behavior, availability, and overall costs. EPA recommends using rats for subchronic, chronic, carcinogenicity, and reproduction studies; mice for carcinogenicity studies; and dogs for subchronic and chronic studies. Rats are routinely used for acute oral and inhalation studies and rabbits for eye and skin irritation studies and acute dermal studies. One exception to this is the use of guinea pigs for dermal sensitization testing. The rat and rabbit are recommended for developmental toxicity (teratogenicity) testing. Justification must be provided for the use of species other than those outlined above.

The number of animals to be tested in each dose group depends on a number of factors, including the purpose of the experiment, the required sensitivity of the study, the reproductive capacity and the fertility of the species, economic aspects, and the availability of animals (IPCS, 1990). Table 4-2 lists the minimum number of animals required by EPA for some toxicity studies. For the most part, these numbers are consistent with those recommended by the International Program Chemical Safety (IPCS).

The selection of dose levels for subchronic studies should be based on the results of acute toxicity testing, on range-finding studies, and on pharmacokinetic (metabolism, including rate in various tissues) data. For subchronic studies, four dose groups of animals should be included: a control group; a low-dose group (a dose that produces no compound related toxicity); a mid-dose group (a dose that elicits some minimal signs of toxicity); and a high-dose group (a dose that results in toxic effects but not in an incidence of fatalities that would prevent a meaningful evaluation; for nonrodents, there should be no fatalities) (EPA, 1984). This same guidance is relevant to chronic toxicity and reproduction studies. For teratology studies, the highest dose tested should elicit some signs of maternal toxicity, but the toxicity should not obscure the results.

The one notable exception to this guidance pertains to carcinogenicity studies. The highest dose levels for these studies should be at a maximum tolerated dose (MTD), as determined in 90-day toxicity studies in the appropriate test species and from pharmacokinetic information on the material being tested. The Committee on Risk Assessment Methodology of the National Research Council (NRC) recently examined the criteria for the MTD and other doses used in carcinogenicity studies (NRC, 1993). The EPA has issued its own guidance for the selection of this dose level. Some of the factors to consider in selecting an MTD are: 10% decrement in body weight gain in 90-day study; observation of potential life-threatening lesions during microscopic examination of organs, e.g., liver necrosis; significant inhibition of cholinesterase activity in two biological compartments, such as brain and plasma; and significant signs of anemia or other biologically relevant effects on blood.



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