The quality and relevance of nanosafety studies constitute major challenges to ensure their key role as a supporting tool in sustainable innovation, and subsequent competitive economic advantage. However, the number of apparently contradictory and inconclusive research results has increased in the past few years, indicating the need to introduce harmonized protocols and good practices in the nanosafety research community. Therefore, we aimed to evaluate if best-practice training and inter-laboratory comparison (ILC) of performance of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay for the cytotoxicity assessment of nanomaterials among 15 European laboratories can improve quality in nanosafety testing. We used two well-described model nanoparticles, 40-nm carboxylated polystyrene (PS-COOH) and 50-nm amino-modified polystyrene (PS-NH2). We followed a tiered approach using well-developed standard operating procedures (SOPs) and sharing the same cells, serum and nanoparticles. We started with determination of the cell growth rate (tier 1), followed by a method transfer phase, in which all laboratories performed the first ILC on the MTS assay (tier 2). Based on the outcome of tier 2 and a survey of laboratory practices, specific training was organized, and the MTS assay SOP was refined. This led to largely improved intra- and inter-laboratory reproducibility in tier 3. In addition, we confirmed that PS-COOH and PS-NH2 are suitable negative and positive control nanoparticles, respectively, to evaluate impact of nanomaterials on cell viability using the MTS assay. Overall, we have demonstrated that the tiered process followed here, with the use of SOPs and representative control nanomaterials, is necessary and makes it possible to achieve good inter-laboratory reproducibility, and therefore high-quality nanotoxicological data.

Biomedical Research Center University of Vigo Campus Lagoas Marcosende 36310 Vigo Spain

Centre for BioNano Interactions University College Dublin Belfield Dublin 4 Ireland

Charles River Laboratories Carrowntreila Ballina Co Mayo Ireland

Department of Chemical and Life Science Engineering Virginia Commonwealth University Richmond VA 23284 USA

Department of Chemicals and Product Safety German Federal Institute for Risk Assessment Max Dohrn Strasse 8 10 10589 Berlin Germany

Department of Toxicology and Carcinogenesis Nofer Institute of Occupational Medicine 91 348 Łódź Poland

ECAMRICERT SRL European Center for the Sustainable Impact of Nanotechnology Corso Stati Uniti 4 35127 Padova Italy

Environmental Research and Innovation 41 rue du Brill L 4422 Belvaux Luxembourg

Experimental Toxicology Unit Institut National de l'Environnement Industriel et des Risques Parc Alata BP2 60550 Verneuil en Halatte France

Faculty of Physics and Center for NanoScience Ludwig Maximilians Universität Geshwister Scholl Platz 1 80539 Munich Germany

Faculty of Safety Engineering VSB Technical University of Ostrava Lumirova 13 70030 Ostrava Vyskovice Czech Republic

Gentian Diagnostics AS Bjørnåsveien 5 1596 Moss Norway

Groningen Research Institute of Pharmacy University of Groningen A Deusinglaan 1 9713AV Groningen The Netherlands

Health Department Flemish Institute for Technological Research Boeretang 200 2400 Mol Belgium

Health Effects Laboratory Department of Environmental Chemistry Norwegian Institute for Air Research Instituttveien 18 2007 Kjeller Norway

Institute for Work and Health University of Lausanne and University of Geneva Route de la Corniche 2 1066 Epalinges Lausanne Switzerland

Institute of Experimental Medicine Czech Academy of Sciences Videnska 1083 14220 Prague 4 Czech Republic

Institute of Toxicology and Genetics Karlsruhe Institute of Technology Hermann von Helmholtz Platz 1 76344 Eggenstein Leopoldshafen Germany

Instituto de Investigación Sanitaria Galicia Sur Hospital Álvaro Cunqueiro Estrada Clara Campoamor 341 Babio Beade 36312 Vigo Spain

Nanoimmunotech SL Edificio CITEXVI Fonte das Abelleiras s n Campus Universitario de Vigo 36310 Vigo Pontevedra Spain

National Center for Drug Research and Evaluation and National Center of Innovative Technologies for Public Health Istituto Superiore di Sanità Viale Regina Elena 299 Rome Italy

Research Unit in Cellular Biology rue de Bruxelles 61 5000 Namur Belgium

School of Life Sciences Heriot Watt University Riccarton Campus Edinburgh EH14 4AS UK

School of Materials Science and Engineering Nanyang Technological University Block N4 1 Nanyang Avenue Singapore 639798 Singapore

Science Foundation Ireland Three Park Place Hatch Street Upper Dublin 2 Ireland

SEQme s r o Dlouha 176 26301 Dobris Czech Republic

Swiss Centre for Occupational and Environmental Health Binzhofstrasse 87 8404 Winterthur Switzerland

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