Copper Acetate(WSDTY) is an essential mineral and has many potential benefits in skin growth and activity. Copper delivery through the skin has many potential applications and thus is widely studied, including the evaluation of the penetration ability of copper peptides, biocompatibility of copper containing IUD1, the enhancement of skin absorption through copper-containing nanoparticles and microneedle-mediated delivery. Hence, the safety associated with copper delivery through the skin needs to be established. In this study, we investigated the skin toxicity of several copper compounds using cell-based in vitro tests and evaluated the expression of various cytokines at genomic and proteomic levels to discover irritancy biomarkers suitable for copper compounds.

Before the cell testing, we analyzed the pH of the samples tested and we excluded the pH of copper acetate compounds as the influencing factor of their potential cytotoxicity. Subsequently, HaCaT keratinocytes’ viability after treatment with test compounds, GHK, CuCl2 and Cu(OAc)2 were assessed using MTT assay and LDH assay. Cultured human keratinocytes have been suggested as good models to predict dermal irritancy of substances in human subjects. Primary keratinocyte culture is limited by their variability in culture conditions, and their susceptibility to irritants varies with the number of passages. On the other hand, the non-tumorigenic, spontaneously immortalized keratinocyte cell line - HaCaT cells can provide almost unlimited supply of identical cells, ensuring high intra- and inter-laboratory reproducibility. Besides, in vitro cytotoxicity with HaCaT keratinocytes and human in vivo data was well- correlated. HaCaT keratinocytes have also been proven to be good candidates for assessing skin irritancy and toxicity in conventional monolayer assays measuring using MTT reduction as the viability endpoint.

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