Enhanced Sensitivity and Human Relevance: a TNF‐α‐Induced HaCaT Keratinocyte Model for Screening Anti‐Inflammatory Cosmetic Materials

A TNF-α-induced inflammatory model using the human keratinocyte HaCaT cell line was developed and validated as an alternative in vitro method for evaluating the anti-inflammatory potential of cosmetic ingredients. In comparison with the conventional lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophage model, the HaCaT-based system demonstrated enhanced sensitivity, detecting significant cytokine inhibition at concentrations up to 1000-fold lower for certain actives. The model reliably distinguished established anti-inflammatory agents (ectoine, troxerutin, and dipotassium glycyrrhizinate) from non-active controls (glycerin, butanediol, and propylene glycol) through the suppression of interleukin-6 (IL-6). Notably, the HaCaT model also identified a potential pro-inflammatory shift at high concentrations of some active ingredients-an effect not observed in the RAW264.7 system. Multi-laboratory verification using the novel cosmetic ingredient β-nicotinamide mononucleotide (NMN) confirmed both the anti-inflammatory activity of NMN and the high reproducibility of the assay. These results support the TNF-α-HaCaT model as a sensitive, human-relevant, and reproducible alternative for screening cosmetic ingredients, contributing to the growing toolbox of nonanimal methods for safety and efficacy assessment.