[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: 40CFR798.3300]
[Page 160-165]
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. 798.3300 Oncogenicity.
(a) Purpose. The objective of a long-term oncogenicity study is to
observe test animals for a major portion of their life span for the
development of neoplastic lesions during or after exposure to various
doses of a test substance by an appropriate route of administration.
(b) Test procedures--(1) Animal selection--(i) Species and strain. A
compound of unknown activity shall be tested on two mammalian species.
Rats and mice are the species of choice because of their relatively
short life spans, the limited cost of their maintenance, their
widespread use in pharmacological and toxicological studies, their
susceptibility to tumor induction, and the availability of inbred or
sufficiently characterized strains. Commonly used laboratory strains
shall be employed. If other species are used, the tester shall provide
justification/reasoning for their selection.
(ii) Age. (A) Dosing of rodents shall begin as soon as possible
after weaning, ideally before the animals 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 shall not exceed 20 percent of the mean
weight for each sex.
(C) Studies using prenatal or neonatal animals may be recommended
under special conditions.
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(iii) Sex. (A) Animals of each sex shall be used at each dose level.
(B) The females shall be nulliparous and non-pregnant.
(iv) Numbers. (A) For rodents, at least 100 animals (50 females and
50 males) shall be used at each dose level and concurrent control.
(B) If interim sacrifices are planned the number shall 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 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 does not fall below 50 percent at
the time of termination.
(2) Control groups. (i) A concurrent control group is required. This
group shall 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.
(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
shall be utilized. The negative control group shall be treated in the
same manner as all other test animals except that this control group
shall not be exposed to either the test substance or any vehicle.
(iii) The use of historical control data (i.e., the incidence of
tumors and other suspect lesions normally occurring 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 3 dose levels shall be used, in addition to the
concurrent control group. Dose levels should be spaced to produce a
gradation of chronic effects.
(ii) The high dose level should elicit signs of minimal toxicity
without substantially altering the normal life span.
(iii) The lowest dose should not interfere with normal growth,
development and longevity of the animal; and it should not otherwise
cause any indication of toxicity. In general, this should not be lower
than ten percent of the high dose.
(iv) The intermediate dose(s) should be established in a mid-range
between the high and low doses, depending upon the toxicokinetic
properties of the chemical, if known.
(v) The selection of these dose levels should be based on existing
data, preferably on the results of subchronic studies.
(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. However, based primarily on
practical considerations, dosing on a 5 day per week basis is considered
to be acceptable.
(5) Observations period. It is necessary that the duration of an
oncogenicity test comprise the majority of the normal life span of the
strain of animals to be used. This time period shall not be less than 24
months for rats and 18 months for mice, and ordinarily not longer than
30 months for rats and 24 months for mice. For longer time periods, and
where any other species are used, consultation with the Agency in regard
to the duration of the test is advised.
(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 shall receive the test substance
in their diet, dissolved in drinking water at levels that do not exceed
the maximum solubility of the test chemical under testing condition.
(B) If the test substance is administered in the drinking water, or
mixed in the diet, exposure shall be continuous.
(C) For a diet mixture, the highest concentration should not exceed
5 percent.
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(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 shall 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 shall 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 shall be tested with
inhalation equipment designed to sustain a minimum 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. To ensure stability of a chamber atmosphere, the
total ``volume'' of the test animals shall 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 shall be withheld during each daily 6-hour
exposure period.
(D) A dynamic inhalation system with a suitable flow control system
shall be used. The rate of air flow shall 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) Observations of animals. (i) Each animal shall be observed daily
and if necessary should be handled to appraise its physical condition.
(ii) Additional observations shall 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 shall be recorded for all
animals. Special attention should be paid to tumor development. The day
of onset, location, dimensions, appearance and progression of each
grossly visible or palpable tumor shall be recorded.
(iv) Body weights shall 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, shall 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 shall 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 shall be monitored continuously and
recorded at intervals of at least once every 30 minutes.
(ii) During each exposure period the actual concentrations of the
test substance shall be held as constant as practicable, monitored
continuously and recorded at least three times during the test period:
at the beginning, at
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an intermediate time and at the end of the period.
(iii) During the development of the generating system, particle size
analysis shall be performed to establish the stability of aerosol
concentrations with respect to particle size. During exposure, analyses
shall be conducted as often as necessary to determine the consistency of
particle size, distribution, and homogeneity of the exposure stream.
(iv) Temperature and humidity shall be monitored continuously, but
shoud be recorded at intervals of at least once every 30 minutes.
(9) Clinical examinations. At 12 months, 18 months, and at
sacrifice, a blood smear shall be obtained from all animals. A
differential blood count shall be performed on blood smears from those
animals in the highest dosage group and the controls. If these data, or
data from the pathological examination indicate a need, then the 12- and
18-month blood smears from other dose levels shall also be examined.
