Copper oxide nanoparticles: from EU-regulatory landscape and mechanistic toxicity towards a cuproptosis-linked adverse outcome pathway

Abstract Copper is an essential trace element, yet concerns regarding its toxic potential have gained increasing attention, particularly with regard to nanoparticulate forms of copper oxides. Both CuO and Cu 2 O are currently authorised in the EU for use in agriculture, biocides, and animal nutrition, but regulatory clarity regarding their nanoforms remains limited. This comprehensive review—combining narrative, bibliometric, and systematic approaches—addressed three key questions: (i) whether CuO and Cu 2 O nanoparticles are sufficiently considered within EU regulations to prevent unintentional oral exposure via the food and feed chain, (ii) whether these nanoparticles can cross the intestinal barrier as intact particles and accumulate in tissues, and (iii) whether they elicit distinct biological responses, including regulated cell death pathways such as cuproptosis. With respect to question (i), a detailed analysis of EU legal frameworks revealed a heterogeneous regulatory landscape and a lack of binding provisions that would preclude unintended presence of copper oxide nanoforms in regulated materials. Although some nano-specific data requirements exist, empirical information on nanoparticulate fractions in commercial products is still lacking. With respect to questions (ii) and (iii), our systematic literature analysis supports consistent intracellular uptake of CuO and Cu 2 O nanoparticles, yet direct evidence for transport of intact particles across the intestinal barrier is scarce. Mechanistic evidence supports cuproptosis as a key pathway for CuO nanoparticle toxicity, while the extent to which these effects differ quantitatively from ionic copper remains unclear due to insufficient data using appropriate experimental controls. Notably, a pronounced data gap was identified for Cu 2 O nanoparticles across all evaluated domains. Finally, to organise and integrate the mechanistic evidence presented, we developed an adverse outcome pathway (AOP), registered as AOP 590 in the AOP Wiki, describing how increased intracellular copper can induce cuproptosis via disruption of energy metabolism. Collectively, these findings highlight the need for coordinated analytical, mechanistic, and regulatory research to support evidence-based risk assessment of CuO and Cu 2 O nanoparticles in the food and feed context.