§ 799.9410 TSCA chronic toxicity.

(a) Scope—(1) Applicability. This section is intended to meet the testing requirement of the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601).

(2) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides and Toxic Substances (OPPTS) harmonized test guideline 870.4100 (August 1998, final guidelines). This source is available at the address in paragraph (h) of this section

(b) 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. A chronic toxicity study should generate data from which to identify the majority of chronic effects and 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 (pathological) effects.

(c) Definitions. The definitions in section 3 of TSCA and in 40 CFR Part 792—Good Laboratory Practice Standards apply to this section. The following definitions also apply to this section.

Chronic toxicity is the adverse effects occurring as a result of the repeated daily exposure of experimental animals to a chemical by the oral, dermal, or inhalation routes of exposure.

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.

Dose in a chronic toxicity study is the amount of test substance administered daily via the oral, dermal or inhalation routes for a period of at least 12 months. Dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of test animal (milligram per kilogram), or as weight of the test substance in parts per million (ppm) in food or drinking water per day. For inhalation exposure, dose is expressed as weight of the test substance per unit volume of air (milligrams per liter) or as parts per million per day. For dermal exposure, dose is expressed as weight of the test substance (grams, milligrams) per unit body weight of the test animal (milligrams per kilogram) or as weight of the substance per unit of surface area (milligrams per square centimeter) per day.

No-observed-effects level (NOEL) is the maximum dose used in a study which produces no adverse effects. The NOEL is usually expressed in terms of the weight of a test substance given daily per unit weight of test animal (milligrams per kilogram per day).

Target organ is any organ of a test animal showing evidence of an effect induced by a test substance.

(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, a full study using three dose levels might not be necessary.

(e) Test procedures—(1) Animal selection—(i) Species and strain. Testing should be performed with two mammalian species, one a rodent and the other a nonrodent. The rat is the preferred rodent species. Commonly used laboratory strains must be employed.

(ii) Age/weight. (A) Testing must be started with young healthy animals as soon as possible after weaning and acclimatization.

(B) Dosing of rodents should generally begin no later than 8 weeks of age.

(C) Dosing of non-rodents should begin between 4 and 6 months of age and in no case later than 9 months of age.

(D) At commencement of the study, the weight variation of animals used should be within 20% of the mean weight for each sex.

(E) 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) Females should be nulliparous and nonpregnant.

(iv) Numbers. (A) For rodents, at least 40 animals (20 males and 20 females) and for nonrodents at least 8 animals (4 females and 4 males) should be used at each dose level and concurrent control group.

(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. The Agency must be notified if excessive early deaths or other problems are encountered that might compromise the integrity of the study.

(D) To avoid bias, the use of adequate randomization procedures for the proper allocation of animals to test and control groups is required.

(E) Each animal should be assigned a unique identification number. Dead animals, their preserved organs and tissues, and microscopic slides should be identified by reference to the unique numbers assigned.

(v) Husbandry. (A) Rodents may be group-caged by sex, but the number of animals per cage must not interfere with clear observation of each animal. The biological properties of the test substance or toxic effects (e.g., morbidity, excitability) may indicate a need for individual caging. Rodents should be housed individually in dermal studies and during exposure in inhalation studies. Caging should be appropriate to the nonrodent species.

(B) The temperature of the experimental animal rooms should be at 22 ±3 °C.

(C) The relative humidity of the experimental animal rooms should be 50 ±20%.

(D) Where lighting is artificial, the sequence should be 12 hours light/12 hours dark.

(E) Control and test animals should be fed from the same batch and lot. The feed should be analyzed to assure adequacy of nutritional requirements of the species tested and for impurities that might influence the outcome of the test. Animals should be fed and watered ad libitum with food replaced at least weekly.

(F) The study should not be initiated until animals have been allowed a period of acclimatization/quarantine to environmental conditions, nor should animals from outside sources be placed on test without an adequate period of quarantine. An acclimation period of at least 5 days is recommended.

(2) Control and test substances. (i) Where necessary, the test substance is dissolved or suspended in a suitable vehicle. If a vehicle or diluent is needed it should not elicit toxic effects itself nor substantially alter the chemical or toxicological properties of the test substance. It is recommended that wherever possible the use of an aqueous solution be the first choice, followed by consideration of solution in oil, and finally, solution in other vehicles.

(ii) One lot of the test substance should be used, if possible, throughout the duration of the study, and the research sample should be stored under conditions that maintain its purity and stability. Prior to the initiation of the study, there should be a characterization of the test substance, including the purity of the test compound, and, if technically feasible, the names and quantities of contaminants and impurities.