Measurement of in vivo mutant frequency in lymphocytes in the mouse

A limiting‐dilution cloning technique for quantifying in vivo mutations at the hypoxanthine phosphoribosyl transferase locus in mouse splenocytes was developed. Mouse splenocytes were cultured in round‐bottom microwells with irradiated feeder cells, concanavalin A, and a source of interleukin 2 at five cells/well in the absence of thioguanine, and at 5 × 10⁴ cells/well in the presence of 2.5 μg/ml thioguanine; mutant frequency was calculated as the ratio of the cloning efficiencies with or without thioguanine. The geometric mean (95% range) for the mutant frequency in 20 mice was 1.54 × 10⁻⁶ (4.7 × 10⁻⁷‐2.6 × 10⁻⁶) and whole‐body X‐irradiation resulted in a dose‐related increase in mutant frequency of up to approximately 20 times the baseline level. The in vivo murine mutation assay should be a useful system for genotoxicity testing and may be of particular value in establishing risk estimates for human populations exposed to genotoxins.