§ 798.6400 Neuropathology.

(a) Purpose. The techniques in this guideline are designed to develop data on morphologic changes in the nervous system for chemical substances and mixtures subject to such testing under the Toxic Substances Control Act. The data will detect and characterize morphologic changes, if and when they occur, and determine a no-effect level for such changes. Neuropathological evaluation should be complemented by other neurotoxicity studies, e.g. behavioral and neurophysiological studies. Neuropathological evaluation may be done following acute, subchronic or chronic exposure.

(b) Definition. Neurotoxicity or a neurotoxic effect is an adverse change in the structure or function of the nervous system following exposure to a chemical agent.

(c) Principle of the test method. The test substance is administered to several groups of experimental animals, one dose being used per group. The animals are sacrificed and tissues in the nervous system are examined grossly and prepared for microscopic examination. Starting with the highest dosage level, tissues are examined under the light microscope for morphologic changes, until a no effect level is determined. In cases where light microscopy has revealed neuropathology, the no effect level may be confirmed by electron microscopy.

(d) Test procedure—(1) Animal selection—(i) Species and strain. Testing shall be performed in the species being used in other tests for neurotoxicity. This will generally be the laboratory rat. The choice of species shall take into consideration such factors as the comparative metabolism of the chemical and species sensitivity to the toxic effects of the test substance, as evidenced by the results of other studies, the potential for combined studies, and the availability of other toxicity data for the species.

(ii) Age. Animals shall be young adults (150-200 gm for rats) at the start of exposure.

(iii) Sex. Both sexes shall be used unless it is demonstrated that one sex is refractory to the effects.

(2) Number of animals. A minimum of six animals per group shall be used. The tissues from each animal shall be examined separately. It is recomse (iv)mended that ten animals per group be used.

(3) Control groups. (i) A concurrent control group(s) is (are) required. This group must be an untreated control group or, if a vehicle is used in administering the test substance, a vehicle control group. If the vehicle used has a known or potential toxic property, both untreated and vehicle control groups are required.

(ii) A satellite group of animals may be treated with the high 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. At least 3 doses, equally spaced on a log scale (e.g., 1⁄2 log units) over a range of at least 1 log unit shall be used in addition to a zero dose or vehicle administration. The data should be sufficient to produce a dose-effect curve.

(i) The highest dose shall produce (A) clear behavioral effects or (B) life-threatening toxicity.

(ii) The data from the lower doses must show either (A) graded dose-dependent effects at two dose levels or (B) no effects at two dose levels, respectively.