[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: 40CFR799.9410]
[Page 391-400]
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. 799.9410 TSCA chronic toxicity.
(a) Scope--(1) Applicability. This section is intended to meet the
testing requirement of the Toxic Substances Control Act (TSCA) (15
U.S.C. 2601).
(2) Source. The source material used in developing this TSCA test
guideline is the Office of Prevention, Pesticides and Toxic Substances
(OPPTS) harmonized test guideline 870.4100 (August 1998, final
guidelines). This source is available at the address in paragraph (h) of
this section
(b) Purpose. The objective of a chronic toxicity study is to
determine the effects of a substance in a mammalian species following
prolonged and repeated exposure. A chronic toxicity study should
generate data from which to identify the majority of chronic effects and
to define long-term dose-response relationships. The design and conduct
of chronic toxicity tests should allow for the detection of general
toxic effects, including neurological, physiological, biochemical, and
hematological effects and exposure-related morphological (pathological)
effects.
(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR
Part 792--Good Laboratory Practice Standards apply to this section. The
following definitions also apply to this section.
Chronic toxicity is the adverse effects occurring as a result of the
repeated daily exposure of experimental animals to a chemical by the
oral, dermal, or inhalation routes of exposure.
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 chronic toxicity study is the amount of test substance
administered daily via the oral, dermal or inhalation routes for a
period of at least 12
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months. 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 (ppm)
in food or drinking water per day. For inhalation exposure, dose is
expressed as weight of the test substance per unit volume of air
(milligrams per liter) or as parts per million per day. For dermal
exposure, dose is expressed as weight of the test substance (grams,
milligrams) per unit body weight of the test animal (milligrams per
kilogram) or as weight of the substance per unit of surface area
(milligrams per square centimeter) 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).
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 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.
Testing should be performed with two mammalian species, one a rodent and
the other a nonrodent. The rat is the preferred rodent species. Commonly
used laboratory strains must be employed.
(ii) Age/weight. (A) Testing must 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 weeks
of age.
(C) Dosing of non-rodents should begin between 4 and 6 months of age
and in no case later than 9 months of age.
(D) At commencement of the study, the weight variation of animals
used should be within 20% of the mean weight for each sex.
(E) Studies using prenatal or neonatal animals may be recommended
under special conditions.
(iii) Sex. (A) Equal numbers of animals of each sex should be used
at each dose level.
(B) Females should be nulliparous and nonpregnant.
(iv) Numbers. (A) For rodents, at least 40 animals (20 males and 20
females) and for nonrodents at least 8 animals (4 females and 4 males)
should be used at each dose level and concurrent control group.
(B) If interim sacrifices are planned, the number should be
increased by the number of animals scheduled to be sacrificed during the
course of the study.
(C) The number of animals at the termination of the study must be
adequate for a meaningful and valid statistical evaluation of chronic
effects. The Agency must be notified if excessive early deaths or other
problems are encountered that might compromise the integrity of the
study.
(D) To avoid bias, the use of adequate randomization procedures for
the proper allocation of animals to test and control groups is required.
(E) 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 unique numbers assigned.
(v) Husbandry. (A) Rodents 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. Rodents should be housed individually in dermal studies and
during exposure in inhalation studies. Caging should be appropriate to
the nonrodent species.
(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.
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(E) Control and test animals should 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. Animals should be fed and watered ad
libitum with food replaced at least weekly.
(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 5 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 it should not elicit toxic effects itself nor
substantially alter the chemical or toxicological properties of the test
substance. It is recommended that wherever possible the use of an
aqueous solution be the first choice, followed by consideration of
solution in oil, and finally, solution in other vehicles.
(ii) One lot of the test substance should be used, if possible,
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 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 40 animals (20 animals per
sex) for rodents and 8 animals (4 animals per sex) for nonrodents may be
treated with the high-dose level for 12 months and observed for
reversibility, persistence, or delayed occurrence of toxic effects for a
post-treatment of appropriate length, normally not less than 28 days. In
addition, a control group of 40 animals (20 animals per sex) for rodents
and 8 animals (4 animals per sex) for nonrodents should be added to the
satellite study.
(5) Dose levels and dose selections. (i) In chronic toxicity tests,
it is desirable to determine a dose-response relationship as well as a
NOEL. Therefore, at least three dose levels with a control group and,
where appropriate, a vehicle control (corresponding to the concentration
of the vehicle at the highest exposure level) should be used. Dose
levels should be spaced to produce a gradation of effects. A rationale
must be provided for the doses selected.
