Detection of Pig-a gene mutants in rat peripheral blood following a single urethane treatment: A comparison of the RBC Pig-a and PIGRET assays

- Urethane was orally administered once to rats and blood was taken at 1, 2, and 4 weeks.
- Pig-a mutant frequency was measured using both the RBC Pig-a and the PIGRET assays.
- Positive responses were observed in PIGRET assay but not in RBC Pig-a assay.
- PIGRET assay might have potential as a tool for short-term mutagenicity screening in vivo.

The rat red blood cell (RBC) Pig-a assay has been recommended by an expert working group of the International Workshop on Genotoxicity Testing as a potential new method to evaluate in vivo gene mutations in regulatory genotoxicity risk assessments. In a collaborative study in Japan, an improved Pig-a assay using reticulocytes (PIGRET assay) with magnetic enrichment of CD71-positive cells was evaluated, and it was revealed that this assay could detect the mutagenicity of chemicals earlier than the RBC Pig-a assay could. To verify further the suitability of the PIGRET assay for an in vivo short-term genotoxicity screening test, a joint research study was conducted by the Japanese Environmental Mutagen Society, and 24 compounds were evaluated. One of the compounds evaluated in this study was urethane, a multi-organ rodent carcinogen. Urethane (250, 500, and 1000 mg/kg body weight) was orally administered once to 8-week-old male Crl:CD (SD) rats. Blood samples were collected at 1, 2, and 4 weeks after the administration and processed for the RBC Pig-a and PIGRET assays. In the PIGRET assay, the Pig-a mutant frequency (MF) significantly increased at both 2 and 4 weeks after the treatment of 1000 mg/kg of urethane. However, in the RBC Pig-a assay, a significant increase in the Pig-a MF was observed only at 1 week after the treatment with 500 mg/kg, but the MF value was within our historical control range; therefore, it was judged to be negative. These results suggest that the PIGRET assay might be useful for evaluating the in vivo mutagenicity more clearly than the RBC Pig-a assay after a single treatment of test compounds.