Evaluation of hexamethylphosphoramide for gene mutations in Salmonella typhimurium using plate incorporation, preincubation, and suspension assays

Hexamethylphosphoramide (HMPA), a potent rat nasal carcinogen by inhalation, and three of its metabolites, pentamethylphosphoramide (PMPA), trimethylphosphoramide (TriMPA), and formaldehyde (HCHO), were assessed in Salmonella typhimurium gene mutation assays using various protocols, including plate incorporation, preincubation and suspension assays. HMPA (tested up to 15 000 μg/plate) was not mutagenic in plate incorporation or preincubation assays with or without metabolic activation. HCHO was mutagenic in the plate incorporation and preincubation assays (tested up to 150 μg/plate). In suspension assays, however, HMPA (tested up to 40 mg/ml), PMPA (up to 44 mg/ml) and HCHO (up to 45 μg/ml), but not TriMPA (up to 29 mg/ml), were mutagenic. HMPA and PMPA were positive only with activation. HMPA's mutagenicity was optimized using a relatively high level of rat liver S9 protein (3.5 mg/plate) in the metabolic activation mixture. Semicarbazide, an HCHO trapping agent, added at concentrations up to 167 μg/ml, markedly inhibited the mutagenic activities of HMPA and PMPA suggesting that HCHO generation may play a role in their mutagenicity. These studies show that HMPA is mutagenic in a modified Salmonella typhimurium reverse mutation assay with metabolic activation. Successive N-demethylation of HMPA eventually eliminates the mutagenic activity which further suggests that HMPA's mutagenic activity is related to the release of HCHO.