(ii) The highest-dose level should elicit signs of toxicity without
substantially altering the normal life span of the animal. The highest
dose should be determined based on the findings from a 90-day study to
ensure that the dose used is adequate to assess the chronic toxicity of
the test substance. Thus, the selection of the highest dose to be tested
is dependent upon changes observed in several toxicological parameters
in subchronic studies. The highest dose tested need not exceed 1,000 mg/
kg/day. If dermal application of the test substance produces severe skin
irritation, then it may be necessary either to terminate the study and
choose a lower high-dose level or to reduce the dose level. Gross
criteria for defining severe irritation would include ulcers, fissures,
exudate/crust(eschar), dead tissue, or anything leading to destruction
of the functional integrity of the epidermis (e.g. caking,
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open sores, fissuring, eschar). Histological criteria for defining
severe irritation would include follicular and interfollicular crust,
microulcer, mild/moderate degeneration/necrosis, moderate/marked
epidermal edema, marked dermal edema, and marked inflammation.
(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. The three main routes of
administration are oral, dermal, and inhalation. The choice of the route
of administration depends upon the physical and chemical characteristics
of the test substance and the form typifying exposure in humans.
(i) Oral studies. Ideally, the animals should be dosed by gavage or
with capsules on a 7-day per week basis for a period of at least 12
months. However, based primarily on practical considerations, dosing by
gavage or capsules on a 5-day per week schedule is acceptable. If the
test substance is administered via in the drinking water or mixed in the
diet, exposure should be on a 7-day per week basis.
(ii) Dermal studies. (A) Preparation of animal skin. Shortly before
testing, fur should be clipped from not less than 10% of the body
surface area for application of the test substance. In order to dose
approximately 10% of the body surface, the area starting at the scapulae
(shoulders) to the wing of the ileum (hipbone) and half way down the
flank on each side of the animal should be shaved. Shaving should be
carried out approximately 24 hours before dosing. Repeated clipping or
shaving is usually needed at approximately weekly intervals. When
clipping or shaving the fur, care should be taken to avoid abrading the
skin which could alter its permeability.
(B) Preparation of test substance. Liquid test substances are
generally used undiluted, except as indicated in paragraph (e)(5)(ii) of
this section. Solids should be pulverized when possible. The substance
should be moistened sufficiently with water or, when necessary, with a
suitable vehicle to ensure good contact with the skin. When a vehicle is
used, the influence of the vehicle on toxicity of, and penetration of
the skin by, the test substance should be taken into account.The volume
of application should be kept constant, e.g., less than 100 [micro]L for
the mouse and less than 300 [micro]L for the rat. Different
concentrations of test solution should be prepared for different dose
levels.
(C) Administration of test substance. The duration of exposure
should be at least for 12 months. Ideally, the animals should be treated
with test substance for at least 6 hours per day on a 7-day per week
basis. However, based on practical considerations, application on a 5-
day per week basis is acceptable. Dosing should be conducted at
approximately the same time each day. The test substance should be
applied uniformly over the treatment site. The surface area covered may
be less for highly toxic substances. As much of the area should be
covered with as thin and uniform a film as possible. For rats, the test
substance may be held in contact with the skin with a porous gauze
dressing and nonirritating tape if necessary. The test site should be
further covered in a suitable manner to retain the gauze dressing plus
test substance and to ensure that the animals cannot ingest the test
substance. The application site should not be covered when the mouse is
the species of choice. The test substance may be wiped from the skin
after the six-hour exposure period to prevent ingestion.
(iii) Inhalation studies. (A) The animals should be exposed to the
test substance for 6 hours per day on a 7-day per week basis, for a
period of at least 12 months. However, based primarily on practical
considerations, exposure for 6 hours per day on a 5-day per week basis
is acceptable.
(B) The animals should be tested in dynamic inhalation equipment
designed to sustain a minimum air flow of 10 air changes per hour, an
adequate oxygen content of at least 19%, and uniform conditions
throughout the exposure chamber. Maintenance of slight negative pressure
inside the chamber will prevent leakage of the test substance into
surrounding areas. It is not
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normally necessary to measure chamber oxygen concentration if airflow is
adequate.
(C) The selection of a dynamic inhalation chamber should be
appropriate for the test substance and test system. When a whole body
chamber is used, individual housing must be used to minimize crowding of
the test animals and maximize their exposure to the test substance. To
ensure stability of a chamber atmosphere, the total volume occupied by
the test animals should not exceed 5% of the volume of the test chamber.
It is recommended, but not required, that nose-only or head-only
exposure be used for aerosol studies in order to minimize oral exposures
due to animals licking compound off their fur. The animals should be
acclimated and heat stress minimized.
