798.2650—Oral toxicity.

(a) Purpose. In the assessment and evaluation of the toxic characteristics of a chemical, the determination of subchronic oral toxicity may be carried out after initial information on toxicity has been obtained by acute testing. The subchronic oral study has been designed to permit the determination of the no-observed-effect level and toxic effects associated with continuous or repeated exposure to a test substance for a period of 90 days. The test is not capable of determining those effects that have a long latency period for development (e.g., carcinogenicity and life shortening). It provides information on health hazards likely to arise from repeated exposure by the oral route over a limited period of time. It will provide information on target organs, the possibilities of accumulation, and can be of use in selecting dose levels for chronic studies and for establishing safety criteria for human exposure.

(b) Definitions. (1) Subchronic oral toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral route for a part (approximately 10 percent) of a life span.

(2) Dose is the amount of test substance administered. Dose is expressed as weight of test substance (g, mg) per unit weight of test animal (e.g., mg/kg), or as weight of test substance per unit weight of food or drinking water.

(3) No-effect level/No-toxic-effect level/No-adverse-effect level/No-observed-effect level is the maximum dose used in a test which produces no observed adverse effects. A no-observed-effect level is expressed in terms of the weight of a substance given daily per unit weight of test animal (mg/kg). When administered to animals in food or drinking water the no-observed-effect level is expressed as mg/kg of food or mg/ml of water.

(4) Cumulative toxicity is the adverse effects of repeated doses occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissue.

(c) Principle of the test method. The test substance is administered orally in graduated daily doses to several groups of experimental animals, one dose level per group, for a period of 90 days. During the period of administration the animals are observed daily to detect signs of toxicity. Animals which die during the period of administration are necropsied. At the conclusion of the test all animals are necropsied and histo-pathological examinations carried out.

(d) Limit test. If a test at one dose level of at least 1,000 mg/kg body weight (expected human exposure may indicate the need for a higher dose level), using the procedures described for this study, produces no observable toxic effects and if toxicity would not be expected based upon data of structurally related compounds, then a full study using three dose levels might not be necessary.

(e) Test procedures— (1) Animal selection— A mammalian species shall be used for testing. A variety of rodent species may be used, although the rat is the preferred species. Commonly used laboratory strains shall be employed. The commonly used nonrodent species is the dog, preferably of a defined breed; the beagle is frequently used. If other mammalian species are used, the tester shall provide justification/reasoning for his or her selection.

(ii) Age— (A) General. Young adult animals shall be employed. At the commencement of the study the weight variation of animals used shall not exceed ±20 percent of the mean weight for each sex.

(B) Rodents. Dosing shall begin as soon as possible after weaning, ideally before the rats are 6, and in any case, not more than 8 weeks old.

(C) Non-rodent. In the case of the dog, dosing shall commence after acclimatization, preferably at 4 to 6 months and not later than 9 months of age.

(iii) Sex. (A) Equal numbers of animals of each sex shall be used at each dose level.

(B) The females shall be nulliparous and nonpregnant.

(iv) Numbers— (A) Rodents. At least 20 animals (10 females and 10 males) shall be used at each dose level.

(B) Non-rodents. At least eight animals (four females and four males) shall be used at each dose level.

(C) If interim sacrifices are planned, the number shall be increased by the number of animals scheduled to be sacrificed before the completion of the study.

(2) Control groups. 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.

(3) Satellite group. (Rodent) A satellite group of 20 animals (10 animals per sex) may be treated with the high dose level for 90 days and observed for reversibility, persistence, or delayed occurrence of toxic effects for a post-treatment period of appropriate length, normally not less than 28 days.

(4) Dose levels and dose selection. (i) In subchronic toxicity tests, it is desirable to have a dose response relationship as well as a no-observed-toxic-effect level. Therefore, at least 3 dose levels with a control and, where appropriate, a vehicle control (corresponding to the concentration of vehicle at the highest exposure level) shall be used. Doses should be spaced appropriately to produce test groups with a range of toxic effects. The data should be sufficient to produce a dose-response curve.

(ii) The highest dose level in rodents should result in toxic effects but not produce an incidence of fatalities which would prevent a meaningful evaluation; for non-rodents 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.

(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; for non-rodents, there should be no fatalities.

(5) Exposure conditions. The animals are dosed with the test substance ideally on a 7-day per week basis over a period of 90 days. However, based primarily on practical considerations, dosing in gavage or capsule studies on a 5-day per week basis is considered to be acceptable.

(6) Observation period. (i) Duration of observation shall be for at least 90 days.

(ii) Animals in the satellite group scheduled for followup observations should be kept for at least 28 days further without treatment to detect recovery from, or persistence of, toxic effects.

(7) Administration of the test substance. (i) The test substance may be administered in the diet or in capsules. In addition, for rodents it may also be administered by gavage or in the drinking water.

(ii) All animals shall be dosed by the same method during the entire experimental period.

(iii) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed, ideally it should not elicit important toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the usage of an aqueous solution be considered first, followed by consideration of a solution of oil and then by possible solution in other vehicles.

(iv) For substances of low toxicity, it is important to ensure that when administered in the diet the quantities of the test substance involved do not interfere with normal nutrition. When the test substance is administered in the diet either a constant dietary concentration (ppm) or a constant dose level in terms of the animals' body weight shall be used; the alternative used shall be specified.

(v) For a substance administered by gavage or capsule, the dose shall be given at approximately the same time each day, and adjusted at intervals (weekly or bi-weekly) to maintain a constant dose level in terms of animal body weight.

