[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: 40CFR799.9305]
[Page 343-349]
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. 799.9305 TSCA Repeated dose 28-day oral toxicity study in rodents.
(a) Scope--(1) Applicability. This section is intended to meet
testing requirements 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 870.3050 (July 2000, final
guidelines). This source is available at the address in paragraph (h) of
this section.
(b) Purpose. (1) In the assessment and evaluation of the toxic
characteristics of a chemical, the determination of oral toxicity using
repeated doses may be carried out after initial information on toxicity
has been obtained by acute testing. This study provides information on
the possible health hazards likely to arise from repeated exposure over
a relatively limited period of time. The method comprises the basic
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repeated dose toxicity study that may be used for chemicals on which a
90-day study is not warranted (e.g., when the production volume does not
exceed certain limits) or as a preliminary to a long term study. The
duration of exposure should normally be 28 days although a 14-day study
may be appropriate in certain circumstances; justification for use of a
14-day exposure period should be provided.
(2) This section places emphasis on neurological effects as a
specific endpoint, and the need for careful clinical observations of the
animals, so as to obtain as much information as possible, is stressed.
The method should identify chemicals with neurotoxic potential, which
may warrant further in-depth investigation of this aspect. In addition,
the method may give an indication of immunological effects and
reproductive organ toxicity.
(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.
Dosage is a general term comprising of dose, its frequency and the
duration of dosing.
Dose is the amount of test substance administered. Dose is expressed
as weight (g, mg) or as weight of test substance per unit weight of test
animal (e.g., mg/kg), or as constant dietary concentrations (parts per
million (ppm)).
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 animals (milligrams per kilograms per day).
(d) Principle of the test. The test substance is orally administered
daily in graduated doses to several groups of experimental animals, one
dose level per group for a period of 28 days. During the period of
administration the animals are observed closely, each day for signs of
toxicity. Animals which die or are sacrificed during the test are
necropsied and at the conclusion of the test surviving animals are
sacrificed and necropsied.
(e) Description of the method--(1) Selection of animal species. The
preferred rodent species is the rat, although other rodent species may
be used. Commonly used laboratory strains of young healthy adult animals
should be employed. The females should be nulliparous and non-pregnant.
Dosing should begin as soon as possible after weaning and, in any case,
before the animals are 9 weeks old. At the commencement of the study the
weight variation of animals used should be minimal and not exceed 20% of the mean weight of each sex. Where a repeated
dose oral study is conducted as a preliminary to a long term study,
preferably animals from the same strain and source should be used in
both studies.
(2) Housing and feeding conditions. The temperature in the
experimental animal room should be 22 [deg]C (3
[deg]C). Although the relative humidity should be at least 30% and
preferably not to exceed 70% other than during room cleaning, the aim
should be 50-60%. Lighting should be artificial, the sequence being 12
hours light, 12 hours dark. 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 a
test substance when administered by this method. Animals may be housed
individually, or be caged in small groups of the same sex; for group
caging, no more than five animals should be housed per cage.
(3) Preparation of animals. Healthy young adult animals must be
randomly assigned to the control and treatment groups. Cages should be
arranged in such a way that possible effects due to cage placement are
minimized. The animals are identified uniquely and kept in their cages
for at least 5 days prior to the start of the study to allow for
acclimatization to the laboratory conditions.
(4) Preparation of doses. (i) The test compound must be administered
by gavage or via the diet or drinking water. The method of oral
administration is dependent on the purpose of the study, and the
physical/chemical properties of the test material.
(ii) Where necessary, the test substance is dissolved or suspended
in a suitable vehicle. It is recommended that, wherever possible, the
use of an
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aqueous solution/suspension be considered first, followed by
consideration of a solution/emulsion in oil (e.g., corn oil) and then by
possible solution in other vehicles. For vehicles other than water the
toxic characteristics of the vehicle must be known. The stability of the
test substance in the vehicle should be determined.
(f) Procedure--(1)Number and sex of animals. At least 10 animals
(five female and five male) should be used at each dose level. 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. Consideration should be given to an additional satellite
group of 10 animals (five per sex) in the control and in the top dose
group for observation of reversibility, persistence, or delayed
occurrence of toxic effects, for at least 14 days post treatment.
