Effect of sample preparation on the in vitro genotoxicity of a light curable glass ionomer cement.

The glass ionomer cement Vitrebond showed a clear genotoxic effect in the in vitro Mammalian Cell Gene Mutation Test (HPRT Test) with CHO cells as well as in the bacterial umu-test with Salmonella typhimurium TA1535/pSK1002. Both DMSO and Ham's F12 cell culture medium extracts according to ISO 10993-12 (Biological evaluation of medical devices-Part 12: sample preparation and reference materials, Geneva, Switzerland) exhibit a clear genotoxic effect in the umu-test. The effect is independent of the extraction volume in a range from 0.5 to 4 ml Ham's F12 cell culture medium. Subsequent extractions of Vitrebond showed no significant difference in the genotoxic response although weight loss and content of 2-hydroxyethyl-methacrylate dropped significantly. In vivo conditions of Vitrebond were simulated by extractions with artificial and collected human saliva. These extracts showed a clear genotoxic effect in the umu-test, even if only a few seconds of extraction time were applied. In conclusion, sample preparations for genotoxicity testing according to ISO 10993-12 reflect the in vivo conditions of Vitrebond applications. This seems to be mostly due to the hydrophilic nature of the genotoxic ingredients.

Toxicological Investigation of Soils with the Solid-phase Flash Assay: Comparison with Other Ecotoxicological Tests.

A new direct-contact toxicity test, the solid-phase flash assay, which utilises photobacteria in direct contact with soil particles during the exposure, was evaluated on four soil samples. Samples HTNT1 and HTNT2 originated from former military sites in Germany, and were highly contaminated with nitroaromatics (approximately 20g/kg), lead and polycyclic aromatic hydrocarbons. Samples LMKW1 and LMKW2, from bioremediation stacks in Germany, were mainly contaminated with mineral oils. The solid-phase flash assay was applied to soil-water slurries, and the results were compared with the toxicity data for soil-water extracts obtained by using various conventional ecotoxicological tests, in which photobacteria, crustaceans, protozoa and algae were used as test organisms. The LMKW1 and LMKW2 samples were not toxic (EC20 > 12.5%) according to all the tests applied, except for the Photobacterium phosphoreum conventional luminescence-inhibition test for LMKW1 (15-minute EC20 = 5.4%(. The HTNT1 and HTNT2 samples were toxic according to all the tests applied, with the majority of EC20 values being lower than 1%. The solid-phase flash assay (1 minute of extraction and 30 seconds of exposure time) gave comparable results to the conventional tests. Therefore, this flash assay could be applied as a fast screening test in parallel with conventional toxicity tests that use soil 24-hour extracts. The flash assay results will be ready by the start of the conventional assays, and could serve as range-finders for these slower and more expensive tests.

Ecotoxicological testing with new kinetic Photorhabdus luminescens growth and luminescence inhibition assays in microtitration scale

Miniaturized luminescence and growth inhibition assays in microtitration plates with the terrestric enthomopathogenic nematode symbiont Photorhabdus luminescens (DSMZ 3368T) are presented and compared with standardized tests with Vibrio fischeri (DSMZ 7151/NRRLB-11177) and Pseudomonas putida (DSMZ 50026). Toxicological parameters (EC and G values) of selected reference toxicants (e.g. heavy metals and environmental samples) were obtained at different temperatures and without sodium chloride supplementation. Kinetic data recordings were compared with results of a cuvette test protocol using integral and endpoint calculation. Growth inhibition experiments with reference samples reveal similar or higher sensitivities as for the Vibrio or Pseudomonas spp. The luminescence inhibition assay shows reduced sensitivities to most of the reference samples compared with the V. fischeri standard assay. But G values obtained with other standardized aquatic assay systems with daphnids, algae and growth inhibition assays with V. fischeri and Ps. putida correspond more closely to data observed with Ph. luminescens. The test procedures are easily to perform and to evaluate and seem to be reliable alternatives to the established protocols at low osmolarities. The sample specifity of the toxic responses of the marine and the terrestric strain recommends to employ both assays to determine the toxic potential of environmental samples. This will support to reduce false positive results in future investigations.

Agonistic and antagonistic toxic effects observed with miniaturized growth and luminescence inhibition assays.

Kinetic data recordings with miniaturized growth and luminescence inhibition assays with prokaryotes (Pseudomonas putida and Vibrio fischeri) reveal typical sample and species specific growth and light emission patterns respectively. Growth inhibiting influences, based on toxicokinetic and toxicodynamic sample specificities appeared in each growth phase. Agonistic and antagonistic effects of combinations of heavy metals like Cd2+, Hg2+ and Pb2+ and other compounds were easily recognized after accurate curve discussions. Some of the influences that made it more difficult to evaluate toxicological results only by restriction on EC values (effective concentrations) were investigated. The findings may lead to a more comprehensive interpretation of toxicological data and a presumption on the composition of complex samples in terms of screening investigations. Therefore, the conclusions demonstrate the possibilities and limits of kinetic data recordings in chronic and acute toxicity testing.