii) Optionally, the following urinalysis determinations could be
performed during the last week of the study using timed urine volume
collection: appearance, volume, osmolality or specific gravity, pH,
protein, glucose and blood/blood cells.
(10) Ophthalmological examination. Ophthalmological examinations
using an ophthalmoscope or an equivalent device must be made on all
animals prior to the administration of the test substance and on all
high dose and control groups at termination. If changes in the eyes are
detected, all animals in the other dose groups must be examined.
(11) Gross necropsy. (i) All animals must 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) The liver, kidneys, adrenals, testes, epididymides, ovaries,
uterus, thymus, spleen, brain, and heart must be trimmed and weighed
wet, as soon as possible after dissection.
(iii) The following organs and tissues, or representative samples
thereof, should be preserved in a suitable medium for possible future
histopathological examination:
(A) Digestive system--salivary glands, esophagus, stomach, duodenum,
jejunum, ileum, cecum, colon, rectum, liver, pancreas, gallbladder (when
present).
(B) Nervous system--brain (including sections of medulla/pons,
cerebellum and cerebrum), pituitary, peripheral nerve (sciatic or
tibial, preferably in close proximity to the muscle), spinal cord (three
levels: cervical, mid-thoracic and lumbar), eyes (retina, optic nerve).
(C) Glandular system--adrenals, parathyroid, thyroid.
(D) Respiratory system--trachea, lungs, pharynx, larynx, nose.
(E) Cardiovascular/hemopoietic system--aorta, heart, bone marrow
(and/or fresh aspirate), lymph nodes (preferably one lymph node covering
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) Others--all gross lesions and masses, skin.
(12) Histopathology. (i) The following histopathology must be
performed:
(A) Full histopathology on the organs and tissues, listed in
paragraph (e)(11)(iii) of this section, of all rodents in the control
and high dose groups, and all rodents that died or were sacrificed
during the study.
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(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(D) When a satellite group is used, histopathology should be
performed on tissues and organs identified as showing effects in the
treated groups.
(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% 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 must 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 observed results, qualitative and
quantitative, should be evaluated by an appropriate and generally
accepted statistical method. Any generally accepted statistical methods
may be used; the statistical methods, including significance criteria,
should be selected during the design of the study.
(2) Evaluation of study results. 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 must 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 also can indicate the
need for an additional longer-term study and provide information on the
selection of dose levels.
(3) Test report. In addition to reporting requirements specified
under EPA Good Laboratory Practice Standards at 40 CFR part 792, subpart
J, the following specific information must be reported:
(i) Test substance characterization should include:
(A) Chemical identification.
(B) Lot or batch number.
(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 regimen including levels, methods, and volume.
(v) Test results should include:
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(1) Number of animals exposed.
(2) Number of animals showing signs of toxicity.
(3) Number of animals dying.
(B) Individual animal data. Data should be presented as summary
(group mean) as well as for individual animals.
(1) Date of death during the study or whether animals survived to
termination.
(2) Date of observation of each abnormal sign and its subsequent
course.
(3) Body weight data.
(4) Feed and water (if 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) Results of ophthalmological examination.
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(7) Results of hematological tests performed.
(8) Results of clinical chemistry tests performed.
(9) Results of urinalysis, if performed.
(10) Necropsy findings, including absolute and relative (to body
weight) organ weight data.
(11) Detailed description of all histopathological findings.
(12) Statistical treatment of results, where appropriate.
(g) Quality control. A system must be developed and maintained to
assure and document adequate performance of laboratory equipment. The
study must be conducted in compliance with 40 CFR Part 792--Good
Laboratory Practice Standards.
(h) References. For additional background information on this test
guideline, the following references should be consulted. These
references are available for inspection at the TSCA Nonconfidential
Information Center, Rm. NE-B607, Environmental Protection Agency, 401 M
St., NW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Boyd, E.M. Chapter 14. Pilot Studies, 15. Uniposal Clinical
Parameters, 16. Uniposal Autopsy Parameters. Predictive Toxicometrics.
Williams and Wilkins, Baltimore (1972).
(2) Fitzhugh, O.G. Subacute Toxicity, 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) Organization for Economic Cooperation and Development. OECD
uidelines for Testing of Chemicals. Guideline 408: Subchronic Oral
Toxicity-Rodent: 90-day Study, Adopted: May 12, 1981.
(4) 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)
[65 FR 78783, Dec. 15, 2000]