[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: 40CFR798.3260]
[Page 155-160]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart D_Chronic Exposure
Sec. 798.3260 Chronic toxicity.
(a) 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. Under the conditions of the chronic
toxicity test, effects which require a long latency period or which are
cumulative should become manifest. The application of this guideline
should generate data on which to identify the majority of chronic
effects and shall serve 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 (pathology) effects.
(b) Test procedures--(1) Animal selection--(i) Species and strain.
Testing should be performed with two mammalian species, one a rodent and
another a non-rodent. The rat is the preferred rodent species and the
dog is the preferred non-rodent species. Commonly used laboratory
strains should be employed. If other mammalian species are used, the
tester should provide justification/reasoning for their selection.
(ii) Age. (A) Dosing of rats should begin as soon as possible after
weaning, ideally before the rats are 6, but in no case more than 8 weeks
old.
(B) Dosing of dogs should begin between 4 and 6 months of age and in
no case later than 9 months of age.
(C) At commencement of the study the weight variation of animals
used should not exceed 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 non-pregnant.
(iv) Numbers. (A) For rodents, at least 40 animals (20 females and
20 males) and for non-rodents (dogs) at least 8 animals (4 females and 4
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 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.
(2) Control groups. (i) A concurrent control group is suggested.
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 strongly
suggested.
(ii) In special circumstances such as in inhalation studies
involving aerosols or the use of an emulsifier of uncharacterized
biological activity in oral studies, a concurrent negative control group
should be utilized. The negative control group should be treated in the
same manner as all other test animals except that this control group
should not be exposed to either the test substance or any vehicle.
(3) Dose levels and dose selections. (i) In chronic toxicity tests,
it is necessary to have a dose-response relationship as well as a no-
observed-toxic-effect level. Therefore, at least three dose levels
should be used in addition to the concurrent control group. Dose levels
should be spaced to produce a gradation of effects.
(ii) The high dose level in rodents should elicit some signs of
toxicity without causing excessive lethality; for non-rodents, there
should be signs of
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toxicity but there should be no fatalities.
(iii) The lowest dose level should not produce any evidence of
toxicity. Where there is a usable estimation of human exposure the
lowest dose level should exceed this even though this dose level may
result in some signs of toxicity.
(iv) Ideally, the intermediate dose level(s) should produce minimal
observable toxic effects. If more than one intermediate dose is used,
the dose level should be spaced to produce a gradation of toxic effects.
(v) For rodents, the incidence of fatalities in low and intermediate
dose groups and in the controls should be low to permit a meaningful
evaluation of the results. For non-rodents, there should be no
fatalities.
(4) Exposure conditions. The animals are dosed with the test
substance ideally on a 7-day per week basis over a period of at least 12
months. However, based primarily on practical considerations, dosing on
a 5-day per week basis is considered to be acceptable.
(5) Observation period. Duration of observation should be for at
least 12 months, and may be concurrent with or subsequent to dosing. If
there is a post-exposure observation period, an interim sacrifice should
be performed on no fewer than half of the animals of each sex at each
dose level immediately upon termination of exposure.
(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. (A) The animals should receive the test substance
in their diet, dissolved in drinking water, or given by gavage or
capsule for a period of at least 12 months.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure is continuous.
(C) For a diet mixture, the highest concentration should not exceed
5 percent.
(ii) Dermal studies. (A) The animals are treated by topical
application with the test substance, ideally for at least 6 hours per
day.
(B) Fur should be clipped from the dorsal area of the trunk of the
test animals. Care must be taken to avoid abrading the skin which could
alter its permeability.
(C) The test substance should be applied uniformly over a shaved
area which is approximately 10 percent of the total body surface area.
With highly toxic substances, the surface area covered may be less, but
as much of the area should be covered with as thin and uniform a film as
possible.
(D) During the exposure period, the test substance may be held if
necessary, in contact with the skin with a porous gauze dressing and
non-irritating tape. The test site should be further covered in a
suitable manner to retain the gauze dressing and test substance and
ensure that the animals cannot ingest the test substance.
(iii) Inhalation studies. (A) The animals should be tested with
inhalation equipment designed to sustain a dynamic air flow of 12 to 15
air changes per hour, ensure an adequate oxygen content of 19 percent
and an evenly distributed exposure atmosphere. Where a chamber is used,
its design should minimize crowding of the test animals and maximize
their exposure to the test substance. This is best accomplished by
individual caging. As a general rule to ensure stability of a chamber
atmosphere, the total ``volume'' of the test animals should not exceed 5
percent of the volume of the test chamber. Alternatively, oro-nasal,
head-only or whole body individual chamber exposure may be used.
(B) The temperature at which the test is performed should be
maintained at 22 [deg]C (2[deg]). Ideally, the
relative humidity should be maintained between 40 to 60 percent, but in
certain instances (e.g., tests of aerosols, use of water vehicle) this
may not be practicable.
(C) Feed and water should be withheld during each daily 6 hour
exposure period.
(D) A dynamic inhalation system with a suitable analytical
concentration control system should be used. The rate of air flow should
be adjusted to ensure that conditions throughout
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the equipment are essentially the same. Maintenance of slight negative
pressure inside the chamber will prevent leakage of the test substance
into the surrounding areas.
