An integrated testing approach to generate validated, lung-relevant toxicity data on graphene nanoplatelets

Graphene nanoplatelets (GFNs) represent a group of carbon-based engineered nanomaterials with special properties, which enable innovation in technology and medicine. Within the general framework of safety assessment of nanomaterials during their lifecycle, the main objective of the PLATOX project (funded by EU FP7-SIINN ERA-NET Programme) is to apply a tiered approach to assess the toxicity of GFNs and finally identify the derived no-effect level (DNEL) parameter for inhalation exposure. A comprehensive physicochemical characterization of six commercially available GFNs (single- and multilayer graphene/graphene oxide, carboxyl graphene, and graphite oxide) was followed by an in vitro toxicity screening using different endpoints and lung-relevant cell models (primary rat alveolar macrophages, murine macrophages and primary human lung fibroblasts). In vitro testing comprised evaluation of the cytotoxic (membrane damage, metabolic activity and cell proliferation), genotoxic (oxidative comet assay and cell cycle dynamics) and pro-inflammatory potentials (cytokine/eicosanoid release). Cytotoxicity results were used to calculate the benchmark dose (BMD30), a parameter subsequently used as starting concentration for the other endpoints. Generally, the in vitro results showed a higher sensitivity of the macrophage models, compared to fibroblasts, and a higher toxicity of single layer graphenes, as compared to the other GFNs. The high toxicity was correlated with a higher surface area of the single layer graphenes. These results allowed us to establish a toxicity ranking and to select two GFNs with low and high toxicity to be tested in a 28-day inhalation toxicity study in rats. All results will be further integrated in the overall risk assessment workflow.