62 Application of simple and robust <i>in vitro</i> pulmonary models to evaluate the safety of carbon-based lubricants

Abstract In vitro pulmonary models are widely used for safety evaluations, simulating human lung responses to inhaled particles or chemicals. These models help detect toxicity early, reducing the need for animal testing. In this study, we used two models: the Alveolar Macrophage Assay (AMA), a simple model, and the tissue model MucilAir™, a more robust system, to evaluate the safety of commercial carbon-based spray lubricants. We also characterized the lubricants to identify their physico-chemical properties but encountered difficulties due to interference from the silicone-based lubricant matrix. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) analysis could not detect carbon-based materials, likely due to the presence of polysiloxanes. Confocal Raman analysis revealed graphite, with minor contributions from graphene, which was consistent with Scanning Electron Microscopy (SEM) findings showing graphite-like particles. Exposure assessment revealed that spray droplets approximately 2.20 µm in size are released during application, suggesting inhalation as a potential exposure route. Hazard assessment using AMA was challenging; direct spray administration onto MucilAir™ caused cell damage or death. The submerged approach with macrophages led to density-driven separation of compounds, but a black material was partially incorporated into cells, triggering a dose-dependent release of lytic enzymes. Despite challenges in characterising and testing graphene-based lubricants, MucilAir™ was shown to be a sensitive model for assessing the safety of such products. A tailored approach, considering specific properties of lubricants, is critical for accurate hazard assessment. The MACRAME project was funded by the EU Horizon Europe (GA no 101092686), Swiss SERI (no 23.00141), and UKRI Innovate (no 10066165).