Tissue-specific mutagenicity of N-nitroso-methylphenylamine assayed by the transgenic rodent assay and duplex sequencing

N -Nitroso-methylphenylamine (NMPA) is a rat esophageal carcinogen that has been found in food, tobacco and drugs. In this study, NMPA was administered via drinking water to Big Blue transgenic rats over 28 days following methods recommended in OECD TG 488; benzo[ a ]pyrene (B[a]P) was administered as the positive control. The esophagus, liver, and bone marrow were collected and analyzed for mutation using both the cII mutation assay and Duplex Sequencing (DS). NMPA treatment induced tissue-specific mutations, with the esophagus exhibiting the highest mutagenic response in both the cII and DS assays, followed by the liver; bone marrow remained relatively unaffected, showing no increase in the cII assay and only a slight elevation when analyzed by DS. The DS mutational spectrum for NMPA was characterized by a broadly distributed elevation across all base substitution types and in all trinucleotide contexts, distinguishing it from other small-molecule nitrosamines. Notably, DS detected fold-increases in NMPA mutagenesis greater than those detected by the transgenic rodent (TGR) assay, with a maximum 7.5-fold increase in genomic mutation frequency in the esophagus compared to the 5.6-fold increase in the cII gene. These data suggest that NMPA is an organ-specific mutagen, and that its mutagenic potency corresponds with its carcinogenicity in the primary target tissue and other tissues.