Assessing the use of known mutagens to calibrate the Salmonella typhimurium mutagenicity assay: II. With exogenous activation

Abstract

In order to determine the usefulness of selected chemicals as potential reference materials for calibrating the Salmonella assay, two laboratories tested a series of Salmonella mutagens that require exogenous activation. When the variance for individual substances within a bioassay is sufficiently low and the rankings of those substances are of acceptable consistency, they can later be evaluated for use as standard control compounds, as audit materials, and as standard reference materials for comparative bioassay efforts. The purpose of this project, therefore, was to evaluate the variability in the mutagenic response of potential reference chemicals that require exogenous metabolic activation in the standard plate-incorporation Salmonella mutagenicity assay, and to develop ranking criteria for mutagenic activity based on these data. Ten indirect-acting mutagens were tested in two laboratories using Salmonella typhimurium TA100 and an Aroclor-induced rat liver S9. Each laboratory conducted four definitive testing rounds. A different batch of S9 was utilized for every two rounds. Of the 10 chemicals tested only 2-anthramine had a mean slope value greater than 1000 revertants/μg. Three chemicals had slope values between 1000 and 100; and five chemicals had slope values between 100 and 10. The remaining compound, 9,10-dimethyl-1,2-benz[ a]anthracene, could not be placed into a single category because it had slope values on either side of 100 revertants per mg. Coefficients of variance were low (i.e., below 25% in most cases). The low variability achieved in this study may be accounted for by two parameters of the study. First, based on Claxton et al. (1991a) and the S9 optimization for three compounds, the amount of S9 was calibrated to a set amount of protein per plate (1.1 mg/plate). Secondly, the 10 test doses were placed in the initial, linear, nontoxic portion of the dose-response curves. The use of ten closely spaced, nontoxic doses allowed for a more accurate estimate of the slope. These data, along with the data generated previously (Claxton et al., 1991b) for 10 direct-acting chemicals, provide a basis for intralaboratory and interlaboratory comparison and ranking of Salmonella typhimurium TA100 mutagenicity data. Concurrent testing (same day, same laboratory, same technician, same bacteria, S9 and media) is a more acceptable method to control variance. However, this is not always possible. The ability to quantify Salmonella mutagenicity data by the use of standard reference materials will assist in potency comparisons of data generated in different laboratories or within the same laboratory on different days.