(D) The temperature at which the test is performed should be
maintained at 22 2 [deg]C. The relative humidity
should be maintained between 40-60%, but in certain instances (e.g., use
of water vehicle) this may not be practicable.
(E) The rate of air flow should be monitored continuously but
recorded at least three times during the exposure.
(F) Temperature and humidity should be monitored continuously but
should be recorded at least every 30 min.
(G) The actual concentrations of the test substance should be
measured in the breathing zone. During the exposure period, the actual
concentrations of the test substance should be held as constant as
practicable, monitored continuously or intermittently depending on the
method of analysis. Chamber concentration may be measured using
gravimetric or analytical methods, as appropriate. If trial run
measurements are reasonably consistent (10% for
liquid aerosol, gas, or vapor; 20% for dry
aerosol), then two measurements should be sufficient. If measurements
are not consistent, three to four measurements should be taken. If there
is some difficulty measuring chamber analytical concentration due to
precipitation, nonhomogeneous mixtures, volatile components, or other
factors, additional analysis of inert components may be necessary.
(H) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol
concentrations with respect to particle size. The mass median
aerodynamic diameter (MMAD) particle size range should be between 1-3
[micro]m. The particle size of hygroscopic materials should be small
enough when dry to assure that the size of the swollen particle will
still be within the 1-3 [micro]m range. Measurements of aerodynamic
particle size in the animal's breathing zone should be measured during a
trial run. If MMAD values for each exposure level are within 10% of each
other, then two measurements during the exposures should be sufficient.
If pretest measurements are not within 10% of each other, three to four
measurements should be taken.
(I) Feed should be withheld during exposure. Water may also be
withheld during exposure.
(7) Observation period. (i) Animals should be observed for a period
of at least 12 months.
(ii) Animals in a 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 should 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 should be made at least once
prior to the initiation of treatment (to allow for within subject
comparisons) and once weekly during treatment in all animals. These
observations should be made outside the home cage, preferably in a
standard arena, and at similar times on each occasion. Effort should be
made to ensure that variations in the observation conditions
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are minimal. Observations should be detailed and carefully recorded,
preferably using scoring systems, explicitly defined by the testing
laboratory. Signs noted should include, but not be limited to, changes
in skin, fur, eyes, mucous membranes, occurrence of secretions and
excretions and autonomic activity (e.g., lacrimation, piloerection,
pupil size, unusual respiratory pattern). Changes in gait, posture and
response to handling as well as the presence of clonic or tonic
movements, stereotypies (e.g., excessive grooming, repetitive circling)
or bizarre behavior (e.g., self-mutilation, walking backwards) should be
recorded.
(iii) Once, near the end of the first year of the exposure period
and in any case not earlier than in month 11, assessment of motor
activity, grip strength, and sensory reactivity to stimuli of different
types (e.g., visual, auditory, and proprioceptive stimuli) should be
conducted in rodents. Further details of the procedures that could be
followed are described in the references listed under paragraphs (h)(2),
(h)(7), (h)(8), and (h)(11) of this section.
(iv) Functional observations conducted towards the end of the study
may be omitted when data on functional observations are available from
other studies and the daily clinical observations did not reveal any
functional deficits.
(v) Exceptionally, functional observations may be omitted for groups
that otherwise reveal signs of toxicity to an extent that would
significantly interfere with functional test performance.
(vi) Body weights should be recorded individually for all animals
once prior to the administration of the test substance, once a week
during the first 13 weeks of study and at least once every 4 weeks
thereafter, unless signs of clinical toxicity suggest more frequent
weighing to facilitate monitoring of health status.
(vii) Measurements of feed consumption should be determined weekly
during the first 13 weeks of the study and at approximately monthly
intervals thereafter unless health status or body weight changes dictate
otherwise. Measurements of water consumption should be determined at the
same intervals if the test substance is administered in the drinking
water.
(viii) Moribund animals should be removed and sacrificed when
noticed and the time of death should be recorded as precisely as
possible. All survivors should be sacrificed at the end of the study
period.
(9) Clinical pathology. Hematology, clinical chemistry, and
urinalysis should be performed on 10 rats per sex per group, and on all
nonrodents. In rodents, the parameters should be examined at
approximately 6 month intervals during the conduct of the study and at
termination. If possible, these collections should be from the same
animals at each interval. In nonrodents, the parameters should be
examined once or twice prior to initiation of treatment, at 6-month
intervals during the conduct of the study, and at termination. If
hematological and biochemical effects were seen in the subchronic study,
testing should also be performed at 3 months. Overnight fasting of
animals prior to blood sampling is recommended.
