[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: 40CFR798.3320]
[Page 165-172]
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. 798.3320 Combined chronic toxicity/oncogenicity.
(a) Purpose. The objective of a combined chronic toxicity/
oncogenicity study is to determine the effects of a substance in a
mammalian species following prolonged and repeated exposure. The
application of this guideline should generate data which identify the
majority of chronic and oncogenic effects and determine dose-response
relationships. The design and conduct should allow for the detection of
neoplastic effects and a determination of oncogenic potential as well as
general toxicity, including neurological, physiological, biochemical,
and hematological effects and exposure-related morphological (pathology)
effects.
(b) Test procedures--(1) Animal selection--(i) Species and strain.
Preliminary studies providing data on acute, subchronic, and metabolic
responses should have been carried out to permit an appropriate choice
of animals (species and strain). As discussed in other guidelines, the
mouse and rat have been most widely used for assessment of oncogenic
potential, while the rat and dog have been most often studied for
chronic toxicity. The rat is the species of choice for combined chronic
toxicity and oncogenicity studies. The provisions of this guideline are
designed primarily for use with the rat as the test species. If other
species are used, the tester should provide justification/reasoning for
their selection. The strain selected should be susceptible to the
oncogenic or toxic effect of the class of substances being tested, if
known, and provided it does not have a spontaneous background too high
for
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meaningful assessment. Commonly used laboratory strains should be
employed.
(ii) Age. (A) Dosing of rats should begin as soon as possible after
weaning, ideally before the rats are 6 weeks old, but in no case more
than 8 weeks old.
(B) At commencement of the study, the weight variation of animals
used should not exceed 20 percent of the mean
weight for each sex.
(C) 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) The females should be nulliparous and nonpregnant.
(iv) Numbers. (A) At least 100 rodents (50 females and 50 males)
should be used at each dose level and concurrent control for those
groups not intended for early sacrifice. At least 40 rodents (20 females
and 20 males) should be used for satellite dose group(s) and the
satellite control group. The purpose of the satellite group is to allow
for the evaluation of pathology other than neoplasia.
(B) If interim sacrifices are planned, the number of animals 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 each phase of the
study should be adequate for a meaningful and valid statistical
evaluation of long term exposure. For a valid interpretation of negative
results, it is essential that survival in all groups not fall below 50
percent at the time of termination.
(2) Control groups. (i) A concurrent control group (50 females and
50 males) and a satellite control group (20 females and 20 males) are
recommended. These groups should be untreated or sham treated control
groups or, if a vehicle is used in administering the test substance,
vehicle control groups. If the toxic properties of the vehicle are not
known or cannot be made available, both untreated and vehicle control
groups are recommended. Animals in the satellite control group should be
sacrificed at the same time the satellite test group is terminated.
(ii) In special circumstances such as 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 the test substance or any vehicle.
(iii) The use of historical control data (i.e., the incidence of
tumors and other suspect lesions normally occuring under the same
laboratory conditions and in the same strain of animals employed in the
test) is desirable for assessing the significance of changes observed in
exposed animals.
(3) Dose levels and dose selection. (i) For risk assessment
purposes, 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 highest dose level in rodents should elicit signs of
toxicity without substantially altering the normal life span due to
effects other than tumors.
(iii) The lowest dose level should produce no 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 levels 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.
(vi) For chronic toxicological assessment, a high dose treated
satellite and a concurrent control satellite group should be included in
the study design. The highest dose for satellite animals should be
chosen so as to produce frank toxicity, but not excessive lethality, in
order to elucidate a chronic toxicological profile of the test
substance. If more than one dose level is selected for satellite dose
groups, the doses should be spaced to produce a gradation of toxic
effects.
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(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 24
months for rats, and 18 months for mice and hamsters, except for the
animals in the satellite groups which should be dosed for 12 months.
(5) Observation period. It is necessary that the duration of the
oncogenicity test comprise the majority of the normal life span of the
animals to be used. It has been suggested that the duration of the study
should be for the entire lifetime of all animals. However, a few animals
may greatly exceed the average lifetime and the duration of the study
may be unnecessarily extended and complicate the conduct and evaluation
of the study. Rather, a finite period covering the majority of the
expected life span of the strain is preferred since the probability is
high that, for the great majority of chemicals, induced tumors will
occur within such an observation period. The following guidelines are
recommended:
(i) Generally, the termination of the study should be at 18 months
for mice and hamsters and 24 months for rats; however, for certain
strains of animals with greater longevity and/or low spontaneous tumor
rate, termination should be at 24 months for mice and hamsters and at 30
months for rats. For longer time periods, and where any other species
are used, consultation with the Agency in regard to duration of the test
is advised.
(ii) However, termination of the study is acceptable when the number
of survivors of the lower doses or of the control group reaches 25
percent. In the case where only the high dose group dies prematurely for
obvious reasons of toxicity, this should not trigger termination of the
study.
