2.7. Hprt Mammalian Gene Mutation Assay

The mammalian in vitro Hprt gene mutation test was performed according to the OECD Guidelines for the Testing of Chemicals 476 [25]. V79-4 cells were cultured in 100 mm diameter Petri dishes; 1 × 10⁶ cells were inoculated per dish in 10 mL medium in duplicate for each concentration, and incubated at 37 °C. On the following day, the cells were exposed to TiO₂ NPs for 24 h, at concentrations from 3 to 75 μg/cm².

Untreated cells cultured in medium for 24 h were used as negative control and cells treated for 3h with 0.1 mM methyl methanesulfonate (MMS; Sigma), served as the positive control.

After exposure, the medium was removed, and cells were washed, trypsinized and re-suspended in 2 mL of medium. They were then seeded in 100 mm diameter Petri dishes at 3 × 10⁵ cells/dish, 3 dishes per concentration. Cells were grown for 8 days, during which they were subcultured three times; duplicate samples were taken at 6 and 8 days after treatment for analysis of mutant frequencies. To detect mutants, cells were inoculated in 100 mm diameter Petri dishes at 2 × 10⁵ cells/dish, 5 dishes per sample giving a total of 10⁶ cells per sample and grown in medium containing 6-thioguanine (Sigma) at 5 μg/mL for 10 days to form colonies. 6-Thioguanine is an analogue of guanine, toxic to cells with functioning Hprt gene, and so only Hprt⁻ cells survive. Mutant colonies were counted manually after staining with 1% methylene blue; only colonies with at least 50 cells were counted.

For each of the two harvests (6 and 8 day duplicate samples), the frequency of surviving cells was assessed using the PE assay, as described above. Treated and untreated cells were seeded in 6-well plates at 50 cells per well, 1 plate per concentration, and incubated for 7 days at 37 °C to form colonies. Cell viability was calculated for each mutant harvest on the basis of the number of colonies as a percentage of the number of inoculated cells. The mutant frequency was determined as previously described [24].