A regenerative erythropoietic response does not increase the frequency of <i>Pig‐a</i> mutant reticulocytes and erythrocytes in Sprague‐Dawley rats

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.

Section: Discussionmentioning

confidence: 99%

Testing of acetaminophen in support of the international multilaboratory in vivo rat Pig‐a assay validation trial

Leede

Weiner

Doninck

et al. 2020

“…The inflammation and increase in cell division associated with IBD might be responsible for the slight elevation in PIG‐A MF. But some rodent Pig‐a studies also indicated that the regenerative erythropoietic response does not lead to increase Pig‐a MFs (Kenyon et al ; Nicolette et al ). Thus, we interpret the results of PIG‐A assay in our study only as a weak positive outcome.…”

Section: Discussionmentioning

confidence: 99%

The potential application of human PIG‐A assay on azathioprine‐treated inflammatory bowel disease patients

Cao

Wang²,

Liu

et al. 2019

The rodent Pig-a assay has been used extensively as a potential regulatory assay for evaluating the in vivo mutagenicity of test substances. Although the assay can be conducted in different mammalian species, there have been only a few reports describing its use in humans, and rarely in genotoxicant-exposed human populations. In this study, PIG-A mutation frequencies (MFs) were evaluated in 36 azathioprine (AZA; human carcinogen)-treated inflammatory bowel disease (IBD) patients and 36 healthy volunteers. IBD patients exhibited a slight but statistically higher MF (6.10 AE 4.44 × 10 −6 ) than healthy volunteers (4.97 AE 2.74 × 10 −6 ) (P = 0.0489). The estimated relative risk for the exposed patients was 1.22 which indicated that AZA is a risk factor for inducing PIG-A mutation. However, the PIG-A MF showed no associations with AZA treatment duration or total AZA exposure. In addition, we performed the cytokinesis-block micronucleus test on the same samples. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in IBD patients (MN: 4.70 AE 2.86‰; NBUD: 1.89 AE 0.95‰) were significantly higher than in healthy volunteers (MN: 1.47 AE 0.77‰, P < 0.001; NBUD: 0.90 AE 0.58‰, P = 0.004). MN frequency also had significant correlations with AZA treatment duration (P = 0.011) and total AZA exposure (P = 0.018).Our findings indicate that AZA-treated IBD patients have only a marginally significant increase in PIG-A MF; in contrast, a much stronger AZA-associated increase in genotoxicity was detected with the lymphocyte MN assay. Environ. Mol. Mutagen. 61:456-464, 2020.

“…However, when blood is collected several days or more after discontinuing treatment, it can manifest as elevated %RET frequencies due to stress erythropoiesis. (Note that Nicolette et al ., 2018 demonstrated that regenerative erythropoietic response does not increase the frequency of MUT RET/RBC in rats.) Test substance‐induced hemolysis is another situation that can manifest as elevated frequencies of reticulocytes and represents evidence of systemic exposure (Kenyon et al ., 2015).…”

Section: Systemic Exposurementioning

confidence: 96%

Recommendations for conducting the rodent erythrocyte Pig‐a assay: A report from the HESI GTTCPig‐a Workgroup

Dertinger

Bhalli

Roberts

et al. 2021

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.