Assessing the Toxicological Relevance of Nanomaterial Agglomerates and Aggregates Using Realistic Exposure In Vitro

Low dose repeated exposures are considered more relevant/realistic in assessing the health risks of nanomaterials (NM), as human exposure such as in workplace occurs in low doses and in a repeated manner. Thus, in a three-week study, we assessed the biological effects (cell viability, cell proliferation, oxidative stress, pro-inflammatory response, and DNA damage) of titanium-di-oxide nanoparticle (TiO₂ NP) agglomerates and synthetic amorphous silica (SAS) aggregates of different sizes in human bronchial epithelial (HBE), colon epithelial (Caco2), and human monocytic (THP-1) cell lines repeatedly exposed to a non-cytotoxic dose (0.76 µg/cm²). We noticed that neither of the two TiO₂ NPs nor their agglomeration states induced any effects (compared to control) in any of the cell lines tested while SAS aggregates induced some significant effects only in HBE cell cultures. In a second set of experiments, HBE cell cultures were exposed repeatedly to different SAS suspensions for two weeks (first and second exposure cycle) and allowed to recover (without SAS exposure, recovery period) for a week. We observed that SAS aggregates of larger sizes (size ~2.5 µm) significantly affected the cell proliferation, IL-6, IL-8, and total glutathione at the end of both exposure cycle while their nanosized counterparts (size less than 100 nm) induced more pronounced effects only at the end of the first exposure cycle. As noticed in our previous short-term (24 h) exposure study, large aggregates of SAS did appear to be similarly potent as nano sized aggregates. This study also suggests that aggregates of SAS of size greater than 100 nm are toxicologically relevant and should be considered in risk assessment.