Compatibility and interference of food simulants and organic solvents with the in vitro toxicological assessment of food contact materials

Ensuring the safety of food contact materials (FCMs) is paramount, governed by regulations such as Regulation (EC) 1935/2004 and Commission Regulation (European Union [EU]) 10/2011 for plastic FCMs. This study evaluates the compatibility of food simulants specified in the latter regulation with in vitro biological testing. HepG2 and Caco-2 cell lines were exposed to various concentrations of ethanol and acetic acid. Maximum tolerable amounts of food simulants A, C, and D1 (10%, 20%, and 50% v/v ethanol) were specified at 0.5%, 1.25%, and 2.5%. Food simulant E, Tenax, requires an additional extraction step for the recovery of migrants. An acetone-methanol mixture was selected for its ability to recover both polar and apolar potential migrants. The mixture exerted cytotoxic effects at combined concentrations above 0.5% v/v (0.25% each). Ethanol (Simulants A, C, and D1) interfered with the neutral red uptake (NRU) assay at concentrations above 0.5% v/v, showing no changes in cell viability despite increasing concentrations. Acetic acid (Simulant B) interfered with the NRU assay at 0.5% v/v and lactate dehydrogenase assay at all concentrations, resulting in negative cytotoxicity values due to pH reduction in the exposure medium. Additionally, sample preparation does not interfere with genotoxicity testing, aligning with the European Food Safety Agency (EFSA)'s mandatory testing requirements.