Development of a standard reference material for diesel mutagenicity in the Salmonella plate incorporation assay

The present study documents the mutagenicity of a new National Institute of Standards and Technology (NIST) standard reference material (SRM) in the Salmonella plate incorporation assay. This study is in response to a previous recommendation by the World Health Organization to develop large batches of new SRMs for biological and chemical research. SRM 1975 is a dichloromethane (DCM) extract of 5.6 kg of filter-collected combustion particulate matter (SRM 2975) from operating forklifts with diesel engines. The mutagenicity and a summary of the related chemical analysis of mutagens in SRM 1975 is presented in this paper, and are available from the NIST. Mutagenicity test conditions were: Salmonella typhimurium TA98, TA100 (standard strains); TA98NR, TA100NR (nitroreductase (NR) gene deficient); and YG1021 and YG1026 (NR gene addition); 10 dose levels in the linear portion of the dose-response curve; duplicate plates per dose; and S9 at 6.4% or 1.1 mg of protein/plate. Four rounds of testing were conducted. Rounds were conducted at least 1 week apart. Slopes (revertants/microg) were calculated by the linear regression rejection model of Bernstein and by the Stead and Krewski models which analyze non-linear data. The GeneTox Manager software package developed at the EPA was used to record the data and calculate the slopes. Results demonstrated: (1) the ranking of slopes without S9 was: YG1021 > TA98 > TA98NR > YG1026 > TA100 > TA100NR in all three statistical models; (2) the mutagenic activity of SRM 1975 was significantly increased by the presence of the NR gene; (3) the slope values for the TA100 series were significantly less than for the TA98 series; (4) in general, the addition of the S9 significantly reduced mutagenic activity; (5) the mutagenic activity of the SRM 1975 was stable over time and variability was low (generally less than 20% in slope values over the 4 rounds); and (6) agreement of the slope values among the three models was excellent due to the linear nature of the data. These data will be useful in ranking other diesel and air samples for mutagenic activity, for quality assurance of data generated in different laboratories, for quality control within a laboratory, and as positive control values for future air and automotive emission studies.