The rat Pig-a assay using an erythroid HIS49 antibody in a single dose study of isopropyl p-toluenesulfonate.

As part of a collaborative study in the Mammalian Mutagenicity Study group of the Japanese Environmental Mutagen Society, we evaluated the in vivo mutagenicity of isopropyl p-toluenesulfonate (IPTS) using a peripheral blood Pig-a assay in rats. Pig-a mutant frequency (MF) data was obtained for both red blood cells (RBCs) and reticulocytes (RETs) at 1, 2 and 4 weeks after a single oral administration of IPTS at doses of 125, 250, or 500mg/kg. The results of the RBC Pig-a assay demonstrated that both the 250 and 500mg/kg treatment groups showed significant increases in Pig-a MF only at 4 weeks after IPTS treatment. In comparison, the PIGRET assay showed a clear and dose-related increase in Pig-a MF at 1 week after treatment, with a continuous increase until 4 weeks after treatment observed in the highest dose group. These results indicate that the both the RBC Pig-a assay and PIGRET assay can detect in vivo IPTS mutagenicity under a single dosing protocol. In particular, the PIGRET assay, which uses magnetic enrichment to analyze greater numbers of RETs in a high-throughput manner, showed an increase in Pig-a MF earlier than the RBC Pig-a assay. The PIGRET assay is also considered to be more sensitive than the RBC Pig-a assay because it exhibits a low spontaneous Pig-a MF. For this reason, the PIGRET assay clearly identified small increases in Pig-a MF as significant at the lower doses than in the RBC Pig-a assay under the conditions in this study.