QUANTITATIVE IN-VITRO ASSESSMENT OF N-ALKYL SULFATE-INDUCED CYTOTOXICITY IN HUMAN KERATINOCYTES (HACAT) - COMPARISON WITH IN-VIVO HUMAN IRRITATION TESTS

A spontaneously immortalized human keratinocyte line, HaCaT, was used as an in vitro model to predict the cutaneous irritation of anionic su rfactants. For this purpose, a number of sodium salts of N-alkyl sulph ates with hydrocarbon chain lengths varying between C-8 and C-16 were studied for possible cytotoxic effects. The endpoints used to assess t oxicity were uptake of the vital dye neutral red (NR) and cell morphol ogy criteria 24 h after dosing. A linear proportionality between kerat inocyte number and NR uptake was established. All tested surfactants h ad cytotoxic effects as demonstrated by a decreased NR uptake, which s howed a clear dose-response relationship. Concentrations resulting in 50% inhibition of NR uptake (IC-50) ranged from 0.15 mmol (sodium laur yl sulphate, C-12) to 1.23 mmol (sodium octyl sulphate, C-8) The in vi tro cytotoxicity data were highly reproducible when the test was repea ted after several weeks. The cytotoxicity data from these assays were compared with the irritant responses (as evaluated by measurement of e rythema and transepidermal water loss) obtained after 24 h application of the same compounds (300 mu l of 20 mmol aqueous solution) to the v olar forearm of human volunteers. There were significant linear correl ations between the IC-50 values and both barrier damage (transepiderma l water loss) and erythema (as evaluated by skin colour reflectance me asurements). For the test substances, however, the sensitivity of the in vitro system was between 10 and 100 times higher than that observed in human skin in vivo. This quantitative difference can largely be as cribed to the effective permeability barrier of normal human skin in v ivo, which protects living keratinocytes from the cytotoxic effects of surfactant molecules. The results indicate that normal human keratino cytes in culture are a promising screening method for predicting the i rritation potential of anionic surfactants. Confirmation, however, has still to be obtained by appropriate in vivo testing in human voluntee rs.