Evaluation of the PIGRET assay as a short-term test using a single dose of diethylnitrosamine

The PIGRET assay, which was developed as the Pig-a assay in reticulocytes, can detect mutagenicity of compounds earlier than the Pig-a assay in total red blood cells (RBC; RBC Pig-a assay). The usefulness of the PIGRET assay as a short-term test has been confirmed in a collaborative study in Japan with 24 chemicals. One of these chemicals, diethylnitrosamine (DEN), which mainly induces liver tumors in both sexes of rats, was tested. To determine the appropriate doses, DEN was dissolved in physiological saline and administered orally with a single dose to male 8-week-old Sprague-Dawley rats in a preliminary dose range finding study. As a result, all of the animals died at doses of 300 and 600 mg/kg. Therefore, 37.5, 75, and 150 mg/kg doses were set for the main study (the RBC Pig-a and PIGRET assays). The results showed no statistically significant increase in the mutant frequency (MF) of CD59-negative cells in the groups treated with DEN for the entire test period; however, the positive control N-ethyl-N-nitrosourea (ENU) produced positive results. Some hematogenic effects were indicated by the significant increase of the percentage of reticulocytes in the medium and at high doses on Day 28. The decrease in the body weight on Days 2-4 in the main study and the mortality in the preliminary dose range-finding study indicated that appropriate doses were used in the main study. Although DEN is a known genotoxic carcinogen in the liver, our negative results in the RBC Pig-a and PIGRET assays indicated that there is no substantial mutagenicity in hematopoietic cells under the conditions using a single dose. The PIGRET assay detected the mutagenicity of ENU one week earlier than the RBC Pig-a assay, indicating the usefulness of the PIGRET assay as a short-term test. (C) 2016 Elsevier B.V. All rights reserved.