92 In Vitro Cytokinesis Block Micronucleus (CBMN) Assay to Evaluate the Genotoxicity of Multicomponent Nanomaterials – a Tiered Testing Approach

Abstract

Abstract The development of nanomaterials has seen exponential growth in the last two decades and, more recently, of complex multicomponent nanomaterials (MCNM), which represent challenges for human hazard testing. This study aims to determine exposure-dose-response relationships and the potential human hazard of industrially relevant MCNMs with a tiered testing approach. Tier1 is a simple in vitro assay, while Tier2 is a more complex in vitro model suitable for mimicking inhalation exposure. The intent was to explore the differences between the complexity of the assays and the genotoxicity response. The cytotoxic and genotoxic potential of selected MCNMs were evaluated with an OECD-approved in vitro Tier1 testing approach with nano-specific modifications. Chromosomal breakage (genotoxicity) was measured with the in vitro cytokinesis-blocked micronucleus (CBMN) assay alongside relative population doubling (RPD) to assess cytotoxicity. For Tier1 testing, the CBMN assay was performed on human lymphoblastoid (TK6) cells; a co-culture of lung epithelial cells (A549) and monocytes (d-THP-1) was utilised for Tier2 genotoxicity testing. MCNMs from four industrially-relevant case studies were analysed. Some materials demonstrated differences in the dose-response relationship between the core components and the MCNM when using Tier1 in vitro tests, suggesting a synergistic effect when the materials were combined. These results are compared across the tier testing system to determine if advanced culture models can provide more physiologically relevant data as compared to standard in vitro systems. This research has received funding from the European Union’s Horizon 2020 research and innovation programme for the SUNSHINE project under grant agreement No 952924.