Report on stage III Pig‐a mutation assays using benzo[a]pyrene

Abstract

Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies.

Report on stage III Pig‐a mutation assays using N‐ethyl‐N‐nitrosourea – comparison with other in vivo genotoxicity endpoints

“…DOI 10.1002/em probably best compared with RBC CD592 mutant frequencies. As was the case for BaP-induced mutation [Bhalli et al, 2011], RBC CD592 mutant frequencies were somewhat higher than Hprt or Pig-a mutant frequencies in lymphocytes, suggesting that erythroid cells are at least as sensitive, if not more sensitive than lymphoid cells for detecting gene mutation. However, Pig-a mutant frequencies in lymphocytes were lower than Hprt lymphocyte mutant frequencies, especially at the 5 mg/kg/day dose.…”

“…These observations suggest that Hprt may have a larger target for ENU-induced gene mutation than does Pig-a, e.g., Hprt may have more nucleotides at risk for ENU-induced mutation that produce a mutant phenotype. It should be noted that that the Pig-a and Hprt lymphocyte assays use different methods to stimulate the target cells to form clones, which produce different lymphocyte cloning efficiencies, and thus different correction factors for calculating mutant frequencies in the assays (see further explanation of differences between the two assays in Bhalli et al [2011]). While it is a reasonable assumption that these corrections produce satisfactory estimates of mutant frequency, as well as that the manifestation kinetics for Pig-a and Hprt lymphocyte mutants are similar, our conclusions must be tempered by these uncertainties.…”

“…As explained previously [Bhalli et al, 2011], Day 56 is a reasonable sampling time for measuring ENU-induced Hprt and Pig-a lymphocyte mutant frequencies, and lymphocyte mutant frequencies are Environmental and Molecular Mutagenesis. DOI 10.1002/em probably best compared with RBC CD592 mutant frequencies.…”

The in vivo Pig-a assay: A report of the International Workshop On Genotoxicity Testing (IWGT) Workgroup

“…2 and Table 3). There is, however, good evidence for a high degree of inter-laboratory transferability and reproducibility based on the results with several potent and weak mutagens (and low doses of potent mutagens) that were tested in a systematic manner as part of the Litron trial [22][23][24][25][26][27]. This trial employed common protocols (refined for each stage of the trial), and common reagents, although the rat strains and flow cytometers used were at the discretion of the participating laboratory.…”

“…The results demonstrated a high level of agreement, and provided confidence in extending the studies to investigate the performance characteristics of the assay further. Stage III utilized a 28-day repeat-dose design, and experiments were performed with ENU [23], dimethylbenz[a]anthracene (DMBA; [24]), N-methyl-N-nitrosourea [25], benzo[a]pyrene (BaP; [26]), and 4-nitroquinoline-1-oxide (4-NQO; [27]) in at least 2 laboratories per agent. Results from these studies also showed a high degree of concordance among laboratories.…”