MUTAGENICITY TESTING OF ORGANIC EXTRACTS OF DIESEL EXHAUST PARTICLES AFTER FRACTIONATION AND RECOMBINATION

A new strategy for the evaluation of mixtures is presented. The mixtur e used was the organic extract of diesel exhaust particles (DEP). Afte r extraction with dichloromethane (DCM), the crude extract was fractio nated according to polarity into five fractions: aliphatic hydrocarbon s, polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, dinitro-PAHs, and polar compounds. After dissolving in dimethylsulphoxide (DMSO), th e three fractions containing the primary muta (fractions 3-5) were rec ombined in different combinations to create new extracts. The blend ma trix was obtained using a mixture design at three dose levels to suppo rt an empirical model with linear, interaction, and quadratic terms (T aylor polynome). The recombined extracts were tested in the Ames Salmo nella assay using strain TA100. Multivariate data analysis was perform ed with projections to latent structures (PLS). The best model describ ing the relation between the mutagenicity (response) and the three fra ctions (variables) contained two interaction terms. The model showed h igh correlation (r(2)) and prediction properties (Q(2)), the latter ob tained after cross validation. Interaction terms are only indications of possible synergism or antagonism and have to be evaluated with resp ect to dose-additivity and response-additivity. The incorporation of d ose in the design reduced the number of samples (recombined extracts) significantly, compared to determining dose-response curves on each sa mple (i.e. the recombined extracts in different dilutions). Furthermor e, instead of running two independent experiments as required in the s tandard procedure for the Ames test, predictions and verifications of a few new samples were used. The principle of fractionation and recomb ination. and the use of mixture design may in principle be extended to an unlimited number of variables. An adaptation of mixture design to the isobole method is discussed.