Evaluation of in vivo mutagenicity of isopropyl methanesulfonate by RBC Pig-a and PIGRET assays

Abstract

A comparison between the original red blood cell (RBC) Pig-a assay, which measures Pig-a mutant cells in RBCs, and the PIGRET assay, which uses reticulocytes, was conducted using the in vivo mutagenesis assay with isopropyl methanesulfonate (iPMS) as a part of a collaborative study by the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen Society. Three dose levels of iPMS (50, 100, and 200mg/kg) were administered once intraperitoneally to 8-week-old male Crl:CD(SD) rats, and peripheral blood was sampled at 0 (1 day before dosing), and 1, 2, and 4 weeks after dosing with iPMS. As a result, a time-dependent increase in the mutant frequency of Pig-a mutant RBCs was observed in the RBC Pig-a assay, and a statistically significant increase was observed from 2 weeks after dosing. In the PIGRET assay, on the other hand, a statistically significant increase in Pig-a mutant frequency was obtained from 1 week after dosing at all dose levels, and the Pig-a mutant frequency at the highest dose level had already reached a plateau on week 1. The maximum Pig-a mutant frequency induced by a single treatment with iPMS at 200mg/kg in the PIGRET assay was approximately two times higher than that in the RBC Pig-a assay. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier and with a higher value in Pig-a mutant frequency compared with the original RBC Pig-a assay, and it can enable judgement of mutagenicity of iPMS within 1 week after a single dose.