Genotoxicity of unmodified and organo-modified montmorillonite

The natural clay mineral montmorillonite (Cloisite® Na+) and an organo-modified montmorillonite (Cloisite® 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-μm pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141 μg/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite® Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24 h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite® 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p < 0.01 and p < 0.001) and (p < 0.05 and p < 0.01), respectively. The unfiltered samples were tested up to concentrations of 170 μg/ml and the filtered samples up to 216 μg/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57 μg/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite® 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite® 30B.