In vitro evaluation of the toxicity mechanisms of two functionalized reduced graphene oxide derivatives

Dodecyl amine functionalized reduced graphene oxide (DA-rGO) and [2-(methacryloyloxy) ethyl] trimethylammonium chloride functionalized rGO (MTAC-rGO) have been developed and characterised for their further use in the food packaging industry as food contact materials. But before their application, an authorization procedure is required in which their safety plays a key role. Therefore, the aim of this work was to evaluate their toxicity with focus on two different toxicity mechanisms: genotoxicity and immunotoxicity. Following the recommendations of the European Food Safety Authority, the mutagenicity and genotoxicity were evaluated by the mouse lymphoma assay and the micronucleus assay, respectively, in L5178Y TK cells. Both assays did not show any effect at the tested concentrations (up to 200 μg/mL). The potential immunotoxicity was evaluated on two human cell lines: THP-1 (monocytes) and Jurkat (lymphocytes). The results showed that the highest cytotoxicity was induced by MTAC-rGO in Jurkat cells. The two functionalized rGO compounds did not significantly affect the differentiation process of monocytes into macrophages. In general, both compounds altered the expression of different cytokines, with the most prominent changes observed with MTAC-rGO in THP-1 cells. Moreover, MTAC-rGO induced the most evident differences in markers of cell death mechanisms. Also, for this graphene derivative, increased levels of IL-1β and TNF-α in THP-1 cell supernatants were observed by ELISA. In conclusion, a case-by-case evaluation is necessary as both functionalized rGO compounds exhibit distinct toxicity profiles that warrant further investigation before their application in the food industry.