Mouse lymphoma thymidine kinase locus gene mutation assay: International Workshop on Genotoxicity Test Procedures Workgroup Report

The Mouse lymphoma Assay (MLA) Workgroup addressed and reached consensus on a number of issues. Discussion focused on Five areas: (1) acceptable assay versions; (2) cytotoxicity measure; (3) 24-hr treatment (4) microwell colo ny counting and sizing; and (5) data acceptability/statistical analysis. Al though the International Conference on Harmonisation (ICH) indicated a pref erence for the microwell over the soft agar method, all of the workgroup me mbers agreed that both versions of the MLA are equally acceptable. The work group agreed that it is desirable for both assay versions to use the same m easure of cytotoxicity to define the acceptable and required concentration range. Currently, laboratories using the microwell version use the relative survival (RS) determined by cloning immediately after the treatment. labor atories using the soft agar method do not obtain an RS but use the relative total growth (RTG), a combination of the relative suspension growth (RSG) during the expression period and the relative cloning efficiency determined at the time of mutant selection. The workgroup agreed to investigate the R SG, the RS, and the RTG and to develop further guidance. In the interim, th e workgroup reached consensus that the RTG be used as the standard measure of cytotoxicity. The ICH recommended a 24-hr treatment in the absence of S9 when negative results are obtained with short (3-4 hr) treatments. The wor kgroup agreed to retain this requirement but acknowledged that more data ar e needed prior to making final recommendations concerning the need for and the specific protocol for the 24-hr treatment. Published 2000 Wiley-Liss, I nc.