N-nitroso-ethylisopropylamine mutagenicity in rat liver using the cII transgenic mutation assay and duplex sequencing analysis of genomic DNA.

N-Nitroso-ethylisopropylamine (NEIPA) is a small-molecule N-nitrosamine impurity that has been found in some sartan drugs. It induced liver and esophageal carcinomas in BD rats and was mutagenic and genotoxic in several in vitro studies. However, its mutagenicity in rat tumor target tissues has not been established. In this study, the mutagenicity of NEIPA was evaluated in the liver of Big Blue transgenic rats using two methods: the transgenic cII mutation assay and duplex sequencing (DS) of rat genomic DNA. Groups of 6 male Big Blue rats were exposed by gavage for 28 days to 0, 12.5, 25 or 50 mg/kg body weight/day NEIPA, while a group of 3 rats was treated with 75 mg/kg/day benzo[a]pyrene (B[a]P) as a positive control. The animals were sacrificed 3 days after the end of treatment and liver DNA was isolated for the cII gene mutation assay and DS. Mutant frequencies showed strong dose-dependent increases in the NEIPA-treated rats, ranging from 20.8 to 868.4 x 10 (vehicle control vs. high NEIPA dose) in the liver cII gene and 6 to 708.5 x 10 basepairs for DS; the B[a]P treatment resulted in frequencies of 134.6 x 10 in the cII gene and 49.7 x 10 basepairs for DS. DS analysis revealed that A:T > G:C transition and A:T > T:A transversion were the predominant mutations in the NEIPA-treated rats; whereas G:C > A:T transition and G:C > T:A transversion were the main types of mutations found in the vehicle and positive control rats, respectively. DS analysis yielded lower BMD values for NEIPA-induced mutagenesis than those obtained from the cII mutation assay. These results indicate that NEIPA is a potent mutagen in the liver of rats. Additionally, the fold-increases and relative BMD values for induced mutation indicate that DS analysis exhibited higher sensitivity than cII assay for detecting the mutations induced by NEIPA.