Standard protocol for the total red blood cell assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society.

The assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The assay on concentrated RETs-the PIGRET assay-has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the assay on total RBCs (the RBC assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC assay in detail.