Improved AMES Test for Genotoxicity Assessment of Drugs: Preincubation Assay Using a Low Concentration of Dimethyl Sulfoxide

The Salmonella /microsome bacterial mutagenicity test (Ames test) is used worldwide as a simple and rapid mutagenicity testing system. Several modified versions of the Ames test have been developed, subsequent to the original “plate incorporation assay” using the Salmonella bacterial tester strains, and rat liver homogenate fraction (S9) for generating reactive metabolites of test compounds. Among the modifications, Ames test with a modified procedure of preincubation (called a preincubation assay) has been the most frequently used. In this assay, dimethyl sulfoxide (DMSO) has been often used at a concentration of 7 or 14 % (particularly in Japan) in the preincubation mixture (0.05 or 0.1 mL of DMSO in 0.7 mL of the mixture), as a vehicle to dissolve a wide range of chemicals. However, DMSO is known to inhibit several kinds of drug-metabolizing enzymes including CYPs involved in the metabolic activation or detoxification of chemical mutagens, even at low concentrations of 1 % or less. Therefore, an improved preincubation assay using a low concentration (e .g ., 1 %) of DMSO is recommended as one option for modification of the Ames test to improve its sensitivity towards the detection of promutagens that require metabolic activation by reducing the inhibiting effect of DMSO on drug-metabolizing enzymes. In genotoxicity tests using mammalian cells, a DMSO concentration of 1 % is often used. In this chapter, a detailed protocol for this slightly modified preincubation assay is presented, together with data useful for the performance of the Ames test and the interpretation of the results.