Regular Article :AATEX 3(4):191-198
The first phase inter-laboratory validation of cytotoxicity tests using established cell lines was conducted by the collaboration of 7-8 laboratories for the purpose to evaluate the test for their relevance as alternative methods to the Draize rabbit eye irritation test (Draize test). The methods studied were the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay in HeLa cells (HeLa-MTT assay) and crystal violet staining assay in CHL/IU cells (CHL/IU-CVS assay). We tested nine surfactants and isotonic sodium chloride solution (phosphate saline) under the common standard operating procedure (SOP) and determined the concentrations of test chemicals that showed 50% absorbance reduction as compared to control (the median effective concentration: EC50).
Inter-laboratory coefficients of variation of the EC50 values obtained for the surfactants obtained were below 0.50 except for polyoxyethylene octylphenylether (10 E.O.: Triton X-100). The correlation coefficients of these EC50 values with maximal average Draize total score (MAS) were -0.902 and -0.817 for the HeLa-MTT and CHL/IC-CVS assays respectively. The cytotoxicity potentials (EC50) of test substances were similar between these two assay methods.
We conclude that the cytotoxicity tests in HeLa-MTT and CHL/IU-CVS assays are promising as alternative methods to the Draize eye test for surfactants. Because surfactants constitute only a minor part of total cosmetic ingredients, these tests should be evaluated with a wider range of chemicals used as cosmetic ingredients.
Key words: validation study, Draize eye irritation test, cytotoxicity test, MTT, crystal violet, HeLa cells, CHL/IU cells, alternatives, in vitro, surfactant.