Comparison of the transgenic rodent mutation assay, error corrected next generation duplex sequencing, and the alkaline comet assay to detect dose-related mutations following exposure to N-nitrosodiethylamine

Abstract

N-Nitrosodiethylamine (NDEA), a well-studied N-nitrosamine, was tested in rats to compare the dose-response relationship of three genotoxicity endpoints. Mutant / mutation frequencies were determined using the transgenic rodent (TGR) gene mutation assay and error corrected next generation sequencing (ecNGS) (i.e., duplex sequencing (DS)), and genetic damage was detected by the alkaline comet assay. Big Blue® (cII Locus) animals (n = 6 per dose group) were administered doses of 0.001, 0.01, 0.1, 1, 3 mg/kg/day NDEA by oral gavage. Samples were collected for cII mutation and DS analyses following 28-days of exposure and 3 days recovery. In a separate study, male Sprague-Dawley (SD) rats (n = 6 per dose group) were administered the same doses by oral gavage for two consecutive days and then samples collected for the alkaline comet assay. A dose-related increase in mutant / mutation frequencies of the liver but not duodenum was observed using the TGR assay and DS with DS resulting in a slightly more sensitive response, with a lower benchmark dose (BMD). In addition, a dose-related increase in percent tail DNA was observed in the liver using the alkaline comet assay. Therefore, DS and comet assays showed good utility for hazard identification and dose-response analysis of a representative N-nitrosamine comparable to the TGR gene mutation assay.