(2) Dosage. (i) Generally, at least three test groups and a control
group should be used, but if from assessment of other data, no effects
would be expected at a dose of 1000 mg/kg bodyweight/per day, a limit
test may be performed. If there are no suitable data available, a range
finding study may be performed to aid the determination of the doses to
be used. Except for treatment with the test substance, animals in the
control group should be handled in an identical manner to the test group
subjects. If a vehicle is used in administering the test substance, the
control group should receive the vehicle in the highest volume used.
(ii) Dose levels should be selected taking into account any existing
toxicity and (toxico-) kinetic data available for the test compound or
related materials. The highest dose level should be chosen with the aim
of inducing toxic effects but not death or severe suffering. Thereafter,
a descending sequence of dose levels should be selected with a view to
demonstrating any dosage related response and NOEL at the lowest dose
level. Two to four fold intervals are frequently optimal for setting the
descending dose levels and addition of a fourth test group is often
preferable to using very large intervals (e.g., more than a factor of
10) between dosages.
(3) Limit test. If a test at one dose level of at least 1000 mg/kg
body weight/day or, for dietary or drinking water administration, an
equivalent percentage in the diet, or drinking water (based upon body
weight determinations), using the procedures described for this study,
produces no observable toxic effects and if toxicity would not be
expected based upon data from structurally related compounds, then a
full study using three dose levels may not be considered necessary. The
limit test applies except when human exposure indicates the need for a
higher dose level to be used.
(4) Administration of doses. (i) The animals are dosed with the test
substance daily 7 days each week for a period of 28 days; use of a 5-day
per week dosing regime or a 14-day exposure period needs to be
justified. When the test substance is administered by gavage, this
should be done in a single dose to the animals using a stomach tube or a
suitable intubation cannula. The maximum volume of liquid that can be
administered at one time depends on the size of the test animal. The
volume should not exceed 1ml/100g body weight, except in the case of
aqueous solutions where 2ml/100g body weight may be used. Except for
irritating or corrosive substances which will normally reveal
exacerbated effects with higher concentrations, variability in test
volume should be minimized by adjusting the concentration to ensure a
constant volume at all dose levels.
(ii) For substances administered via the diet or drinking water it
is important to ensure that the quantities of the test substance
involved do not interfere with normal nutrition or water balance. When
the test substance is administered in the diet either a constant dietary
concentration (parts per million (ppm)) or a constant dose level in
terms of the animals' body weight may be used; the alternative used must
be specified. For a substance administered by gavage, the dose should be
given at similar times each day, and adjusted as necessary to maintain a
constant dose level in terms of animal body weight. Where a repeated
dose study is used as a preliminary to a long term study, a similar diet
should be used in both studies.
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(5) Observations. (i) The observation period should be 28 days,
unless the study duration is 14 days (see paragraph (b)(1) of this
section). Animals in a satellite group scheduled for follow-up
observations should be kept for at least a further 14 days without
treatment to detect delayed occurrence, or persistence of, or recovery
from toxic effects.
(ii) General clinical observations should be made at least once a
day, preferably at the same time(s) each day and considering the peak
period of anticipated effects after dosing. The health condition of the
animals should be recorded. At least twice daily, all animals are
observed for morbidity and mortality.
(iii) Once before the first exposure (to allow for within-subject
comparisons), and at least once a week thereafter, detailed clinical
observations should be made in all animals. These observations should be
made outside the home cage in a standard arena and preferably at the
same time, each time. They should be carefully recorded, preferably
using scoring systems, explicitly defined by the testing laboratory.
Effort should be made to ensure that variations in the test conditions
are minimal and that observations are preferably conducted by observers
unaware of the treatment. 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 behaviour (e.g., self-mutilation, walking
backwards) should also be recorded.
(iv) In the fourth exposure week sensory reactivity to stimuli of
different types (see paragraph (h)(2) of this section) (e.g., auditory,
visual and proprioceptive stimuli), assessment of grip strength and
motor activity assessment should be conducted. Further details of the
procedures that could be followed are given in the respective
references. However, alternative procedures than those referenced could
also be used. Examples of procedures for observation are described in
the references in paragraphs (h)(1), (h)(2), (h)(3), (h)(4), and (h)(5)
of this section.
