Towards a harmonized testing strategy for nanofibers by integrating toxicological screening and proteomic profiling.

Nanofibers, particularly multi-walled carbon nanotubes, have attracted attention for their exceptional properties, but concerns remain about their potential health hazards due to their fiber-like morphology. Although bio-durable nanofibers may cause cancer upon inhalation, only rigid nanofibers may exhibit morphology-driven pathogenicity. Since no validated methods exist for assessing their rigidity, alternative approaches are needed that comply with the 3R principles (Replacement, Reduction, Refinement) and the European Commission efforts to foster alternatives to animal testing. This study aims to advance the development of a harmonized test method for nanofibers toxicity by comparing effects of selected carbon-based nanomaterials (NMs) with different morphologies: a nanofiber (Mitsui-7-JRCNM40011a), an elongated material (NM-400) and a particle (Printex-90). Therefore, in vitro toxicological screening and proteomic investigations were employed using differentiated THP-1 (dTHP-1) macrophage-like cells. First, we evaluated cytotoxicity and pro-inflammatory responses of the different dTHP-1 phenotypes (M0, M1 and M2) to evaluate their sensitivity, and thus selected the M0 phenotype for further oxidative and lysosomal investigations: Mitsui-7-JRCNM40011a caused, besides increased cytotoxicity and pro-inflammatory effects, oxidative stress and lysosomal dysfunction. Moreover, decreased levels of 25 lysosomal proteins, including five cathepsins, were detected. These findings deepen the understanding of nanofiber-related toxicity, supporting the development of a reliable in vitro testing strategy.