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The use of uninduced rat liver S-9 to supplement BALB/3T3 cells in the in vitro transformation assay.

Because of limited inherent capacity to metabolize chemicals to their reactive form, the BALB/3T3 transformation assay using clone A31-1-1 cells requires metabolic supplementation with rodent liver homogenate preparations (S-9). Activation by S-9 is limited, however, by its cytotoxicity to the cells, thus necessitating a reduction in treatment time from the usual 24-72 to 1-4 hr. With cyclophosphamide (CP) as a test chemical, we were able to increase the treatment time to 24 hr by using lower concentrations of cofactors and S-9 prepared from the livers of untreated rats. Statistically significant increases in transformed foci were induced in cultures treated with 50 or 100 micrograms CP/ml in the presence of 100 or 200 micrograms of uninduced rat liver S-9/ml and 76 or 380 micrograms each of NADH, NADP, NADPH, and glucose-6-phosphate per ml of incubation medium.