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Zinc pyrithione is an active component incorporated in an extensive range of topically applied commercial products that are used worldwide. Despite its prevalence, no published study has investigated the penetration of zinc from the zinc pyrithione complex into human skin. Zinc is crucial for healthy skin function however an elevated concentration of labile zinc is toxic outside a narrow concentration range. Synchrotron X-ray fluorescence microscopy in conjunction with X-ray absorption near edge structure spectroscopy was used to map the deposition of zinc, quantitate the amount of zinc within the skin and to identify a change in the chemical form of zinc after application. This study has demonstrated a ˜3.8 fold increase in zinc concentration within the viable epidermis (VE) after 24 h topical application of zinc pyrithione that increased significantly by ˜250 fold after 48 h when compared to control skin. Confocal microscopy using a labile zinc specific dye, ZinPyr-1, showed that zinc pyrithione disrupted the skin cells zinc homeostasis and significantly increased the intracellular zinc concentration leading to cell toxicity. Overall, this study demonstrates that topical application of zinc pyrithione formulations leads to an increase in zinc penetration in human skin, consequently, raising concerns for potential localised toxicity to occur.Zn penetration in ex-vivo human skin was quantitatively mapped.ZnPt undergoes a chemical change and the Zn concentration increases in the viable epidermis.Toxicity was observed at concentrations sufficient to disrupt Zn homeostasis.Toxicity concerns are strongly time dependant.