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 (R) (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/m(3) with an in vivo-ranking of lung toxicity: SLG > GNP > Printex 90 (R). 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.