Can In-Vitro Assays Predict Chemically-Induced Skin Damage?

Abstract

Eleven chemicals, with a known potential to damage skin as corrosives, irritants or non-irritants, have been tested for their ability to affect the function and viability of mammalian cells in three in vitro assays. The assays measured total protein content by Coomassie Blue R binding (CB), lysosomal integrity by Neutral Red uptake (NR), or mitochondrial function by MIT uptake/reduction. Two cell lines, Chinese Hamster Ovary cells (CHO) and Swiss Albino Mouse Fibroblasts (3T3), were compared.

The results of all three assays agreed well with one another and with the observed number and morphology of the cells for each chemical tested. The CB tended to be the least sensitive and the NR the most variable of the three assays. Overall the MTT assay was most reliable, but may give anomalous results if used alone. CHO and 3T3 cells gave similar results, although 3T3 cells tended to be less sensitive than CHO cells. This may depend principally on the rate of division and proportion of dividing cells.

Using the results from the MTT assay, corrosive chemicals were cytotoxic, since concentrations of less than 10 µg/ml reduced dye uptake by 50%. Chemicals which were skin irritants were moderately cytotoxic, since concentrations in the order of 50–500 µg/ml reduced dye uptake by 50%. Non-irritant chemicals were not cytotoxic up to 5 mg/ml. In decreasing order of cytotoxicity, the chemicals were ranked: tributyltin chloride, dibutyltin dichloride, silver nitrate, benzalkonium chloride, zinc monoglycerolate, sodium dodecyl sulphate, benoxaprofen, fenclofenac, n-hexane, butan-1-ol, 2-methoxyethanol. The categorization and ranking of the degree of skin irritation concurred with the degree of cell kill. These results indicate that such simple in-vitro tests could be used instead of animals to predict skin irritants at an early stage of hazard identification.