Methods: BALB3T3 and ARPE19 cell lines were cultured in 96-well plates for direct contact cytotoxicity test of 22%, 59%, and 83% contact areas and 2.5, 12, and 24 h contact times. Cell morphology was graded by light microscopy. Cell viability was quantified by MTT assay in ARPE19 cells and neutral red uptake viability assay for BALB3T3. 1-H perfluorooctane (1H PFO) and purified perfluoro-n-octane (PFO) were used as positive and negative controls, respectively.
Results: Qualitative evaluation showed that positive control induced the presence of severe reactivity zones, resulting in 2.2 to 3.0 grading score, and cytotoxicity according to ISO 10993-5 in all tested conditions. Quantitative evaluation of 1H PFO applied on 22%, 59%, and 83% contact areas corresponded to 14–26%, 84–96%, and 86–99% cell mortality ranges, respectively. No cytotoxicity according to ISO 10993-5 was detected in tested cell lines, when 1H PFO was applied on a 22% area at the tested time intervals. The negative control was not cytotoxic with both approaches in all tested conditions.
Conclusions: Direct contact cytotoxicity test according to ISO 10993-5 of PFCLs was validated quantitatively and qualitatively, using ARPE19 and BALB 3T3 cell lines and covering 59% of the cell surface areas for 24 h of contact time. Test conditions using a smaller contact area could not be validated.