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— 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.

(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.

(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 °C (±2°). 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.

(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 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.

(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. Data shall 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, 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 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).

Code of Federal Regulations

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19075, May 20, 1987; 54 FR 21064, May 16, 1989]