(7) Observation of animals. (i) Each animal should be handled and
its physical condition appraised at least once each day.
(ii) Additional observations should be made daily with appropriate
actions taken to minimize loss of animals to the study (e.g., necropsy
or refrigeration of those animals found dead and isolation or sacrific
of weak or moribund animals).
(iii) Clinical signs of toxicity including suspected tumors and
mortality should be recorded as they are observed, including the time of
onset, the degree and duration.
(iv) Cage-side observations should include, but not be limited to,
changes in skin and fur, eyes and mucous membranes, respiratory,
circulatory, autonomic and central nervous systems, somatomotor activity
and behavior pattern.
(v) Body weights should be recorded individually for all animals
once a week during the first 13 weeks of the test period and at least
once every 4 weeks thereafter unless signs of clinical toxicity suggest
more frequent weighings to facilitate monitoring of health status.
(vi) When the test substance is administered in the feed or drinking
water, measurements of feed or water consumption, respectively, should
be determined weekly during the first 13 weeks of the study and then at
approximately monthly intervals unless health status or body weight
changes dictate otherwise.
(vii) At the end of the study period all survivors should be
sacrificed. Moribund animals should be removed and sacrificed when
noticed.
(8) Physical measurements. For inhalation studies, measurements or
monitoring should be made of the following:
(i) The rate of air flow should be monitored continuously, but
should be recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the
test substance should be held as constant as practicable, monitored
continuously and measured at least three times during the test period:
at the beginning, at an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size
analysis should be performed to establish the stability of aerosol
concentrations. During exposure, analysis should be conducted as often
as necessary to determine the consistency of particle size distribution
and homogeneity of the exposure stream.
(iv) Temperature and humidity should be monitored continuously, but
should be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. The following examinations should be made
on at least 10 rats of each sex per dose and on all non-rodents.
(i) Certain hematology determinations (e.g., hemoglobin content,
packed cell volume, total red blood cells, total white blood cells,
platelets, or other measures of clotting potential) should be performed
at termination and should be performed at 3 months, 6 months and at
approximately 6 month intervals thereafter (for studies extending beyond
12 months) on blood samples collected from all non-rodents and from 10
rats per sex of all groups. These collections should be from the same
animals at each interval. If clinical observations suggest a
deterioration in health of the animals during the study, a differential
blood count of the affected animals should be performed. A differential
blood count should be performed on samples from those animals in the
highest dosage group and the controls. Differential blood counts should
be performed for the next lower group(s) if there is a major discrepancy
between the highest group and the controls. If hematological effects
were noted in the subchronic test, hematological testing should be
performed at 3, 6, 12, 18, and 24 months for a two year study and at 3,
6, and 12 months for a 1-year study.
(ii) Certain clinical biochemistry determinations on blood should be
carried out at least three times during the test period: just prior to
initiation of dosing (base line data), near the middle and at the end of
the test period. Blood
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samples should be drawn for clinical chemistry measurements from all
non-rodents and at least ten rodents per sex of all groups; if possible,
from the same rodents at each time interval. Test areas which are
considered appropriate to all studies: 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. Suggested chemical
determinations: calcium, phosphorus, chloride, sodium, potassium,
fasting glucose (with period of fasting appropriate to the species),
serum glutamic-pyruvic transaminase (now known as serum alanine
aminotransferase), serum glutamic oxaloacetic transaminase (now known as
serum aspartate aminotransferase), ornithine decarboxylase, gamma
glutamyl transpeptidase, blood urea nitrogen, albumen, blood creatinine,
creatinine phosphokinase, total cholesterol, total bilirubin and total
serum protein measurements. Other determinations which may be necessary
for an adequate toxicological evaluation include analyses of lipids,
hormones, acid/base balance, methemoglobin and cholinesterase activity.
Additional clinical biochemistry may be employed where necessary to
extend the investigation of observed effects.
(iii) Urine samples from rodents at the same intervals as the
hematological examinations under paragraph (b)(9)(i) of this section
should be collected for analysis. The following determinations should be
made from either individual animals or on a pooled sample/sex/group for
rodents: appearance (volume and specific gravity), protein, glucose,
ketones, bilirubin, occult blood (semi-quantitatively); and microscopy
of sediment (semi-quantitatively).
(iv) Ophthalmological examination, using an ophthalmoscope or
equivalent suitable equipment, should be made prior to the
administration of the test substance and at the termination of the
study. If changes in eyes are detected all animals should be examined.
(10) Gross necropsy. (i) A complete gross examination should be
performed on all animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) The liver, kidneys, adrenals, brain and gonads should be
weighed wet, as soon as possible after dissection to avoid drying. For
these organs, at least 10 rodents per sex per group and all non-rodents
should be weighed.
