Acetamide (CAS 60-35-5) is classified by IARC as a Group 2B, possible human carcinogen, based on the induction of hepatocellular carcinomas in rats following chronic exposure to high doses. Recently, acetamide was found to be present in a variety of human foods, warranting further investigation. The regulatory body JECFA has previously noted conflicting reports on acetamide's ability to induce micronuclei (MN) in mice in vivo. To better understand the potential in vivo genotoxicity of acetamide, we performed acute MN studies in rats and mice, and a subchronic study in rats, the target species for liver cancer. In the acute exposure, animals were gavaged with water vehicle control, 250, 1000, or 2000 mg/kg acetamide, or the positive control (1 mg/kg mitomycin C). In the subchronic assay, bone marrow of rats gavaged at 1000 mg/kg/day (limit dose) for 28 days was evaluated. Both acute and subchronic exposures showed no change in the ratio of polychromatic to total erythrocytes (P/E) at any dose, nor was there any increase in the incidence of micronucleated polychromatic erythrocytes (MN-PCE). Potential mutagenicity of acetamide was evaluated in male rats gavaged with vehicle control or 1500 mg/kg/day acetamide using the in vivoPig-a gene mutation assay. There was no increase in mutant red blood cells or reticulocytes in acetamide-treated animals. In both acute and sub-chronic studies, elevated blood plasma acetamide in treated animals provided evidence of systemic exposure. We conclude based on this study that acetamide is not clastogenic, aneugenic, or mutagenic in vivo in rodent hematopoietic tissue warranting a formal regulatory re-evaluation.

Background: Acorus calamus (sweet flag) is widely used in traditional medicine, yet its dermal safety profile remains insufficiently defined under modern regulatory standards. Objective: To comprehensively evaluate the skin irritation, corrosion, and sensitisation potential of A. calamus rhizome oil using new approach methodologies’ (NAMs) test batteries under GLP conditions. Results: The A. calamus 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% (w/w) for leave-on products and up to 7.46% (w/w) for rinse-off formulations. Conclusions: The combined evidence from the NAM-based assays showed that A. calamus 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.