Evaluation of carbendazim for gene mutations in the Salmonella/Ames plate-incorporation assay: the role of aminophenazine impurities

Benomyl (methyl [1-[(butylamino)carbonyl]-1 H-benzimidazol-2-yl]carbamate) and its major metabolite carbendazim (methyl 2-benzimidazolecarbamate) are major agricultural systemic fungicides. These compounds inhibit fungal microtubular function and thereby cause nondisjunction of chromosomes at cell division. Several investigators have proposed that these compounds can also cause gene mutations (base-pair substitutions). In this laboratory, no mutagenic activity was observed with either benomyl (analytical grade) or Benlate® (samples tested up to 500 and 1200 μg/plate, respectively, the limit of cytotoxicity) in the Salmonella/Ames plate-incorporation test in either base-pair substitution (TA100 and TA1535) or frameshift-sensitive (TA98 and TA1537) strains with or without S9 metabolic activation. However, some carbendazim preparations caused mutations in frameshift-sensitive strains at very high concentrations (≥ 5000 μg/plate) with metabolic activation. The mutagenic activity was not due to the major carbendazim metabolite, methyl (5-hydroxy-1 H-benzimidazol-2-yl)carbamate (5-OH MBC), since 5-OH MBC was not mutagenic with (up to 20 000 μg/plate) or without (up to 16 000 μg/plate) activation. Subsequently, two highly mutagenic contaminants, 2,3-diaminophenazine (DAP) and 2-amino-3-hydroxyphenazine (AHP) were detected in mutagenic carbendazim samples. In those samples, DAP and AHP contaminant levels ranged as high as 46.5 and 11.6 ppm, respectively. No evidence of mutagenicity could be detected in preparations in which the DAP content was < 1.8 ppm. The mutagenic activity of these two contaminants was further investigated in strain TA98. Without activation, DAP and AHP were positive at test concentrations as low as 5 and 10 μg/plate, respectively. In the presence of S9, mutations were detected at much lower concentrations (beginning at 0.025 and 0.05 μg/plate, respectively). These results indicate that carbendazim samples containing DAP or AHP at levels as low as 5 or 10