An approach towards the standardization of the mammalian spot test.

The mammalian spot test, which was developed for radiation experiments at the Oak Ridge National Laboratory more than two decades ago, has recently been demonstrated to be a promising method for the detection of presumed somatic mutations induced by chemicals. The results reported in this paper are part of our efforts directed towards the validation and standardization of this test system for mutagenicity testing. (T x HT)F1 embryos, heterozygous for the recessive coat color alleles ln, pa, b, c(ch), p, d, and pe were exposed in utero to a single dose of the test compound between day 8 and 11 post conception. To compare the sensitivities of different genotypes of embryos an additional experiment was carried out with (C57BL/6JHan x T)F1 embryos, which are heterozygous for b, c(ch), p, and d. The offspring were screened for the presence of color spots at the age of 12-14 days and at weaning. The chemical mutagens used in the present experiment were mitomycin C (MC), methyl methanesulfonate (MMS), and procarbazine. The lowest doses tested in these experiments which induced coat color spots (presumed somatic mutations) were 1 mg/kg MC, 50 mg/kg MMS, and 50 mg/kg procarbazine, when the females were treated on day 9 of pregnancy. The frequency of white midventral spots (WMVS), which are caused by an inactivation of melanocyte precursor cells, was increased in the higher dose groups of MC and procarbazine. These doses also reduced the average litter size and caused malformations. The type of malformations depended on the day of treatment. The data obtained with 50 mg/kg procarbazine indicate comparable sensitivities of the two genotypes (T x HT)F1 and (C57BL/6JHan x T)F1. The lowest doubling doses calculated for somatic mutations were 0.3 mg/kg MC, 6.6 mg/kg MMS and 11 mg/kg procarbazine. For germinal gene mutations they were 0.6 mg/kg MC, 1.2 mg/kg MMS and 34 mg/kg procarbazine. The comparison between the expected and observed distribution of coat color spots between the head and body of the mice supports randomness of distribution. Further analysis of the data revealed that the scoring of spots only in the dorsal and lateral regions does not alter the sensitivity of the test. Thereby one can avoid any ambiguity arising from the distribution of midventral white gray and light gray spots