Abstract
The rapid development of nanotechnologies and increased production and use of nanomaterials raise concerns about their potential toxic effects for human health and environment. To evaluate the biological effects of nanomaterials, a set of reliable and reproducible methods and development of standard operating procedures (SOPs) is required. In the framework of the European FP7 NanoValid project, three different cell viability assays (MTS, ATP content, and caspase-3/7 activity) with different readouts (absorbance, luminescence and fluorescence) and two immune assays (ELISA of pro-inflammatory cytokines IL1-β and TNF-α) were evaluated by inter-laboratory comparison. The aim was to determine the suitability and reliability of these assays for nanosafety assessment. Studies on silver and copper oxide nanoparticles (NPs) were performed, and SOPs for particle handling, cell culture, and in vitro assays were established or adapted. These SOPs give precise descriptions of assay procedures, cell culture/seeding conditions, NPs/positive control preparation and dilutions, experimental well plate preparation, and evaluation of NPs interference. The following conclusions can be highlighted from the pan-European inter-laboratory studies: Testing of NPs interference with the toxicity assays should always be conducted. Interference tests should be designed as close as possible to the cell exposure conditions. ATP and MTS assays gave consistent toxicity results with low inter-laboratory variability using Ag and CuO NPs and different cell lines and therefore, could be recommended for further validation and standardization. High inter-laboratory variability was observed for Caspase 3/7 assay and ELISA for IL1-β and TNF-α measurements.
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Acknowledgements
This research was funded by the European Community’s Seventh Framework Programme (FP7/2007-2013) under Grant Agreement No. 263147 (NanoValid Project—development of reference methods for hazard identification, risk assessment and LCA of engineered nanomaterials). NICPB authors were also supported from Institutional funding IUT 23-5. Authors would like to thank the project QualityNano INFRASTRUCTURE-2010-1.1.31-262163 for kindly sharing protocols for the A549 cell culture and MTS-ATP assays. O. Toussaint is a senior Research Associate of the F.N.R.S., Belgium. O. Toussaint thanks European Commission and the DG06 (Direction générale opérationnelle de l’Economie, de l’Emploi & de la Recherche) of the Walloon Region of Belgium for the Large-scale Integrating Collaborative project “Nanovalid” (#NMP-FP7-2010-1.3-1) and QualityNano INFRASTRUCTURE-2010-1.1.31-262163 project. J.-P. Piret is senior post-doc for these projects. Ana R. Ribeiro and Paulo Emilio C. Leite acknowledge INMETRO scholarship Pronametro Nos. 52600.017263/2013 and 052375/2012-31, respectively.
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Piret, JP., Bondarenko, O.M., Boyles, M.S.P. et al. Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles. Arch Toxicol 91, 2315–2330 (2017). https://doi.org/10.1007/s00204-016-1897-2
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DOI: https://doi.org/10.1007/s00204-016-1897-2