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Review
. 2021 Mar;62(3):227-237.
doi: 10.1002/em.22427. Epub 2021 Mar 2.

Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup

Stephen D Dertinger  1 , Javed A Bhalli  2 , Daniel J Roberts  3 , Leon F Stankowski Jr  3 , B Bhaskar Gollapudi  4 , David P Lovell  5 , Leslie Recio  6 , Takafumi Kimoto  7 , Daishiro Miura  7 , Robert H Heflich  8
Affiliations
Review

Recommendations for conducting the rodent erythrocyte Pig-a assay: A report from the HESI GTTC Pig-a Workgroup

Stephen D Dertinger et al. Environ Mol Mutagen. 2021 Mar.

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.

Keywords: Pig-a gene; flow cytometry; genotoxicity; mutagen; mutation assay.

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Figures

FIGURE 1
FIGURE 1
Reticulocyte (RET), and mutant reticulocyte (MUT RET) and mutant erythrocyte (MUT RBC) frequencies are graphed for male and female rats (39 each) that had been exposed to one of several common vehicles. Blood samples were collected when the rats were 7 weeks old. Whereas each circle represents an individual animal, the ranges are denoted by the length of horizonal lines, and group means are indicated by a vertical tick mark. These data were evaluated by Restricted Maximum Likelihood analysis and demonstrate that variation in %RET is dominated by sex (~75%, with males > females). On the other hand, MUT RET and MUT RBC variation is mainly attributable to inter‐animal variation (~72–91%), with much lower contributions from sex (~2–8%) and study number (~8–21%)
FIGURE 2
FIGURE 2
Mutant reticulocyte (MUT RET) and mutant erythrocyte (MUT RBC) frequencies are graphed for the same vehicle‐exposed male and female rats portrayed in Figure 1 (i.e., 78 individuals). In this case, the results are plotted on control charts according to the order that analyses occurred (13 studies over 14 months). Zones A, B, and C signify values that are within 3, 2, and 1 SD from the mean, respectively. Nelson rules violations (numbered 1–8) are superimposed on data points when alerts are triggered. In the current example, “1” indicates a value is greater than 3 SDs from the mean, while “2” signifies that nine or more points in a row are on the same side of the mean. Overall, the low number of violations gives one confidence that the mutant cell scoring process is “under control.” While these charts were produced using the JMP software (v12.0.1), other packages such as Minitab can produce similar charts, as can packages in the R statistical programming language
See this image and copyright information in PMC

References

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