Differential blood counts shall be performed for the next lower group(s)
if there is a major discrepancy between the highest group and the
controls. If clinical observations suggest a deterioration in health of
the animals during the study, a differential blood count of the affected
animals shall be performed.
(10) Gross necropsy. (i) A complete gross examination shall be
performed on all animals, including those which died during the
experiment or were killed in moribund conditions.
(ii) The following organs and tissues or representative samples
thereof, shall be preserved in a suitable medium for possible future
histopathological examination: All gross lesions and tumors of all
animals shall be preserved; brain--including sections of medulla/pons,
cerebellar cortex and cerebral cortex; pituitary; thyroid/parathyroid;
thymus; lungs; trachea; heart; spinal cord at three levels--cervical,
midthoracic and lumbar; 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); mammary
gland; skin; musculature; peripheral nerve; and eyes. In inhalation
studies, the entire respiratory tract shall be preserved, including
nasal cavity, pharynx, larynx and paranasal sinuses. In dermal studies,
skin from sites of skin painting shall be examined and preserved.
(iii) 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 required for
appropriate and valid histopathological examination.
(iv) If other clinical examinations are carried out, the information
obtained from these procedures shall be available before microscopic
examination, since they may provide significant guidance to the
pathologist.
(11) Histopathology. (i) The following histopathology shall be
performed:
(A) Full histopathology on organs and tissues listed above of all
animals in the control and high dose groups and all animals that died or
were killed during the study.
(B) All gross lesions in all animals.
(C) Target organs in all animals.
(ii) If a significant difference is observed in hyperplastic, pre-
neoplastic or neoplastic lesions between the highest dose and control
groups, microscopic examination shall be made on that particular organ
or tissue of all animals in the study.
(iii) If excessive early deaths or other problems occur in the high
dose group, compromising the significance of the data, the next lower
dose level shall be examined for complete histopathology.
(iv) 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 neoplastic response, the next lower dose
level shall be examined fully as described in this section.
(v) An attempt shall be made to correlate gross observations with
microscopic findings.
(c) Data and reporting--(1) Treatment of results. (i) Data shall be
summarized in tabular form, showing for each test group the number of
animals at the
[[Page 164]]
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, shall be
evaluated by an appropriate statistical method. Any generally accepted
statistical method may be used; the statistical methods shall be
selected during the design of the study.
(2) Evaluation of study results. (i) The findings of an oncogenic
toxicity study shall 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 shall 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.
(iii) In order for a negative test to be acceptable, it shall 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 should be 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 shall 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 shall 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 shall be described.
(B) Exposure data. These shall be tabulated and presented with mean
values and a measure of variability (e.g., standard deviation) and shall
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) 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).
(2) Food and Drug Administration Advisory Committee on Protocols for
[[Page 165]]
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).
(3) International Union Against Cancer. ``Carcinogenicity Testing,''
IUCC Technical Report Series. Vol. 2., Ed. I. Berenblum. (Geneva:
International Union Against Cancer, 1969).
(4) Leong, B.K.J., Laskin, S. ``Number and Species of Experimental
Animals for Inhalation Carcinogenicity Studies'' Paper presented at
Conference on Target Organ Toxicity, September 1975, Cincinnati, Ohio.
(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 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).
(7) National Center for Toxicological Research. ``Appendix B,''
Report of Chronic Studies Task Force Committee. April 13-21 (Rockville:
National Center for Toxicological Research, 1972).
(8) Page, N.P. ``Chronic Toxicity and Carcinogenicity Guidelines,''
Journal of Environmental Pathology and Toxicology. 1:161-182 (1977).
(9) Page, N.P. ``Concepts of a Bioassay Program in Environmental
Carcinogenesis,'' Advances in Modern Toxicology Vol. 3, Ed. Kraybill and
Mehlman. (Washington, DC: Hemisphere Publishing Corporation, 1977) pp.
87-171.
(10) Sontag, J.M., Page N.P., 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).
(11) United States Pharmaceutical Manufacturers Association.
Guidelines for the Assessment of Drug and Medical Device Safety in
Animals. (1977).
(12) World Health Organization. ``Principles for the Testing and
Evaluation of Drugs for Carcinogenicity,'' WHO Technical Report Series
No. 426. (Geneva: World Health Organization, 1969).
(13) World Health Organization. ``Part I. Environmental Health
Criteria 6,'' Principles and Methods for Evaluating the Toxicity of
Chemicals. (Geneva: World Health Organization, 1978).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19075, May 20, 1987;
54 FR 21064, May 16, 1989]