Cytotoxicity of antimicrobial wound irrigation solutions and antiseptics: an in vitro and ex vivo study

Introduction Topical antiseptics must control wound bioburden without damaging cells required for tissue repair, yet cytocompatibility data obtained from isolated active compounds or monolayer exposure may not reflect finished formulations under clinically relevant conditions. Methods This study compared the cytotoxic effects of six commercial antiseptics and wound-irrigation solutions across complementary in-vitro and ex-vivo models. An octenidine-based antiseptic, two polyhexamethylene biguanide formulations (0.1 and 0.04%) and three chlorine-based solutions (0.005% sodium hypochlorite/hypochlorous acid, 0.08% sodium hypochlorite, and 0.03% hypochlorous acid) were tested in HaCaT keratinocytes and CRL-2522 human dermal fibroblasts. Cell viability was assessed after 24 h using an ISO 10993-5-based assay and after 1, 5 and 15 min using an application-oriented exposure-and-washout assay. In addition, the cell viability and membrane integrity was assessed by a qualitative LIVE/DEAD staining. Repeated topical treatment was additionally evaluated over 15 days in a human full-thickness ex-vivo skin wound model. Results Cytotoxicity was strongly dependent on formulation, exposure time, concentration, and cell type. Octenidine/Phenoxyethanol caused the greatest and most rapid loss of viability, with substantial effects after 1 min and the most pronounced epithelial disorganization ex-vivo . Both polyhexamethylene biguanide formulations showed cytotoxicity during continuous 24 h exposure and, despite better short-term tolerance, impaired epithelial continuity after repeated treatment. The 0.005% sodium hypochlorite/hypochlorous acid and 0.08% sodium hypochlorite formulations maintained viability above the 70% cytotoxicity threshold under all tested conditions and preserved epithelial architecture. The 0.03% hypochlorous acid formulation showed time-, concentration-and cell-specific toxicity, including marked keratinocyte damage after 15 min in LIVE/DEAD analysis. Discussion Cytocompatibility cannot be inferred from antiseptic class or nominal active-agent concentration alone. Finished formulations should be evaluated across various clinically relevant exposure conditions and tissue-level models and product selection should balance antimicrobial efficacy with preservation of regenerative cells and tissue.