P14-28 In vivo Pig-a mutant frequencies in Wistar Rats following exposure to N-Nitroso-N-Ethylurea and Benzo(a) Pyrene via oral gavage

The Pig-a assay is a flow cytometry-based method used to detect mutations in the endogenous X-linked reporter gene, phosphatidyl inositol glycan class A (PIG-A), in mammalian cells, specifically red blood cells in rodents [1] . This assay is gaining recognition for its advantages, including minimal blood volume requirements and the ability to collect blood samples without euthanizing the animal. This approach aligns with ethical principles in animal testing. This exploratory study focuses on assessing the potential to cause Pig-a gene mutations in Wistar rats following exposure to N-Nitroso-Ethyl (ENU) urea and Benzo(a) Pyrene, the proven mutagens after three days of consecutive dosing. Six Wistar-Han rats were exposed to ENU and Benzo(a)Pyrene, for three consecutive days. Blood samples were collected from the lateral tail vein on days 29 from ENU-treated animals. We extended this study to 90 days for the B(a)P-treated animals to evaluate the mutant frequency after acute treatment of of ENU. Samples were processed and analysed using Litron's Muta-Flow® kit with a BD FACSLyric™. Statistical analysis of the Pig-a gene mutation assay data was performed using GraphPad Prism. The experiment demonstrated that treatment with ENU as well as Benzo(a)Pyrene led to a significant increase in the mean population of RET CD59− and RBC CD59− in blood collected on Day 29 for both male and female rats in comparison to the control animals. Blood samples taken on Day 90 revealed an increase in the mean RBC CD59− population compared to the negative control group in both male and female rats, although no significant difference in the mean RET CD59− population was noted. The results indicated responses in the mutant red blood cell (RBC) populations compared to mutant reticulocytes at later time points, as noted previously by Torous et al. , 2012, they recorded a drop in RET CD59− from day 15-45 was also observed by with 3 days of treatment with Benzo(a)pyrene. While RBC CD59− was achieved on 30 th day, the drop in the RBC CD59− mutant frequency from 30 th day to 45 th day was not as sudden as compared to the drop in RET CD59− frequency 2 . As the absence of variation may be linked to the brief exposure duration and the prolonged sampling interval on Day 90 also the life spam of RETs versus RBCs.The study highlighted differences in the kinetics of CD59- mutant population induction between the RBC and RET cohorts, with increases in mutant frequencies observed on Day 29 but not on Day 90 for both the cell types. Although these results align with the OECD TG470 guideline suggesting blood sampling on Day 29. This study also supports validity of acute expousre period for detection of mutant frequncy.