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The rodent Pig‐a assay is a flow cytometric, phenotype‐based method used to measure in vivo somatic cell mutation. An Organization for Economic Co‐operation and Development (OECD) test guideline is currently being developed to support routine use of the assay for regulatory purposes (OECD project number 4.93). This article provides advice on best practices for designing and conducting rodent Pig‐a studies in support of evaluating test substance safety, with a focus on the rat model. Various aspects of assay conduct, including laboratory proficiency, minimum number of animals per dose group, preferred treatment and blood sampling schedule, and statistical analysis are described. [ABSTRACT FROM AUTHOR]

Acetaminophen, a nonmutagenic compound as previously concluded from bacteria, in vitro mammalian cell, and in vivo transgenic rat assays, presented a good profile as a nonmutagenic reference compound for use in the international multilaboratory Pig‐a assay validation. Acetaminophen was administered at 250, 500, 1,000, and 2,000 mg·kg−1·day−1 to male Sprague Dawley rats once daily in 3 studies (3 days, 2 weeks, and 1 month with a 1‐month recovery group). The 3‐Day and 1‐Month Studies included assessments of the micronucleus endpoint in peripheral blood erythrocytes and the comet endpoint in liver cells and peripheral blood cells in addition to the Pig‐a assay; appropriate positive controls were included for each assay. Within these studies, potential toxicity of acetaminophen was evaluated and confirmed by inclusion of liver damage biomarkers and histopathology. Blood was sampled pre‐treatment and at multiple time points up to Day 57. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as CD59‐negative RBC and CD59‐negative RET frequencies, respectively. No increases in DNA damage as indicated through Pig‐a, micronucleus, or comet endpoints were seen in treated rats. All positive controls responded as appropriate. Data from this series of studies demonstrate that acetaminophen is not mutagenic in the rat Pig‐a model. These data are consistent with multiple studies in other nonclinical models, which have shown that acetaminophen is not mutagenic. At 1,000 mg·kg−1·day−1, Cmax values of acetaminophen on Day 28 were 153,600 ng/ml and 131,500 ng/ml after single and repeat dosing, respectively, which were multiples over that of clinical therapeutic exposures (2.6–6.1 fold for single doses of 4,000 mg and 1,000 mg, respectively, and 11.5 fold for multiple dose of 4,000 mg) (FDA 2002). Data generated were of high quality and valid for contribution to the international multilaboratory validation of the in vivo Rat Pig‐a Mutation Assay. [ABSTRACT FROM AUTHOR]

TP53 is one of the most commonly mutated genes in human tumours. Variations in the types and frequencies of mutations at different tumour sites suggest that they may provide clues to the identity of the causative mutagenic agent. A useful model for studying human TP53 mutagenesis is the partial human TP53 knock-in (Hupki) mouse containing exons 4–9 of human TP53 in place of the corresponding mouse exons. For an in vitro assay, embryo fibroblasts from the Hupki mouse can be examined for the generation and selection of TP53 mutations because mouse cells can be immortalized by mutation of Tp53 alone. Thus far, four environmental carcinogens have been examined using the Hupki embryo fibroblast immortalization assay: (a) UV light, which is linked to human skin cancer; (b) benzo[ a]pyrene, which is associated with tobacco smoke-induced lung cancer; (c) 3-nitrobenzanthrone, a suspected human lung carcinogen linked to diesel exposure; and (d) aristolochic acid, which is linked to Balkan endemic nephropathy-associated urothelial cancer. In each case, a unique TP53 mutation pattern was generated that corresponded to the pattern found in human tumours where exposure to these agents has been documented. Therefore, the Hupki embryo fibroblast immortalization assay has sufficient specificity to make it applicable to other environmental mutagens that putatively play a role in cancer aetiology. Despite the utility of the current Hupki embryo fibroblast immortalization assay, it has several limitations that could be addressed by future developments, in order to improve its sensitivity and selectivity. [ABSTRACT FROM AUTHOR]