Rat Pig-a mutation assay responds to the genotoxic carcinogen ethyl carbamate but not the non-genotoxic carcinogen methyl carbamate

Abstract

Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500 mg/kg/day) or EC (250 mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9×10(-6) on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2×10(-6) on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action.

Assessment of the Pig‐a, micronucleus, and comet assay endpoints in rats treated by acute or repeated dosing protocols with procarbazine hydrochloride and ethyl carbamate

“…It is noteworthy that EC caused a significant induction of Pig‐a mutant cells: increased RET CD59− and RBC CD59− frequencies were noted in both the 3‐day and 28‐day studies. The kinetics and magnitude of the mutant cell responses in the 3‐day and 28‐day studies were very similar to those shown in an experiment conducted by Labash et al () in male and female rodents exposed to 600 mg/kg/day EC for 3 consecutive days and the research of Bemis et al () in rats dosed orally for 28 consecutive days with 250 mg/kg/day EC. However, the maximal mutation frequency of RET in our 28‐day treatment study with EC (150–300 mg/kg/day), an ~13.2‐fold increase over the control, was lower than the ~74.0‐fold increase in a similar study conducted by Stankowski et al (), where SD rats were treated orally with 25–250 mg/kg/day using the same dosing protocol; while the maximal ~26.4‐fold increase over the control for mutant RBC in our study is greater than the ~8.1‐fold increase in the study of Stankowski et al ().…”

Induction of Pig-a mutant erythrocytes in male and female rats exposed to 1,3-propane sultone, ethyl carbamate, or thiotepa

“…Treatments were based on previous studies (11-13) with male rats that showed these dose levels are tolerated and significant increases in Pig-a mutant phenotype cells are induced.…”

Scientific opinion on the re‐evaluation of dimethyl dicarbonate (DMDC, E 242) as a food additive

“…The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to 0, 125, 250 or 500 mg MC/kg bw/day in male Sprague-Dawley rats (Bemis et al, 2015). Peripheral blood was collected on days −5 (i.e.…”

“…MC proved to be negative in several in vitro genotoxicity assays and in vivo in the SCE assay in mice (Cheng et al, 1981), the Pig A assay and in the peripheral blood micronucleated reticulocytes assay in rats (Bemis et al, 2015),. The Panel noted that, in contrast to the chemically related ethyl carbamate (urethane), the methyl group of MC does not readily undergo biotransformation to an epoxide.…”