§ 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 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.