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Carbon-based nanoplatelets (CNP) represent a new class of 2-D nanostructures in multiple variants and with interesting functional properties (e.g. material enforcement and electrical conductivity). A very high toxicity among members of the carbon family is not expected for nanoplatelets, however, hazard characterization is still incomplete. Commercial CNP candidates (ACS Material, USA) were selected, covering single layer or multi layer graphene, carboxyl graphene, single layer graphene oxide, and graphite oxide. Technical soot (Printex 90) served as particulate, non-platelet reference. The CNP were analyzed on sterility and endotoxin content; morphology (SEM pictures) and the specific surface area (BET method) were re-evaluated. As in vitro screening models both, primary rat alveolar macrophages (AM) and MRC-5 human lung fibroblast cells were analyzed on membrane damage (LDH release) and metabolic activity (AlamarBlue test). Interestingly, the two single layer graphene samples induced marked concentration-dependent membrane damage in AM after 24h of incubation, with a BMD30 of 3.2 and 2.5 µg/cm2, whereas no such effect was observed for MRC-5 cells. Some LDH release was also observed for single layer graphite oxide (BMD30: 39.3 µg/cm2). The other materials were nearly inactive. Significant effects on metabolic activity were not observed. In AM, single layer graphene CNP additionally induced direct DNA damage and release of PGE2. In conclusion, single layer graphene showed a (geno) toxic potential in vitro in AM, but not in lung fibroblasts. Based on the in vitro screening data and for validation, a single layer (highest) and a multilayer (lowest toxic potential) were selected for in vivo investigations. In a dose range finding (DRF) test, with dosing by intratracheal instillation (0.02 and 0.2 mg/rat, each) single layer graphene was confirmed as the most inflammogenic sample in bronchoalveolar lavage fluid (BALF) inducing the recruitment of neutrophils and eosinophils. In the subsequent 4-week nose-only inhalation study with the same total doses (predicted by MPPD model) the inflammatory response of single layer graphene was weaker and no eosinophils were detected in BALF. Histopathological examination is underway. PLATOX funding: FP7- ERA-NET SIINN. |
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