Cytotoxicity testing using neutral red and MTT assays on a three-dimensional human skin substrate

The use of a three-dimensional dermal culture system as a substrate in cytotoxicity assays is described. This substrate consists of several layers of dermal fibroblasts, derived from human foreskin, grown on pretreated nylon mesh. The physiological model of the human dermis has been used in conjunction with the neutral red assay and the MTT assay to assess the in vitro toxicity of a panel of 15 test agents from several different classes. NR50 and MTT50 endpoints (test agent concentrations yielding 50% viability) were obtained for compounds/formulations from the following groups: surfactants, alcohols, antimicrobial preservatives, metal chlorides and pesticidies. In addition, the carboxylic ionophore, monensin, was tested in both assays. Limited comparisons of the in vitro neutral red and MTT results, using the three-dimensional culture system, with existing in vivo rabbit ocular irritancy data look promising. The three-dimensional model may afford several advantages over monolayer cultures.