(iii) The following organs and tissues, or representative samples
thereof, should be preserved in a suitable medium for possible future
histopathological examination: All gross lesions and tumors; brain--
including sections of medulla/pons, cerebellar cortex, and cerebral
cortex; pituitary; thyroid/parathyroid; thymus; lungs; trachea; heart;
sternum and/or femur with bone marrow; salivary glands; liver; spleen;
kidneys; adrenals; esophagus; stomach; duodenum; jejunum; ileum; cecum;
colon; rectum; urinary bladder; representative lymph nodes; pancreas;
gonads; uterus; accessory genital organs (epididymis, prostate, and, if
present, seminal vesicles; female mammary gland; aorta; gall bladder (if
present); skin; musculature; peripheral nerve; spinal cord at three
levels--cervical, midthoracic, and lumbar; and eyes. In inhalation
studies, the entire respiratory tract, including nose, pharynx, larynx,
and paranasal sinuses should be examined and preserved. In dermal
studies, skin from sites of skin painting should be examined and
preserved.
(iv) 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.
(v) If other clinical examinations are carried out, the information
obtained from these procedures should be available before microscopic
examination, since they may provide significant guidance to the
pathologist.
(11) Histopathology. (i) The following histopathology should be
performed:
(A) Full histopathology on the organs and tissues, listed above, of
all non-rodents, of all rodents in the control and high dose groups and
of all rodents that died or were killed during the study.
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(B) All gross lesions in all animals.
(C) Target organs in all animals.
(D) Lungs, liver and kidneys of all animals. Special attention to
examination of the lungs of rodents should be made for evidence of
infection since this provides an assessment of the state of health of
the animals.
(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) In case the results of an experiment give evidence of
substantial alteration of the animals' normal longevity or the induction
of effects that might affect a toxic response, the next lower dose level
should be examined fully, as described under paragraph (b)(11)(i) of
this section.
(iv) An attempt should be made to correlate gross observations with
microscopic findings.
(c) 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) All observed results, quantitative and incidental, should be
evaluated by an appropriate 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 study results. (i) 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, 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.
(ii) In any study which demonstrates an absence of toxic effects,
further investigation to establish absorption and bioavailability of the
test substance should be considered.
(3) Test report. (i) In addition to the reporting requirements as
specified under 40 CFR part 792 subpart J, the following specific
information should be reported:
(A) Group animal data. Tabulation of toxic response data by species,
strain, sex and exposure level for:
(1) Number of animals dying.
(2) Number of animals showing signs of toxicity.
(3) Number of animals exposed.
(B) Individual animal data. (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 consumption data, when collected.
(5) Results of ophthalmological examination, when performed.
(6) Hematological tests employed and all results.
(7) Clinical biochemistry tests employed and all results.
(8) Necropsy findings.
(9) Detailed description of all histopathological findings.
(10) Statistical treatment of results, where appropriate.
(ii) In addition, for inhalation studies the following should be
reported:
(A) Test conditions. (1) Description of exposure apparatus including
design, type, dimensions, source of air, system for generating
particulates 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 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) Nominal concentration (total amount of test substance fed into
the inhalation equipment divided by volume of air).
[[Page 160]]
(4) Actual concentration in test breathing zone.
(5) Particle size distribution (e.g., median aerodynamic diameter of
particles with standard deviation from the mean).
(d) References. For additional background information on this test
guideline the following references should be consulted:
(1) Benitz, K.F. ``Measurement of Chronic Toxicity,'' Methods of
Toxicology. Ed. G.E. Paget. (Oxford: Blackwell Scientific Publications,
1970) pp. 82-131.
(2) D'Aguanno, W. ``Drug Safety Evaluation--Pre-Clinical
Considerations,'' Industrial Pharmacology: Neuroleptics. Vol. I, Ed. S.
Fielding and H. Lal. (Mt. Kisco: Futura Publishing Co. 1974) pp. 317-
332.
(3) Fitzhugh, O.G. Third Printing: 1975. ``Chronic Oral 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. 36-45.
(4) Goldenthal, E.I., D'Aguanno, W. ``Evaluation of Drugs,''
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. 60-67.
(5) 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
auspices of the Committee on Toxicology, National Research Council,
National Academy of Sciences, Washington, DC (1977).
(6) National Center for Toxicological Research. ``Appendix B,''
Report of Chronic Studies Task Force Committee, April 13-21, 1972.
(Rockville: National Center for Toxicological Research, 1972).
(7) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,''
Journal of Environmental Pathology and Toxicology, 1:161-182 (1977).
(8) Schwartz, E. ``Toxicology of Neuroleptic Agents,'' Industrial
Pharmacology: Neuroleptics Ed. S. Fielding and H. Lal. (Mt. Kisco,
Futura Publishing Co., 1974) pp. 203-221.
(9) United States Pharmaceutical Manufacturers Association.
Guidelines for the Assessment of Drug and Medical Device Safety in
Animals. (1977).
(10) World Health Organization. ``Guidelines for Evaluation of Drugs
for Use in Man,'' WHO Technical Report Series No. 563. (Geneva: World
Health Organization, 1975).
(11) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
(12) World Health Organization. ``Principles for Pre-Clinical
Testing of Drug Safety,'' WHO Technical Report Series No. 341. (Geneva:
World Health Organization, 1966).
[50 FR 39397, Sept. 27, 1985, as amended at 54 FR 21064, May 16, 1989]