Comprehensive In Vitro Safety Assessment of Acorus calamus Rhizome Oil Using OECD-Compliant New Approach Methods: Classification as a GHS Category 1B Sensitiser and Category 2 Irritant

<b>Background</b>: <i>Acorus calamus</i> (sweet flag) is widely used in traditional medicine, yet its dermal safety profile remains insufficiently defined under modern regulatory standards. <b>Objective</b>: To comprehensively evaluate the skin irritation, corrosion, and sensitisation potential of <i>A. calamus</i> rhizome oil using new approach methodologies' (NAMs) test batteries under GLP conditions. <b>Results</b>: The <i>A. calamus</i> rhizome oil was predicted as a Category 2 skin irritant, non-corrosive and GHS Category 1B skin sensitiser. Chemical analysis revealed β-asarone as the major constituent (~40.75%). The reconstructed human epidermis models established reversible irritation without corrosion. Mechanistic concordance across the Direct Peptide Reactivity Assay, KeratinoSens™, and Human Cell Line Activation Test showed activation of the three key events of the skin sensitisation adverse outcome pathway. Using the "2-out-of-3" Defined Approach with the KE 3/1 sequential strategy allowed for hazard classification into GHS Category 1B. Quantitative risk modelling using SARA-ICE models and SCCS parameters yielded conservative safe-use concentrations ranging from 0.13 to 0.78% (<i>w</i>/<i>w</i>) for leave-on products and up to 7.46% (<i>w</i>/<i>w</i>) for rinse-off formulations. <b>Conclusions</b>: The combined evidence from the NAM-based assays showed that <i>A. calamus</i> rhizome oil is a moderate sensitiser and irritant but not corrosive, providing critical data for risk assessment and regulatory decision-making, which was previously unknown. The SARA-ICE PoD-derived safe-use concentrations provide guidance for cosmetic formulators to ensure consumer safety, particularly in leave-on applications such as face and hand creams, where sensitisation risk is highest. This study demonstrates the utility of NAMs for botanical safety assessment and regulatory decision-making.