(8) Observation of animals. (i) Each animal shall be observed daily and, if necessary, 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) Signs of toxicity shall be recorded as they are observed including the time of onset, degree and duration.

(iv) Cage-side observations shall 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) Measurements shall be made weekly of feed consumption or water consumption when the test substance is administered in the feed or drinking water, respectively.

(vi) Animals shall be weighed weekly.

(vii) At the end of the 90-day period all survivors in the nonsatellite treatment groups shall be sacrificed. Moribund animals shall be removed and sacrificed when noticed.

(9) Clinical examinations. (i) The following examinations shall be made on all animals of each sex in each group for rodents and all animals when non-rodents are used as test animals.

(A) Certain hematology determinations shall be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Hematology determinations which are appropriate to all studies: Hematocrit, hemoglobin concentration, erythrocyte count, total and differential leukocyte count, and a measure of clotting potential such as clotting time, prothrombin time, thromboplastin time, or platelet count.

(B) Certain clinical biochemistry determinations on blood should be carried out at least two times during the test period on all groups of animals including concurrent controls: After 30 days of test and just prior to terminal sacrifice at the end of the test period. Clinical biochemistry 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. Suggested 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, urea nitrogen, albumen, blood creatinine, 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 examinations shall be made on high dose and control groups. If changes in the eyes are detected, all animals should be examined.

(A) Ophthalmological examination, using an ophthalmoscope or equivalent suitable equipment, shall be made prior to the administration of the test substance and at the termination of the study.

(B) Urinalysis is not recommended on a routine basis, but only when there is an indication based on expected and or observed toxicity.

(10) Gross necropsy. (i) All animals shall be subjected to a full gross necropsy which includes examination of the external surface of the body, all orifices, and the cranial, thoracic and abdominal cavities and their contents.

(ii) At least the liver, kidneys, adrenals, and gonads shall be weighed wet, as soon as possible after dissection to avoid drying. In addition, for the rodent, the brain; for the non-rodent, the thyroid with parathyroids also shall be weighed wet.

(iii) The following organs and tissues, or representative samples thereof, shall be preserved in a suitable medium for possible future histopathological examination: All gross lesions; lungs—which should be removed intact, weighed, and treated with a suitable fixative to ensure that lung structure is maintained (perfusion with the fixative is considered to be an effective procedure); nasopharyngeal tissues; brain—including sections of medulla/pons, cerebellar cortex, and cerebral cortex; pituitary; thyroid/parathyroid; thymus; trachea; heart; sternum with bone marrow; salivary glands; liver; spleen; kidneys; adrenals; pancreas; gonads; uterus; accessory genital organs (epididymis, prostate, and, if present, seminal vesicles); aorta; (skin); gall bladder (if present); esophagus; stomach; duodenum; jejunum; ileum; cecum; colon; rectum; urinary bladder; representative lymph node; (mammary gland); (thigh musculature); peripheral nerve; (eyes); (femur—including articular surface); (spinal cord at three levels—cervical, midthoracic, and lumbar); and (zymbal and exorbital lachrymal glands); and (rodent-zymbal glands).

(11) Histopathology. The following histopathology shall be performed:

(i) Full histopathology on the organs and tissues, listed above, of all rodents in the control and high dose groups, all non-rodents, and all rodents that died or were killed during the study.

(ii) All gross lesions in all animals.

(iii) Target organs in all animals.

(iv) The tissues mentioned in brackets (listed above) if indicated by signs of toxicity of target organ involvement.

(v) Lungs, liver and kidneys of all animals. Special attention to examination of the lungs of rodents shall be made for evidence of infection since this provides a convenient assessment of the state of health of the animals.

(vi) When a satellite group is used (rodents), histopathology shall be performed on tissues and organs identified as showing effects in the treated groups.

(f) 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, 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 the study results. (i) The findings of a subchronic oral toxicity study should be evaluated in conjunction with the findings of preceding studies and considered in terms of the toxic effects and the necropsy and histopathological findings. The evaluation will include the relationship between the dose of the test substance and the presence or absence, the 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. A properly conducted subchronic test should provide a satisfactory estimation of a no-effect level.

(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. In addition to the reporting requirements as specified under EPA Good Laboratory Practice Standards, 40 CFR part 792, subpart J, the following specific information shall be reported:

(i) Group animal data. Tabulation of toxic response data by species, strain, sex and exposure level for:

(A) Number of animals dying.

(B) Number of animals showing signs of toxicity.

(ii) Individual animal data. (A) Date of death during the study or whether animals survived to termination.

(B) Date of observation of each abnormal sign and its subsequent course.

(D) Feed consumption data when collected.

(E) Hematological tests employed and all results.

(F) Clinical biochemistry tests employed and all results.

(G) Necropsy findings.

(H) Detailed description of all histopathological findings.

(I) Statistical treatment of results where appropriate.

(g) References. For additional background information on this test guideline the following references should be consulted:

(1) Boyd, E.M. “Chapter 14—Pilot Studies, 15—Uniposal Clinical Parameters, 16—Uniposal Autopsy Parameters.” Predictive Toxicometrics. (Baltimore: Williams and Wilkins, 1972).

(2) Fitzhugh, O.G. “Subacute 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. 26-35.

(3) Food Safety Council. “Subchronic Toxicity Studies,” Proposed System for Food Safety Assessment. (Columbia: Food Safety Council, 1978) pp. 83-96.

(4) 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).

(5) 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 19074, May 20, 1987; 53 FR 49150, Dec. 6, 1988; 54 FR 21064, May 16, 1989]