(v) Functional observations conducted in the fourth exposure week
may be omitted when the study is conducted as a preliminary study to a
subsequent subchronic (90-day) study. In that case, the functional
observations should be included in this follow-up study. On the other
hand, the availability of data on functional observations from the
repeated dose study may enhance the ability to select dose levels for a
subsequent subchronic study.
(vi) Exceptionally, functional observations may also be omitted for
groups that otherwise reveal signs of toxicity to an extent that would
significantly interfere with the functional test performance.
(6) Body weight and food/water consumption. All animals should be
weighed at least once a week. Measurements of food consumption should be
made at least weekly. If the test substance is administered via the
drinking water, water consumption should also be measured at least
weekly.
(7) Hematology. (i) The following hematological examinations should
be made at the end of the test period: hematocrit, hemoglobin
concentration, erythrocyte count, total and differential leukocyte
count, platelet count and a measure of blood clotting time/potential.
(ii) Blood samples should be taken from a named site just prior to
or as part of the procedure for sacrificing the animals, and stored
under appropriate conditions.
(8) Clinical Biochemistry. (i) Clinical biochemistry determinations
to investigate major toxic effects in tissues and, specifically, effects
on kidney and liver, should be performed on blood samples obtained of
all animals just prior to or as part of the procedure for sacrificing
the animals (apart from those found moribund and/or intercurrently
sacrificed). Overnight fasting of the animals prior to blood sampling is
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recommended.\1\ Investigations of plasma or serum shall include sodium,
potassium, glucose, total cholesterol, urea, creatinine, total protein
and albumin, at least two enzymes indicative of hepatocellular effects
(such as alanine aminotransferase, aspartate aminotransferase, alkaline
phosphatase, gamma glutamyl transpeptidase, and sorbitol dehydrogenase).
Measurements of additional enzymes (of hepatic or other origin) and bile
acids may provide useful information under certain circumstances.
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\1\ For a number of measurements in serum and plasma, most notably
for glucose, overnight fasting would be preferable. The major reason for
this preference is that the increased variability which would inevitably
result from non-fasting, would tend to mask more subtle effects and make
interpretation difficult. On the other hand, however, overnight fasting
may interfere with the general metabolism of the animals and,
particularly in feeding studies, may disturb the daily exposure to the
test substance. If overnight fasting is adopted, clinical biochemical
determinations should be performed after the conduct of functional
observations in week 4 of the study.
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(ii) 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 and blood cells.
(iii) In addition, studies to investigate serum markers of general
tissue damage should be considered. Other determinations that should be
carried out if the known properties of the test substance may, or are
suspected to, affect related metabolic profiles include calcium,
phosphate, fasting triglycerides, specific hormones, methemoglobin and
cholinesterase. These must to be identified for chemicals in certain
classes or on a case-by-case basis.
(iv) Overall, there is a need for a flexible approach, depending on
the species and the observed and/or expected effect with a given
compound.
(v) If historical baseline data are inadequate, consideration should
be given to determination of hematological and clinical biochemistry
variables before dosing commences.
(9) Pathology--(i)Gross necropsy. (A) All animals in the study must
be subjected to a full, detailed gross necropsy which includes careful
examination of the external surface of the body, all orifices, and the
cranial, thoracic and abdominal cavities and their contents. The liver,
kidneys, adrenals, testes, epididymides, thymus, spleen, brain and heart
of all animals (apart from those found moribund and/or intercurrently
sacrificed) should be trimmed of any adherent tissue, as appropriate,
and their wet weight taken as soon as possible after dissection to avoid
drying.
