National Toxicology Program

This mammalian cell mutagenicity assay is used to determine whether a chemical is capable of inducing a change in cultured mammalian cells (mouse lymphoma L5178Y cells) measured as acquired resistance to trifluorothymidine (the result of forward mutation at the thymidine kinase locus). In brief, cells are treated with the test chemical and then placed into suspension cultures with selective medium for replication and fixation of induced mutations. Cells are then plated for colony growth, and after several days, colony numbers and colony size are recorded. The number of mutant colonies is a measure of the ability of the test chemical to induce a genetic change at the thymidine kinase locus in these transformed cells. The mouse lymphoma L5178Y/tk+/- assay can detect both point mutations and chromosomal alterations.

All study chemicals tested by the NTP in this assay were supplied as coded aliquots from Radian Corporation. The highest dose of test chemical was determined by solubility or toxicity, and did not exceed 5 mg/kg in the absence of dose-limiting toxicity. Mouse lymphoma L5178Y TK+/- cells were maintained at 37° C as suspension cultures in Fischer's medium supplemented with 2 mM l-glutamine, 110 ug/mL sodium pyruvate, 0.05% luronic F68, antibiotics, and heat-inactivated horse serum; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring trifluorothymidine (TFT) resistant cells, subcultures were exposed once to medium containing thymidine, hypoxanthine, methotrexate, and glycine for one day; to thymidine, hypoxanthine, and glycine for one day; and to normal medium for 3 to 5 days. For cloning, horse serum content was increased and Noble agar was added.

All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated. Treated cultures contained 6 x 10 6 cells in 10 mL of medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with the test chemical continued for 4 hours, at which time the medium plus chemical was removed and the cells were resuspended in 20 mL of fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, 3 x 10⁶ cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells (TK-/-) and in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37 C. in 5% CO₂ for 10 to 12 days. At the end of incubation, colonies were counted with an automated counter. The test was initially performed without S9. If a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of either Aroclor 1254-induced or non-induced male Fischer 344 rats.

Typically, 4 solvent control cultures and 3 positive control cultures were included in each experiment. Two or three cell cultures were used for each concentration of test chemical that was studied in a single experiment. Five to six concentration levels were tested per experiment. All experiments were replicated at least once.

Quality control factors (minimum criteria for accepting an experiment as valid) included such factors as cloning efficiencies within acceptable parameters for control and treated cultures, relative total growth of treated cultures above 1%, absence of test chemical precipitate, and two or more acceptable cultures per dose set. A detailed description of quality control factors and of the statistical analysis and data evaluation procedures are presented in Caspary et al. (1988) [Environ. Mol. Mutagen. 12 (Suppl. 13): 19-36]. All data were evaluated statistically for trend and peak responses. Both responses had to be significant (P < 0.05) for a chemical to be considered capable of inducing TFT resistance; a single significant response led to a "questionable" conclusion, and the absence of both a trend and a peak response resulted in a "negative" call.

Further protocol details can be found in Mitchell et al. (1988), Evaluation of the L5178Y Mouse Lymphoma Cell Mutagenesis Assay: Methods Used and Chemicals Evaluated. Environ. Mol. Mutagen. 12 (Suppl. 13): 1-18.