Use of the mouse spot test in chemical mutagenesis: interpretation of past data and recommendations for future work.

The mouse spot test, developed 23 years ago, is an in vivo assay capable of detecting genetic effects of several kinds, including intragenic mutations, minute deficiencies, deletions (through breakage or nondisjunction) of various amounts of chromosomal material, and somatic crossing-over. The method involves exposing embryos that are heterozygous for a number of coat-color markers to the test agent, and, 3 weeks later, looking for clones of mutant cells, i.e., spots of color expressing the recessive marker in an otherwise black fur. Spots having other causes may also be induced, specifically white midventral spots due to cytotoxic effects, and certain spots resulting from misdifferentiation. Spot-test results have, to date, been reported from 7 laboratories. Because the control results for any one cross and solvent were found to be reasonably consistent between the laboratories, we pooled these to develop a "historical" control with which experimental results for the same cross and solvent were compared. Experimental results were classified as positive, negative, or inconclusive on the basis of a multiple-decision procedure produced by the testing of the following 2 hypotheses: (1) the mutation frequency (induced + spontaneous) in treated mice is not higher than the mutation frequency in the appropriate pooled control, and (2) the induced mutation frequency of the treated mice is no less than 4 times as high as the observed mutation frequency in the appropriate pooled control. Each hypothesis was tested at the 5% significance level. To date, 30 substances have been employed in the spot test, including 3 that are solvents for some of the others. Of the remaining 27 (26 compounds and 1 mixture), 16 were positive, 6 negative, and 5 inconclusive. The 26 compounds fell into 27 chemical classifications (using a system provided for use by the GENE-TOX program). The inadequacies in the design and reporting of some past experiments indicate a need for a carefully specified protocol. When properly done, the spot test will fulfill a useful role in mutagenicity testing programs because (1) it is an in vivo mammalian assay, (2) it detects genetic effects of many kinds, and (3) it is relatively rapid. Since the test appears well suited to the identification of potent mutagens, its main value should be in screening large numbers of substances and singling out the potentially worst offenders to be further studied in germ-line mutagenesis tests.