(iii) The satellite groups and the concurrent satellite control
group should be retained in the study for at least 12 months. These
groups should be scheduled for sacrifice for an estimation of test-
substance-related pathology uncomplicated by geriatric changes.
(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 24 months for rats and 18 months for
mice and hamsters.
(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 should 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 as possible 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
nonirritating 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, to 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
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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 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 sacrifice
of weak or moribund animals).
(iii) Clinical signs and mortality should be recorded for all
animals. Special attention should be paid to tumor development. The time
of onset, location, dimensions, appearance and progression of each
grossly visible or palpable tumor should be recorded.
(iv) 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.
(v) 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.
(vi) At the end of the study period, all survivors are 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 airflow 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 recorded 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, analyses 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. (i) The following examinations should be
made on at least 20 rodents of each sex per dose level:
(A) 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 those groups on test for
longer than 12 months) on blood samples collected from 20 rodents 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 animals in the highest dosage group
and the controls. Differential blood counts should be performed for the
next lower group(s) if
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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 year study.
(B) Certain clinical biochemistry determinations on blood should be
carried out at least three times during the test period: Just prior to
initiation of dosing (baseline data), near the middle and at the end of
the test period. Blood samples should be drawn for clinical measurements
from 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, 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.
(ii) The following should be performed on at least 10 rodents of
each sex per dose level:
(A) Urine samples from the same rodents at the same intervals as
hematological examination above, 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).
(B) 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 the 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 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.
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(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.
(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 the 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 as described above.
(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 combined
chronic toxicity/oncogenicity 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 bioavailablity of the
test substance should be considered.
(iii) In order for a negative test to be acceptable, it should meet
the following criteria: No more than 10 percent of any group is lost due
to autolysis, cannibalism, or management problems; and survival in each
group is no less than 50 percent at 18 months for mice and hamsters and
at 24 months for rats.
(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.
(11) Historical control data, if taken into account.
(ii) In addition, for inhalation studies the following should be
reported:
[[Page 171]]
(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).
(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, New York: Futura Publishing Co., 1974)
pp. 317-332.
(3) Department of Health and Welfare. The Testing of Chemicals for
Carcinogenicity, Mutagenicity, Teratogenicity. Minister of Health and
Welfare. (Canada: Department of Health and Welfare, 1975).
(4) Fitzhugh, O.G. ``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.
(5) Food and Drug Administration Advisory Committee on Protocols for
Safety Evaluation: Panel on Carcinogenesis. ``Report on Cancer Testing
in the Safety of Food Additives and Pesticides,'' Toxicology and Applied
Pharmacology. 20:419-438 (1971).
(6) Goldenthal, E.I., and 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.
(7) International Union Against Cancer. ``Carcinogenicity Testing,''
IUCC Technical Report Series Vol. 2, Ed. I. Berenblum. (Geneva:
International Union Against Cancer, 1969).
(8) Leong, B.K.J., and Laskin, S. ``Number and Species of
Experimental Animals for Inhalation Carcinogenicity Studies,'' Paper
presented at Conference on Target Organ Toxicity. September, 1975,
Cincinnati, Ohio.
(9) 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).
(10) National Cancer Institute. Report of the Subtask Group on
Carcinogen Testing to the Interagency Collaborative Group on
Environmental Carcinogenesis. (Bethesda: United States National Cancer
Institute, 1976).
(11) National Center for Toxicological. Report of Chronic Studies
Task Force Research Committee. ``Appendix B, (Rockville: National Center
for Toxicological Research, 1972)).
(12) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,''
Journal Environmental Pathology and Toxicology. 1:161-182 (1977).
(13) Page, N.P. ``Concepts of a Bioassay Program in Environmental
Carcinogenesis,'' Advances in Modern Toxicology Volume 3, Ed. Kraybill
and Mehlman. (Washington, D.C.: Hemisphere Publishing Corp., 1977) pp.
87-171.
(14) Schwartz, E. 1974. ``Toxicology of Neuroleptic Agents,''
Industrial Pharmacology: Neuroleptics. Ed. S. Fielding and H. Lal. (Mt.
Kisco, New York: Futura Publishing Co, 1974) pp. 203-221.
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(15) Sontag, J.M., Page, N.P., and Saffiotti, U. Guidelines for
Carcinogen Bioassay in Small Rodents. NCI-CS-TR-1 (Bethesda: United
States Cancer Institute, Division of Cancer Control and Prevention,
Carcinogenesis Bioassay Program, 1976).
(16) United States Pharmaceutical Manufacturers Association.
Guidelines for the Assessment of Drug and Medical Device Safety in
Animals. (1977).
(17) World Health Organization. ``Principles for the Testing and
Evaluation of Drugs for Carcinogenicity,'' WHO Technical Report Series
No. 426. (Geneva: World Health Organization, 1969).
(18) World Health Organization. ``Guidelines for Evaluation of Drugs
for Use in Man,'' WHO Technical Report Series No. 563. (Geneva: World
Health Organization, 1975).
(19) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
(20) 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]