Microwell mutation assays: evaluation of ethylmethanesulphonate, benzo[a]pyrene and benzidine using the tk locus in...

As part of the third UKEMS collaborative trial, ICI Central Toxicology Laboratory (CTL) tested the three study chemicals ethylmethanesulphonate (EMS), benzo[a]pyrene (B[a]P) and benzidine (BZD) in a microwell adaptation of the L5178Y gene mutation assay using the thymidine kinase (tk) locus. Cofactors for metabolic activation were optimized prior to testing the chemicals under differing expression times (48 and 72 h) and S9 levels (2,5 and 10%). The results for zero hour survival, relative total growth and mutant frequency were subjected to extensive statistical analyses. It was concluded that the methods described produce a robust and reliable assay and that for routine use a single 72 h expression time and 5% S9 level are sufficient, provided true independent replicates are used.