(338d) Assessing Organomodified Nanoclay Pulmonary Toxicity across Its Life Cycle Using Integrated in Vitro / In Vivo Approaches

Incorporation of organomodified nanoclays (ONC), montmorillonite coated with different quaternary ammonium compounds, into different types of organic polymers continues to fuel expansion of nanoclay composite technologies. Based on projected widespread use in numerous industrial sectors and commercial products, recent forecasts suggest increased airborne occupational exposures are likely to occur; however, little is known about ONC pulmonary health risks along its life cycle. Here, we characterized physicochemical properties of several different ONCs across their life cycle including as-produced, breakdown products during nanoclay composite machining, and incinerated by-products. We used an integrated in vitro / in vivo approach to assess pulmonary toxicity following ONC exposure to compare human macrophage, epithelial, and fibroblast cell model responses to animal model responses to identify key cellular events to help understand mode of action and potential adverse outcomes. By using high-throughput in vitro techniques, including multiplex fluorescent high content screening and electrical cell impedance sensing, pristine uncoated nanoclay (UC) exposure elicited increased membrane damage, mitochondrial dysfunction, and apoptosis in epithelial cells while pre-incinerated ONCs (CC) primarily exhibited an apoptotic and necrotic cell death resulting in a loss in epithelial monolayer integrity. Next, UC caused inflammasome activation, cell membrane damage, and apoptosis initiation in macrophages while CC caused large-scale macrophage necrosis with no indication of inflammasome activation. Incinerated ONC byproduct exposure caused acute toxicity, loss of epithelial monolayer integrity, and inflammation activation in macrophages only at high doses. Both UC and CC exposure to fibroblasts indicated their pro-fibrotic potential with increased proliferation, collagen, and reticular fiber deposition. In vivo exposure in an animal model indicated that pre- and post-incinerated ONCs caused a low grade, persistent inflammation with pro-fibrotic signaling at Day 28 post-exposure, which correlated with key events identified in the in vitro models. In summary, presence of organic coating and incineration status influences potential mode of action of the pulmonary response. The ONC coating protected against an acute silica-associated inflammatory response, but caused membrane damage, low grade inflammation, and a stimulation of extracellular matrix proteins associated with fibrosis. Current research efforts include composite synthesis, generation, and characterization of nanoclay-enabled composite dusts following controlled machining experiments to understand physicochemical characteristics of released particles and how their exposure affects our high-throughput in vitro screening models. Linking physicochemical properties to adverse effects in both in vitro and in vivo models allows for rapid assessment and information to inform and improve prevention-by-design strategies.