OECD 476, EG 440/2008 B.17
The "Mouse Lymphoma Assay“ enables the testing for genetic mutations caused by chemical substances and assesses the two endpoints gene mutation and chromosome damage.
The permanent Mouse Lymphoma cell line L5178Y TK+/- 3.7.2C is usually applied.
Its thymidine-kinase locus (tk1) is heterocygotically expressed on chromosome 11. The inactivation of the tk+ allele is inducing a trifluorothymidine (TFT) resistance. In this way, the tk-/- mutants can be selected in a background of tk+/- non-mutated cells. Mutated cell strains show a bimodal size distribution with strains that grow at the normal growth rate of tk+/- cells, and strains that grow considerably more slowly. It could be shown that the small colonies are frequently associated with aberrations of chromosome 11, while mutants of the bigger colonies show no cytogenetic changes, but are due to genetic mutations.
In our laboratory the „Mouse Lymphoma Assay“ is applied as a micro-well method. According to standard guidelines, at least 8 significant concentrations or 4 concentrations each as duplicate, a double negative control line as well as a positive control, each with and without metabolic activation (S9) are tested. Negative or non-consistent results are independently repeated. Incubation time with regard to the test substance varies usually between 3 and 4 hours. The incubation time for the testing of pharmaceuticals has to be extended to 24 hours by an additional test without metabolic activation (according to ICH S2B). By this, an improved detection of nucleoside and base analogues as well as of aneuploidy is obtained.
A positiv effect is an increase of mutants of at least 100 per 106 cells compared to the rate of the control. Additional a significance test with ANOVA has to be performed.
Test substance properties
The Mouse Lymphoma Assay is applicable for solid, soluble substances as well as liquid substances and water samples.