In Vitro Mouse Lymphoma Assay TK

According to OECD Guidelines for Testing of Chemicals, Test No. 490, the in vitro mouse lymphoma assay (MLA) is used to detect induced point mutations or clastogenic events of the thymidine kinase (TK) locus of L5178Y TK+/– mouse lymphoma cells by measuring resistance to the lethal nucleoside analogue triflurothymidine (TFT).

STEMart provides in vitro mouse lymphoma assay to assess the capacity of leachable from a medical device to induce gene mutations or chromosomal events. This assay is available under either GLP (Good Laboratory Practice) or Non-GLP conditions.

Standard

OECD 490

Test Cell

L5178Y TK^+/- -3.7.2C mouse lymphoma cell line

Test Method

Cells are incubated with addition of at least four analysable concentrations of the test substance for 3 to 4 hours in the presence and absence of metabolic activation (S9) and for 24 hours in the absence of S9.

Cells are subcultured for 48 hours following the end of treatment to allow phenotypic expression of induced mutations.

Cells are seeded into 96-well plates in media with and without trifluorothymidine (TFT) for mutant selection and measurement of cloning efficiency.

Mutant colonies are characterized by size. Small colonies indicate gene mutations while large colonies indicate chromosomal events.

Negative vehicle control and positive controls are included in each study.

Criteria

Test sample with mutagenic potential causes the lack of TK activity of TK proficient cells. Thus, mutant cells are resistant to the cytostatic effects of the pyrimidine analogue trifluorothymidine (TFT) and able to proliferate in the presence of TFT.

Fig. 1 Principle of the mouse lymphoma assay. (Mei, N., 2014)

A positive result is an increase of mutants of at least 100 per 10⁶ cells compared to the rate of the control.

Final Report

The final report including information on the methodology, raw data, analysis, and interpretation of the results will be provided for customer.

Advantages

• Rapid growth in suspension culture to high cell density

Provide very large numbers of cells for a statistically valid test to be treated and subcultured for the required expression time before being subjected to mutant selection.

• Relatively short time for expression of newly induced mutants

Make the assay more cost effective and reduce fluctuations in the relative proportion of mutants due to clonal expansion and variable fitness of some newly induced mutants.

If you have additional questions about in vitro mouse lymphoma assay, or would like to find out more about our services, please feel free to contact us.

References

• US Food and Drug Administration. "Red Book 2000, Toxicological Principles for the Safety of Food Ingredients: IV. C. 1. c. Mouse Lymphoma Thymidine Kinase Gene Mutation Assay." (2001).
• OECD. "OECD Guidelines for the Testing of Chemicals/Section 4: Health Effects." (1997).
• Cole, J., et al. "The mouse lymphoma assay in the wake of ICH4—where are we now?" Mutagenesis 14.3: 265-270 (1999).
• Mei, N., et al. "Methods for using the mouse lymphoma assay to screen for chemical mutagenicity and photo-mutagenicity." Optimization in drug discovery. Humana Press, Totowa, NJ, 561-592 (2014).