Genetic Toxicology Studies - Study Design of In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene (OECD 490)
The In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene (OECD 490) is a sensitive and widely used assay to assess the mutagenic potential of chemicals in cultured mammalian cells. It focuses on the thymidine kinase (tk) gene, which plays a crucial role in cell growth and survival.
Objectives:
- Determine if a test substance can induce mutations in the tk gene of mammalian cells.
- Evaluate the dose-response relationship for tk mutations.
- Classify the substance according to its mutagenic potential for regulatory purposes.
Test System:
- Cell line: Two specific heterozygous tk-deficient cell lines are used:
- L5178Y tk +/– 3.7.2C for the mouse lymphoma assay (MLA).
- TK6 tk +/– for the TK6 assay.
- Selection and counter-selection: Mutant cells lacking functional tk cannot utilize thymidine for DNA synthesis and will die in the presence of selective agents (e.g., bromodeoxyuridine, BrdU). However, they can survive and grow in the presence of counter-selection agents (e.g., thymidine).
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 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 tk 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 490 criteria (e.g., mutagenic, non-mutagenic).
Benefits:
- Sensitive in detecting point mutations in the tk gene.
- Allows for both forward and reverse mutation analysis, providing a broader picture of mutagenic potential.
- 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
- Test No. 490: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene: https://www.oecd.org/publications/test-no-490-in-vitro-mammalian-cell-gene-mutation-tests-using-the-thymidine-kinase-gene-9789264264908-en.htm
- The Use of the Thymidine Kinase Gene Mutation Assay in Genotoxicity Testing: A Review: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4628933/
- In Vitro Mammalian Cell Gene Mutation Test Using the Thymidine Kinase Gene (OECD 490): A Practical Guide: https://www.intechopen.com/chapters/53375
