A combined testing protocol for assessing genotoxicity in individual animals: Application to environmental toxicology

A multiple end‐point approach to assessing genetic toxicity (the combined testing protocol, CTP) was evaluated in male and female CD‐1 mice exposed subacutely (3 and 6 weeks) to low levels of a custom‐blended gas mixture (epichlorohydrin, benzene, chloroprene and xylene, at 50, 100, 100, and 100 ppb, respectively, as the low dose, with concentration levels 10‐fold and 100‐fold higher as the intermediate and high doses, or 0.1, 1 and 10 ppm of benzene). Urine mutagenicity was tested in the Salmonella/microsome assay, chromosome aberrations were examined in bone marrow and spleen lymphocytes, micronuclei were measured in bone marrow and peripheral erythrocytes, and cytochrome P450 and glutathione S‐transferases were measured in the liver. Structural aberrations in alveolar macrophages and spermatocytes, and thioguanine resistance in spleen lymphocytes were examined for their suitability for incorporation into the overall protocol. Spleen lymphocytes were the most sensitive indicator cells, and showed a dose‐related increase (P<0.01) in structural chromosome aberrations and in cytotoxicity after 6 weeks of exposure. Analysis of micronucleus formation and metaphase aberrations in the bone marrow, and micronuclei in peripheral erythrocytes showed an overall statistically non‐significant but positive trend at the high dose. No mutagenicity was detected in pooled urine samples. Liver microsomal cytochrome P450 was not increased, but cytosolic glutathione S‐transferases were significantly increased in a dose‐related manner. Since the probability of detecting a genotoxic effect increases with the number of endpoints and tissues examined, this approach should be applicable to many situations without having to perform separate experiments for each tissue examined. The multiple endpoints evaluated and the flexibility of the CTP with respect to which endpoints are included, plus the reduction in experimental variability, give it advantages over batteries of separate assays.