Methods: Material samples were first set in Teflon rings (0.5cm diameter) for 6h at 37°C and then extracted in culture medium for 24h at 37°C. The formation of micronuclei (MN) to indicate genotoxicity was tested in Chinese hamster fibroblasts (V79). Ethyl methanesulfonate (EMS) was used as a positive control substance. V79 cells were seeded onto microscopic glass slides and continuously exposed to extracts or EMS for 24h at 37°C. Then, cells were fixed in ethanol and stained with Schiff reagent. Genomic DNA-containing structures (nuclei and micronuclei) were analyzed microscopically in 1000 cells/slide of two parallel cultures per extract concentration in two independent experiments (n=4). Differences of median values between the numbers of MN induced in treated and untreated cell cultures were statistically analyzed using the non-parametric Mann-Whitney-U test (p<0.05).
Results: Extracts of Acroseal, Epiphany and AHPlus significantly increased the number of micronuclei. Acroseal was the most genotoxic material tested, since even diluted extracts were as effective as 5mM EMS. The number of micronuclei increased 6-fold from 11 MN/1000 cells in untreated controls to 61 MN/1000 cells in cultures treated with Acroseal (1:64 dilution). Epiphany (1:8 dilution) caused a 2-fold increase of the number of micronuclei, which was only slightly increased by AHPlus extracts (1:8 dilution). A significant increase of the number of micronuclei caused by COP extracts was not detected.
Conclusions: The lack of the formation of micronuclei by extracts of COP is a good basis for further studies on its biocompatibility.