Genetic Toxicology Studies - Study Design of In Vitro Mammalian Cell Gene Mutation Test Using the Hprt and xprt genes (OECD 476)
The In Vitro Mammalian Cell Gene Mutation Test Using the Hprt and xprt Genes (OECD 476) is a robust assay designed to assess the mutagenic potential of chemicals in cultured mammalian cells. Here’s a detailed breakdown of the study design:
Objectives:
- Determine whether a test substance can induce mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) and xanthine-guanine phosphoribosyltransferase (xprt) genes of mammalian cells.
- Evaluate the dose-response relationship for gene mutations.
- Classify the test substance according to its mutagenic potential for regulatory purposes.
Target Genes:
- Hprt: Crucial for salvaging purines, necessary for DNA synthesis and cell growth. Loss of Hprt function leads to cell death in the presence of selective agents.
- Xprt: Similar function to Hprt, but offers a different mutational spectrum for a more comprehensive assessment.
Test System:
- Cell line: Typically, established mammalian cell lines like Chinese hamster ovary (CHO) cells are used.
- Selection and counter-selection: Mutant cells lacking Hprt function cannot survive in the presence of selective agents (e.g., 6-thioguanine). Conversely, mutant cells lacking xprt function can survive in the presence of counter-selection agents (e.g., aminopterin).
Test Procedure:
- Exposure: Cells are exposed to the test substance at various concentrations, both with and without metabolic activation (using S9 mix).
- Selection and subculture: Cells are plated in media with selective agents and counter-selection agents to identify mutant cells. Surviving cells are subcultured to allow expression of mutations.
- Mutant frequency determination: The number of mutant colonies at each dose level is counted and compared to controls. Mutant frequency is calculated as the ratio of mutant to total cells.
Endpoints:
- Mutant frequency at different dose levels.
- Dose-response relationship for gene mutations.
- Relative total growth (RTG) as a measure of cytotoxicity.
- Mutagenic index (ratio of mutant frequency to control frequency).
- Classification of the test substance according to OECD 476 criteria (e.g., mutagenic, non-mutagenic).
Benefits:
- Sensitive in detecting gene mutations.
- Allows for assessment of different mutational spectra with Hprt and xprt genes.
- Relatively rapid and cost-effective compared to in vivo studies.
- Standardized and internationally accepted method.
Limitations:
- Cannot detect all types of genetic damage (e.g., chromosomal aberrations).
- Does not directly assess mutagenic risk in humans.
- May not capture all potential mutagens due to limitations in metabolic activation.
Additional Resources
- OECD Test Guideline 476: https://www.oecd.org/environment/test-no-476-in-vitro-mammalian-cell-gene-mutation-tests-using-the-hprt-and-xprt-genes-9789264243088-en.htm
- The HPRT and XRPT Gene Mutation Assays in Genotoxicity Testing: A Review: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3173198/
- In Vitro Mammalian Cell Gene Mutation Test Using the Hprt and xprt Genes (OECD 476): A Practical Guide: https://www.intechopen.com/chapters/53376
