PLATOX: Integrated In Vitro/In Vivo Approach for Screening of Adverse Lung Effects of Graphene-Related 2D Nanomaterials.

Graphene-related two-dimensional nanomaterials possess very technically promising characteristics, but gaps exist regarding their potential adverse health effects. Based on their nano-thickness and lateral micron dimensions, nanoplates exhibit particular aerodynamic properties, including respirability. To develop a lung-focused, in vitro/in vivo screening approach for toxicological hazard assessment, various graphene-related nanoplates, i.e., single-layer graphene (SLG), graphene nanoplatelets (GNP), carboxyl graphene, graphene oxide, graphite oxide and Printex 90® (particle reference) were used. Material characterization preceded in vitro (geno)toxicity screening (membrane integrity, metabolic activity, proliferation, DNA damage) with primary rat alveolar macrophages (AM), MRC-5 lung fibroblasts, NR8383 and RAW 264.7 cells. Submerse cell exposure and material-adapted methods indicated material-, cell type-, concentration-, and time-specific effects. SLG and GNP were finally chosen as in vitro biologically active or more inert graphene showed eosinophils in lavage fluid for SLG but not GNP. The subsequent 28-day inhalation study (OECD 412) confirmed a toxic, genotoxic and pro-inflammatory potential for SLG at 3.2 mg/m3 with an in vivo-ranking of lung toxicity: SLG > GNP > Printex 90®. The in vivo ranking finally pointed to AM (lactate dehydrogenase release, DNA damage) as the most predictive in vitro model for the (geno)toxicity screening of graphene nanoplates.