Integrated In silico and In vitro toxicological characterization of citrus essential oil nano-emulsions for food additives

Essential oils (EOs) are gaining attention as potential natural food additives; however, their safety data remain limited. This study combined predictive in silico modelling with EFSA-compliant toxicology assays to evaluate nano-emulsions of bergamot, lemon, orange, and tacle® EOs. Ten major constituents produced 66 predicted metabolites, with dehydration dominant for linalyl acetate, linalool, and terpineol, and desaturation for sabinene, β-cis-ocimene, and γ-/α-terpinene. Guided by these predictions, bergamot EO nano-emulsions were tested in vitro . At 0.001–0.023%, no mutagenic or genotoxic effects were detected in Ames or micronucleus assays. In contrast, TA98 strain in the Ames Test showed metabolism-dependent positives at 0.3–5% with S9 activation, indicating that biotransformation can drive genotoxic risk at higher concentrations. These results demonstrate the value of integrating metabolic transformation into risk assessment and provide mechanism-based evidence to guide the safe use of EO nano-emulsions in food. • Toxicological assessment of CEONEs using in silico and in vitro approaches were performed. • A total of 187 metabolites were identified in BeNEs biotransformation. • Desaturation was predominant for sabinene, β-ocimene, γ-terpinene, and α-terpinene. • No genotoxic effects were observed in micronucleus assays at tested doses. • Positive Ames test results in TA98 with S9 highlight biotransformation’s toxicological role.