(B) The following tissues should be preserved in the most
appropriate fixation medium for both the type of tissue and the intended
subsequent histopathological examination: all gross lesions, brain
(representative regions including cerebrum, cerebellum and pons), spinal
cord, stomach, small and large intestines (including Peyer's patches),
liver, kidneys, adrenals, spleen, heart, thymus, thyroid, trachea and
lungs (preserved by inflation with fixative and then immersion),
ovaries, uterus, testes, epididymides, accessory sex organs (e.g.,
prostate, seminal vesicles), urinary bladder, lymph nodes (preferably
one lymph node covering the route of administration and another one
distant from the route of administration to cover systemic effects),
peripheral nerve (sciatic or tibial) preferably in close proximity to
the muscle, and a section of bone marrow (or, alternatively, a fresh
mounted bone marrow aspirate). The clinical and other findings may
suggest the need to examine additional tissues. Also any organs
considered likely to be target organs based on the known properties of
the test substance should be preserved.
(ii) Histopathology. (A) Full histopathology should be carried out
on the preserved organs and tissues of all animals in the control and
high dose groups. These examinations should be extended to animals of
all other dosage groups, if treatment-related changes are observed in
the high dose group.
(B) All gross lesions must be examined.
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(C) When a satellite group is used, histopathology should be
performed on tissues and organs identified as showing effects in the
treated groups.
(g) Data and reporting--(1) Data. (i) Individual data should be
provided. Additionally, all 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 found dead during the test or sacrificed for
humane reasons and the time of any death or humane sacrifice, the number
showing signs of toxicity, a description of the signs of toxicity
observed, including time of onset, duration, and severity of any toxic
effects, the number of animals showing lesions, the type of lesions and
the percentage of animals displaying each type of lesion.
(ii) When possible, numerical results should be evaluated by an
appropriate and generally acceptable statistical method. The statistical
methods should be selected during the design of the study.
(2) Test report. The test report must include the following
information:
(i) Test substance:
(A) Physical nature, purity and physicochemical properties.
(B) Identification data.
(ii) Vehicle (if appropriate): Justification for choice of vehicle,
if other than water.
(iii) Test animals:
(A) Species/strain used.
(B) Number, age and sex of animals.
(C) Source, housing conditions, diet, etc.
(D) Individual weights of animals at the start of the test.
(iv) Test conditions:
(A) Rationale for dose level selection.
(B) Details of test substance formulation/diet preparation, achieved
concentration, stability and homogeneity of the preparation.
(C) Details of the administration of the test substance.
(D) Conversion from diet/drinking water test substance concentration
(parts per million (ppm)) to the actual dose (mg/kg body weight/day), if
applicable.
(E) Details of food and water quality.
(v) Results:
(A) Body weight/body weight changes.
(B) Food consumption, and water consumption, if applicable.
(C) Toxic response data by sex and dose level, including signs of
toxicity.
(D) Nature, severity and duration of clinical observations (whether
reversible or not).
(E) Sensory activity, grip strength and motor activity assessments.
(F) Hematological tests with relevant base-line values.
(G) Clinical biochemistry tests with relevant base-line values.
(H) Body weight at sacrificing and organ weight data.
(I) Necropsy findings.
(J) A detailed description of all histopathological findings.
(K) Absorption data if available.
(L) Statistical treatment of results, where appropriate.
(vi) Discussion of results.
(vii) Conclusions.
(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) Tupper, D.E., Wallace, R.B. (1980). Utility of the Neurologic
Examination in Rats. Acta Neurobiological Exposure, 40:999-1003.
(2) Gad, S.C. (1982). A Neuromuscular Screen for Use in Industrial
Toxicology. Journal of Toxicology and Environmental Health, 9:691-704.
(3) Moser, V.C., McDaniel, K.M., Phillips, P.M. (1991). Rat Strain
and Stock Comparisons Using a Functional Observational Battery: Baseline
Values and Effects of Amitraz. Toxicology and Applied Pharmacology,
108:267-283.
(4) Meyer O.A., Tilson H.A., Byrd W.C., Riley M.T. (1979). A Method
forthe Routine Assessment of Fore- and Hindlimb Grip Strength of Rats
and Mice. Neurobehavioral Toxicology, 1:233-236.
(5) Crofton K.M., Howard J.L., Moser V.C., Gill M.W., Reiter L.W.,
Tilson
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H.A., MacPhail R.C. (1991). Interlaboratory Comparison of Motor Activity
Experiments: Implication for Neurotoxicological Assessments.
Neurotoxicology and Teratology, 13:599-609.
[65 FR 78780, Dec. 15, 2000]