Overview

Secondary tier assays like Pig-a offer significant value both for understanding a compound’s mode of action and for following up on a positive in vitro genetic toxicology assay.


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Additional Support and Regulatory Follow-Up

  • This assay is used for scoring the frequency of mutant phenotype erythrocytes (RBCs) and mutant phenotype immature erythrocytes (reticulocytes, or RETs) by measuring the induction of mutations at the Pig-a gene.
    • Mutations in the Pig-a gene can prevent production of glycosylphosphatidylinositol (GPI) anchor proteins on the cell surface. Thus, cells without these cell surface markers represent a reliable phenotypic marker of Pig-a mutation.
  • Advantages
    • Easily integrates into standard toxicity studies
    • Provides fast, quantifiable results via flow cytometry
    • Uses very low volumes of blood
    • 3Rs compliant
    • Replaces transgenic rodent mutation assays
Flow cytometry scatter plots showing the increase in incidence of Pig-a mutant cells after treatment with a positive control

Figure: Flow cytometry scatter plots showing the increase in incidence of Pig-a mutant cells after treatment with a positive control.

When to Perform

  • IND-enabling studies
    • To determine mutagenic potential
  • Additional support
    • To assess mutagenicity potential when the Ames assay is not appropriate (e.g., test item is toxic to bacteria)
    • Follow-up on a positive result in the Ames assay
    • Additional information for compounds with predicted mutagenic properties