Genotoxic effects of N-nitrosodicyclohexylamine in isolated human lymphocytes

Dicyclohexylamine x nitrite is classified as an "experimental equivocal tumorigenic agent" by the National Toxicology Program. Since no genotoxic effects of the substance itself are known, the reported tumorigenic potential of dicyclohexylamine x nitrite could be due to generation of N-nitrosodicyclohexylamine (N-NO-DCHA), which occurs under conditions of use and can be detected in foils that contain dicyclohexylamine x nitrite. Therefore, we investigated possible mutagenic properties of N-NO-DCHA in the Ames test and the cytokinesis-block micronucleus assay with human lymphocytes. Since N-NO-DCHA is not commercially available, the substance was synthesized and purified by thin-layer chromatography. Identity was confirmed by gas chromatography/mass spectroscopy (GC/MS) and 1H- and 13C-NMR. More than 97% purity was achieved. Stability and availability in the solvent were checked by GC/MS. N-NO-DCHA induced micronuclei in isolated human lymphocytes at a dose range of 15-100 micrograms/ml (= 71.4-476.2 microM), exceeding the base rate significantly at one or two nontoxic concentrations in four out of six experiments. For the Ames test, arochlor-1254-, beta-naphthoflavone/phenobarbital- and pyrazole-induced S9-fractions were used with Salmonella typhimurium TA100, TA1535, TA98 and TA104. No effects were seen in the Ames test, with the exception of microcolony induction at doses higher than 250 micrograms (= 1.2 mmol) N-NO-DCHA/plate using TA104 and 20% arochlor-1254 induced S9 at pH 6.5. In conclusion, N-NO-DCHA was negative in the Ames test using TA98, TA100 and TA1535, inconclusive using TA104, and weakly genotoxic in the in vitro micronucleus test with isolated human lymphocytes. With regard to the tumorigenicity of the majority of nitrosamines, our data underline the necessity of further studies on possible genotoxic effects of N-NO-DCHA.