The dispersion method does not affect the in vitro genotoxicity of multi-walled carbon nanotubes despite inducing surface alterations

Multi-walled carbon nanotubes (MWCNTs) are a desirable class of high aspect ratio nanomaterials (HARNs) owing to their extensive applications. Given their demand, the growing occupational and consumer exposure to these materials has warranted an extensive investigation into potential hazards they may pose towards human health. This study utilised both the in vitro mammalian cell gene mutation and the cytokinesis-blocked micronucleus (CBMN) assays to investigate genotoxicity in human lymphoblastoid (TK6) and 16HBE14o^- human lung epithelial cells, following exposure to NM-400 and NM-401 MWCNTs for 24 h. To evaluate the potential for secondary genotoxicity, the CBMN assay was applied on a co-culture of 16HBE14o^- with differentiated human monocytic (dTHP-1) cells. In addition, two dispersion methods (NanoGenoTox vs. high shear mixing) were utilised prior to exposures and in acellular experiments to assess the effects on MWCNT oxidative potential, aspect ratio and surface properties. These were characterized in chemico as well as by electron microscopy and Raman spectroscopy. Structural damage of NM-400 was observed following both dispersion approaches; Raman spectra highlighted greater oxidative transformation under probe sonication as opposed to high shear mixing. Despite the changes to the oxidative potential of the MWCNTs, no statistically significant genotoxicity was observed under the conditions applied. There was also no visible signs of cellular internaliation of NM-400 or NM-401 into either cell type under the test conditions, which may support the negative genotoxic response. Whilst these HARNs may have oxidative potential, cells have natural protective mechanisms for repairing transient DNA damage. Therefore, it is crucial to evaluate biological endpoints which measure fixed DNA damage to account for the impact of DNA repair mechanisms.