Evaluation of the in vivo mutagenicity of melamine by the RBC Pig-a assay and PIGRET assay

Abstract

The Pig-a assay is a new in vivo genotoxicity test for detecting mutagens in the bodies of animals, using the endogenous Pig-a gene as the target. There are two types of Pig-a assays: the red blood cell (RBC) Pig-a assay, which uses RBCs, and the PIGRET assay, which uses reticulocytes. The Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group collaborative study of the Pig-a assay was carried out to investigate the usefulness of the PIGRET assay. The mutagenicity of melamine was evaluated as part of this study. Eight-week-old male Crl:CD (SD) rats were administered a single gavage dose of melamine as a non-genotoxic bladder carcinogen. Blood samples were collected at the first, second and fourth weeks after administration, and the RBC Pig-a assay and PIGRET assays were conducted using these samples. Three dose levels were used in the study: the highest dose was 2000mg/kg, which is generally used as the maximum dose in in vivo genotoxicity testing, and 1000 and 500mg/kg were also used. As a positive control, a group of rats was administered a single dose of N-nitroso-N-ethylurea (ENU) by gavage at 40mg/kg. The Pig-a mutant frequencies (Pig-a MFs) did not increase in any of the melamine groups throughout the experimental period in either the RBC Pig-a assay or the PIGRET assay. Both the RBC Pig-a and PIGRET assays revealed significant increases in the Pig-a MFs in the ENU group, starting at day 7 after a single administration. Therefore, these two assays, when evaluated after a single administration, can be used to determine that melamine is non-mutagenic.