Use of the spiral salmonella assay to detect the mutagenicity of complex environmental mixtures

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Abstract
The success demonstrated by the spiral Salmonella assay in a recent study of 20 pure prompted us to examine the effectiveness of this automated bacterial mutagenicity assay for testing complex environmental mixtures. Three sets of combustion emissions were selected for evaluation: automotive diesel exhaust, woodsmoke, and a coal combustion emission. Each sample was tested in the Salmonella mutagenicity assay according to standard protocol (plate incorporation) and spiral assay techniques. In the spiral assay, a specialized plating instrument dispenses the bacteria, test agent, and S9 mix in a spiral pattern onto a minimal agar plate supplemented with histidine and biotin. The components of the assay are administered in such a way that a uniform density of bacteria is exposed to a concentration gradient of the test agent on a single plate. When results are analyzed, a dose‐response curve comprised of 13 data points is generated. A comparison of results from the two assays demonstrated the following: 1) Diesel exhaust was generally the most mutagenically potent sample in both assays, followed closely by the coal combustion emission. The woodsmoke sample was only weakly mutagenic in the standard assay but demonstrated higher mutagenic activity in the spiral assay. 2) Samples were more mutagenic on rev/μg basis in the spiral assay, especially when metabolic activation was added. This disparity presumably was due to differences in the relative amounts of S9 administered across the dose range. 3) The spiral assay required 1/20 the sample mass of the standard assay to test equivalent doses; in addition, for some samples, 50 times more sample mass was required by the standard assay to generate a comparable dose response. 4) Dichloromethane extracts of the complex mixtures could be tested for mutagenicity in the spiral assay, thereby precluding solvent exchange (to dimethylsulfoxide) required by the standard assay for sample/bioassay compatibility.