In Vitro Mammalian Cell Gene Mutation Test (OECD 490)

Creative Bioarray provides in vitro mammalian cell gene mutation test (OECD 490¹, originally described in OECD 476²) to identify chemicals that has the potential to induce gene mutations in mammalian cells by measuring forward mutations in reporter genes (e.g. thymidine kinase gene). This assay is available under either GLP (Good Laboratory Practice) or Non-GLP conditions.

This test can be conducted on two established cell lines: The L5178Y TK^+/--3.7.2C mouse lymphoma cell line (L5178Y) and the TK6 human lymphoblastoid cell line (TK6). Under effect of mutagens, mutant cells deficient in thymidine kinase enzyme activity are generated due to a mutation from TK^+/- to TK^-/-. Those cells are resistant to trifluorothymidine (TFT, a pyrimidine analogue), which inhibits cellular metabolism and halts cell division, thus are able to proliferate in the presence of TFT and form visible colonies, whereas un-mutated heterozygous TK^+/- cells, which contain thymidine kinase and are sensitive to TFT, are not. Therefore, mutant frequency can be calculated by counting the visible colonies and determining the cloning efficiency.

Test System:

•	L5178Y TK^+/--3.7.2C mouse lymphoma cell line (L5178Y)
•	TK6 Human Lymphoblastoid cell line (TK6)

Test Method:

The in vitro mammalian cell micronucleus test is carried out in compliance with the OECD Guideline 490. Proliferating mammalian cell cultures are exposed to the test chemical for a suitable length of time (3-6 hours or customized) both in the presence and in the absence of an exogenous source of metabolic activation (Aroclor-1254 induced rat liver S9). After an appropriate period of exposure to the test articles (at least 3 concentrations with duplicates/triplicates), the cells are cultured for sufficient time to allow optimal phenotypic expression of induced mutants. Cells are then suspended in medium with and without selective agent (TFT) to determine the numbers of mutants and cloning efficiency respectively. Early appearing colonies are counted after culturing for 10-14 days. For slow growing TK6 mutants, the cells will be maintained for an additional 7-10 days. Colonies are then counted and the number of mutant colonies adjusted by the cloning efficiency to calculate the mutant frequency.

Creative Bioarray can provide in vitro mammalian cell micronucleus test following GLP principles³. Typically, a draft study plan is prepared and reviewed by the sponsor and our quality assurance (QA) personnel after the collection of the characterization and formulation information of the test articles. The study can get started after the approval of the study plan by both parties.

For test compounds with no toxicity or solubility information, we will perform range-finding tests before the main assay (additional fees may be charged).

After the study, a draft report of the results will be prepared for the sponsor and the quality assurance group to review. After the final approval, the final report will be provided with a QA statement.

To find out more about our service with more information, please feel free to leave a message below, or contact us. Our well-experienced experts will be more than happy to help.

References:

1.	OECD Guideline for Testing of Chemicals: In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene. TG 490. Adopted 29 July 2016.
2.	OECD Guideline for Testing of Chemicals: In Vitro Mammalian Cell Gene Mutation Test. TG 476. Adopted 21st July 1997.
3.	OECD Principles of Good Laboratory Practice (as revised in 1997). OECD Environmental Health and Safety Publications. OECD. 1. 1998.

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