Bioassay-directed fractionation and sub-fractionation for mutagenicity and chemical analysis of diesel exhaust particles

Several types of diesel exhaust particles (DEPs) have been used for toxicology studies, including a highorganic automobile DEP (A-DEP) from Japan, and a low-organic forklift DEP developed by the National Institute of Standards and Technology (N-DEP). However, these DEPs were not characterized extensively for chemical composition or sub-fractionated and tested extensively for mutagenicity. We collected a compressor-generated DEP (C-DEP) and characterized it by conducting bioassay-directed fractionation of the extractable organics in Salmonella and correlating the results by hierarchical clustering with the concentrations of 32 polycyclic aromatic hydrocarbons (PAHs). Relative to A- and N-DEP, the mutagenic potency of C-DEP was intermediate in TA100 1S9 (PAH mutagenicity) but was lowest in TA98 – S9 (nitroarene mutagenicity). More than 50% of the mass of the extractable organics of C-DEP eluted in the nonpolar fraction 1, and only -20% eluted in the moderately polar Fractions 2 and 3. However, most of the mutagenicity eluted in Fractions 2 and 3, similar to A-DEP but different from N-DEP. HPLC-derived mutagrams of 62 sub-fractions per fraction confirmed that most of the mutagenicity was due to moderately polar compounds. The diagnostic strains identified a strong role for PAHs, nitroarenes, aromatic amines, and oxy-PAHs in the mutagenicity of C-DEP. Hierarchical clustering confirmed the importance of oxy-PAHs but not that of nitroarenes. To our knowledge this is the first use of hierarchical clustering to correlate chemical composition with the mutagenicity of a complex mixture. The chemical analysis and mutagenicity of C-DEP described here makes C-DEP suitable for additional toxicological studies.