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Methylchloroisothiazolinone (MCI) and methylisothiazolinone (MI) are biocides used in many types of products such as cosmetics, paints, and cleaning agents. Skin contact is often encountered when using these products. Although MCI and MI are strong allergens and cause skin irritation, no scientific skin permeation study has been reported except for some unpublished data. Therefore, this study assessed the permeation of MCI and MI both separately and as a mixture through freshly dermatomed human skin (800 µm) in a flow-through diffusion cell system. Different concentrations of aqueous standards (1.5/1, 70/50, 150/35, and 750/175 µg/mL of MCI/MI) and various commercial products were assessed after 15-20 h of exposure. In parallel, the dose-dependent irritant effects of MCI/MI and MI were estimated by histology following 6- or 24-h exposure. Overall results show that MI in formulations or in aqueous standard solutions quickly permeated the skin with time lags less than 15 min while MCI was much slower (>3.5 h). MCI in formulations had permeation rates up to five times greater than that for MI in the same product, and in two tested creams were not found to permeate skin. Some signs of irritation were observed by histology; especially at the highest MCI/MI concentrations (750/250 µg/mL) in aqueous solutions. This confirms that MCI reacts readily with skin and may induce local irritation. The MCI and MI permeations are also greatly influenced by the topical vehicle. It is, therefore, more relevant to test exposures to formulations than aqueous standard solutions.