§ 799.9430 TSCA combined chronic toxicity/carcinogenicity.

(a) Scope. This section is intended to meet the testing requirements under section 4 of the Toxic Substances Control Act (TSCA). The objective of a combined chronic toxicity/carcinogenicity study is to determine the effects of a substance in a mammalian species following prolonged and repeated exposure. The application of this section should generate data which identify the majority of chronic and carcinogenicity effects and determine dose-response relationships. The design and conduct should allow for the detection of neoplastic effects and a determination of the carcinogenic potential as well as general toxicity, including neurological, physiological, biochemical, and hematological effects and exposure-related morphological (pathology) effects.

(b) Source. The source material used in developing this TSCA test guideline is the Office of Prevention, Pesticides, and Toxic Substances (OPPTS) harmonized test guideline 870.4300 (August 1998, final guideline). This source is available at the address in paragraph (h) of this section.

(c) Definitions. The following definitions apply to this section.

Carcinogenicity is the development of neoplastic lesions as a result of the repeated daily exposure of experimental animals to a chemical by the oral, dermal, or inhalation routes of exposure.

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 dose occurring as a result of prolonged action on, or increased concentration of, the administered test substance or its metabolites in susceptible tissues.

Dose in a combined chronic toxicity/carcinogenicity study is the amount of test substance administered via the oral, dermal, or inhalation routes for a period of up to 24 months. Dose is expressed as weight of the test substance per unit body weight of test animal (milligrams per kilogram), or as weight of the test substance in parts per million (ppm) in food or drinking water. When exposed via inhalation, 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 application, 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 surface area (milligrams per square centimeter) per day.

No-observed-effects level (NOEL) is the maximum dose used in a study which produces no observed 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 or if toxic effects 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—(i) Species and strain. Preliminary studies providing data on acute, subchronic, and metabolic responses should have been carried out to permit an appropriate choice of animals (species and strain). As discussed in other guidelines, the mouse and rat have been most widely used for assessment of carcinogenic potential, while the rat and dog have been most often studied for chronic toxicity. For the combined chronic toxicity/carcinogenicity study via the oral and inhalation routes, the rat is the species of choice and for the dermal route, the mouse is species of choice. If other species are used, the tester must provide justification/reasoning for their selection. The strain selected should be susceptible to the carcinogenic or toxic effect of the class of substances being tested, if known, and provided it does not have a spontaneous background incidence too high for meaningful assessment. 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 should generally begin no later than 8 weeks of age.

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

(D) Studies using prenatal or neonatal animals may be recommended under special conditions.

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

(B) Females must be nulliparous and nonpregnant.

(iv) Numbers. (A) At least 100 rodents (50 males and 50 females) must be used at each dose level and concurrent control group. At least 20 additional rodents (10 males and 10 females) should be used for satellite dose groups and the satellite control group. The purpose of the satellite group is to allow for the evaluation of chronic toxicity after 12 months of exposure to the test substance.

(B) For a meaningful and valid statistical evaluation of long term exposure and for a valid interpretation of negative results, the number of animals in any group should not fall below 50% at 15 months in mice and 18 months in rats. Survival in any group should not fall below 25% at 18 months in mice and 24 months in rats.

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

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

(v) Husbandry. (A) Animals 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.

(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 uniform distribution and 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 five days is recommended.