(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, calcium (nonrodent), phosphorus (nonrodent), chloride
(nonrodent), glucose, total cholesterol, urea nitrogen, creatinine,
total protein, total bilirubin (nonrodent), and albumin. More than two
hepatic enzymes, (such as alanine aminotransferase, aspartate
aminotransferase, alkaline phosphatase, sorbitol dehydrogenase, or
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gamma glutamyl transpeptidase) should also be measured. Measurements of
additional 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) Urinalysis. Urinalysis for rodents should be performed at the
end of the study using timed urine collection. Urinalysis for nonrodents
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 specific
gravity, pH, protein, glucose, and blood/blood cells.
(10) Ophthalmological examination. Examinations should be made of
all animals using an ophthalmoscope or equivalent device prior to the
administration of the test substance and at termination of the study on
10 rats of each sex in the high-dose and control groups and preferably
in all nonrodents, but at least the control and high-dose groups should
be examined. If changes in eyes are detected, all animals 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, kidneys, adrenals, testes, epididymides,
ovaries, uterus, nonrodent thyroid (with parathyroid), spleen, brain,
and heart should be weighed wet as soon as possible after dissection to
avoid drying. The lungs should be weighed if the test substance is
administered by the inhalation route.
(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 (multiple sections, including cerebrum,
cerebellum and medulla/pons), 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/hematopoietic 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.
(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.
(iv) In inhalation studies, the entire respiratory tract, including
nose, pharynx, larynx, and paranasal sinuses should be examined and
preserved. In dermal studies, skin from treated and adjacent control
skin sites should be examined and preserved.
(v) Inflation of lungs and urinary bladder with a fixative is the
optimal method for preservation of these tissues. The proper inflation
and fixation of the lungs in inhalation studies is considered essential
for appropriate and valid histopathological examination.
(vi) Information from clinical pathology and other in-life data
should be considered before microscopic examination, since they may
provide significant guidance to the pathologist.
(12) Histopathology. (i) The following histopathology should be
performed:
(A) Full histopathology on the organs and tissues (listed under
paragraph (e)(11)(iii) of this section) of all rodents and nonrodents in
the control and high-dose groups, and all rodents
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and nonrodents that died or were sacrificed during the study. The
examination should be extended to all animals in all dosage groups if
treatment-related changes are observed in the high-dose group.
(B) All gross lesions in all animals.
(C) Target tissues in all animals.
(ii) If the results show substantial alteration of the animal's
normal life span, or other effects that might compromise the
significance of the data, the next lower levels should be examined fully
as described in paragraph (e)(12)(i) of this section.
(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 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 observed results (quantitative and
qualitative) should be evaluated by an appropriate 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 chronic toxicity
study should be evaluated in conjunction with the findings of preceding
studies and considered in terms of the toxic effects as well as the
necropsy and histopathological findings. The evaluation will include the
relationship between the dose of the test substance and the presence,
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.
(3) Test report. In addition to the 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.
(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) Time of death during the study or whether animals survived to
termination.
(2) Time 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 examinations.
(7) Results of hematological tests performed.
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(8) Results of clinical chemistry tests performed.
(9) Urinalysis tests performed and results.
(10) Results of observations made.
(11) Necropsy findings, including absolute and relative (to body
weight) organ weight data.
(12) Detailed description of all histopathological findings.
(13) Statistical treatment of results, where appropriate.
(vi) In addition, for inhalation studies the following should be
reported:
(A) Test conditions. The following exposure conditions must be
reported:
(1) Description of exposure apparatus including design, type,
dimensions, source of air, system for generating particulate and
aerosols, method of conditioning air, treatment of exhaust air and the
method of housing the animals in a test chamber.
(2) The equipment for measuring temperature, humidity, and
particulate aerosol concentrations and size should be described.
(B) Exposure data. These data should be tabulated and presented with
mean values and a measure of variability (e.g., standard deviation) and
should include:
(1) Airflow rates through the inhalation equipment.
(2) Temperature and humidity of air.
(3) Actual (analytical or gravimetric) concentration in the
breathing zone.
(4) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
(5) Particle size distribution, calculated MMAD, and geometric
standard deviation.
(6) Explanation as to why the desired chamber concentration and/or
particle size could not be achieved (if applicable) and the efforts
taken to comply with this aspect of the guidelines.
(g) 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 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., SW., Washington, DC, 12 noon to 4 p.m., Monday through Friday,
except legal holidays.
(1) Benitz, K.F. Measurement of Chronic Toxicity. Methods of
Toxicology. Ed. G.E. Paget. Blackwell, Oxford. pp. 82-131 (1970).
(2) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Leiter L.W.,
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