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

Abstract

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.

Human genetic risk of treatment with antiviral nucleoside analog drugs that induce lethal mutagenesis: the special case of molnupiravir

“…ICH and OECD test guidelines for 14+ days of exposure FDA Guidance for Industry M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals recommend dosing at 1000 mg/kg/day unless limited by toxicity, or other factors (Dertinger et al 2021). According to the HESI GTTC Pig‐a Workgroup, “after an appropriate phenotypic expression time that allows elevated mutant reticulocyte (MUT RET) and mutant erythrocyte (MUT RBC) frequencies to appear in the peripheral blood compartment, blood from the exposed rodent and a sex/age‐matched negative control animal (either naïve or vehicle treated) are collected (Dertinger et al 2021). ” In the Big Blue ( cII locus) rat assay performed by Merck/Ridgeback, only one rapidly dividing tissue (bone marrow) and one slowly dividing tissue (liver) were sampled.…”

Test No. 470: Mammalian Erythrocyte Pig-a Gene Mutation Assay

“…The main components and procedures for the Pig-a assay are described below. Further guidance for conducting the test, including detailed laboratory protocols that are compliant with the recommendations in this TG, can be found in these and other publications (4) (5) (6) (7).…”

“…In addition to collecting data on Pig-a mutant induction, the fraction of RET among total RBC can be used as a measure of toxicity to the erythroid cell compartment. Percent RET (%RET) data can be useful for establishing that the bone marrow has been exposed to biologically significant levels of the test chemical or its metabolites ((2) (7); see also paragraphs 49 and 55). When blood samples are collected within approximately 3 days following cessation of dosing, bone marrow toxicity often manifests as a reduction in %RET relative to %RET in concurrent vehicle/solvent control animals.…”

“…Data collected from testing diverse genotoxicants suggest that sampling on Day 28-31 gives sufficient time for manifestation of MUT RET and MUT RBC responses and that the exact timing of sample collection is not critical. This schedule has the advantage of facilitating integration of the Pig-a assay with commonly utilised general toxicology studies and other genetic toxicology tests (paragraphs [7][8]. Note that an extra administration of the test chemical on Day 29, followed by sample collection several hours later, is permissible for facilitating tissue harvest for conducting the in vivo comet test.…”

“…The following criteria determine the acceptability of the test:  The concurrent negative control frequencies are consistent with the distribution described by the laboratory historical negative control data base. It is important that the historical negative control data base was constructed appropriately (for instance as recommended in paragraph 22 and Annex 4, section 4.1), and the historical negative control mutant frequencies and their distribution are consistent with literature values (1) (3) (7).…”