A comparison of the soft agar and microtitre methodologies for the L5178Y tk +/- mouse lymphoma assay

The L5178Y tk +/- mouse lymphoma assay (MLA) has been validated as a sensitive and specific test system for the detection of mutagens/clastogens. There are currently two methodologies for performing the MLA: the original soft agar procedure and the newer microtitre procedure. While both procedures are considered acceptable, a limited amount of comparative information exists for the two methods. In this report the two methods were compared with regard to: (1) spontaneous and induced mutant frequencies; (2) cloning efficiencies; and (3) colony size distributions for mutants. In addition, small and large mutant colonies from microtitre wells were rechallenged for trifluorothymidine (TFT) resistance. In a majority of the cases, cloning efficiency values were higher for the microtitre as were the spontaneous and induced mutation frequency (MF) values. Nevertheless, when responses were compared according to mutation index (fold increase over background MF) the results from the two systems were often similar. More spontaneous small colonies were observed in the microtitre assay. While colony size distribution for induced mutant colonies was compound specific, generally, more small colonies were counted in microtitre. All mutant clones that were rechallenged with TFT demonstrated resistance. Aside from the differences mentioned above, both the microtitre and the soft agar procedures appear equally capable of identifying mutagenic agents.