title: Pan-European inter-laboratory studies on a panel of in vitro cytotoxicity and pro-inflammation assays for nanoparticles.
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.
authors.affiliation: Research Unit in Cellular Biology (URBC), Namur Nanosafety Center (NNC), NAmur Research Institute for Life Sciences (NARILIS), University of Namur (UNamur), Namur, Belgium. jean-pascal.piret@unamur.be.
authors.forename: Jean-Pascal
authors.initials: JP
authors.lastname: Piret
authors.affiliation: Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics (NICPB), Tallinn, Estonia.
authors.forename: Olesja M
authors.initials: OM
authors.lastname: Bondarenko
authors.affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Matthew S P
authors.initials: MSP
authors.lastname: Boyles
authors.affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Himly
authors.affiliation: Directory of Life Sciences Applied Metrology, National Institute of Metrology, Quality and Technology, Rio de Janeiro, Brazil.
authors.forename: Ana R
authors.initials: AR
authors.lastname: Ribeiro
authors.affiliation: ECSIN-European Center for the Sustainable Impact of Nanotechnology, Veneto Nanotech S.C.p.A., Rovigo, Italy.|ECSIN-European Center for the Sustainable Impact of Nanotechnology, ECAMRICERT srl, Rovigo, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Benetti
authors.affiliation: Straticell Screening Technologies, S.A., Les Isnes, Belgium.
authors.forename: Caroline
authors.initials: C
authors.lastname: Smal
authors.affiliation: Department of Biochemistry and Immunology, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
authors.forename: Braulio
authors.initials: B
authors.lastname: Lima
authors.affiliation: Fraunhofer-Institut für Keramische Technologien und Systeme (IKTS), Dresden, Germany.
authors.forename: Annegret
authors.initials: A
authors.lastname: Potthoff
authors.affiliation: Laboratory of Nanobiotechnology, National Institute for Research and Development in Microtechnologies - IMT Bucharest, Bucharest, Romania.
authors.forename: Monica
authors.initials: M
authors.lastname: Simion
authors.affiliation: Research Unit in Cellular Biology (URBC), Namur Nanosafety Center (NNC), NAmur Research Institute for Life Sciences (NARILIS), University of Namur (UNamur), Namur, Belgium.
authors.forename: Elise
authors.initials: E
authors.lastname: Dumortier
authors.affiliation: Directory of Life Sciences Applied Metrology, National Institute of Metrology, Quality and Technology, Rio de Janeiro, Brazil.
authors.forename: Paulo Emilio C
authors.initials: PEC
authors.lastname: Leite
authors.affiliation: Directory of Life Sciences Applied Metrology, National Institute of Metrology, Quality and Technology, Rio de Janeiro, Brazil.
authors.forename: Luciene Bottentuit
authors.initials: LB
authors.lastname: Balottin
authors.affiliation: Directory of Life Sciences Applied Metrology, National Institute of Metrology, Quality and Technology, Rio de Janeiro, Brazil.
authors.forename: José Mauro
authors.initials: JM
authors.lastname: Granjeiro
authors.affiliation: Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics (NICPB), Tallinn, Estonia.|Future Industries Institute, University of South Australia, Mawson Lakes, Australia.
authors.forename: Angela
authors.initials: A
authors.lastname: Ivask
authors.affiliation: Laboratory of Environmental Toxicology, National Institute of Chemical Physics and Biophysics (NICPB), Tallinn, Estonia.
authors.forename: Anne
authors.initials: A
authors.lastname: Kahru
authors.affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Isabella
authors.initials: I
authors.lastname: Radauer-Preiml
authors.affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Tischler
authors.affiliation: Division of Allergy and Immunology, Department of Molecular Biology, University of Salzburg, Salzburg, Austria.
authors.forename: Albert
authors.initials: A
authors.lastname: Duschl
authors.affiliation: Research Unit in Cellular Biology (URBC), Namur Nanosafety Center (NNC), NAmur Research Institute for Life Sciences (NARILIS), University of Namur (UNamur), Namur, Belgium.
authors.forename: Christelle
authors.initials: C
authors.lastname: Saout
authors.affiliation: Centre for BioNano Interactions, University College Dublin, Belfield, Dublin, 4, Ireland.
authors.forename: Sergio
authors.initials: S
authors.lastname: Anguissola
authors.affiliation: Department of Chemical and Product Safety, German Federal Institute for Risk Assessment (BfR), Berlin, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Haase
authors.affiliation: Environmental Risk and Health Unit, Flemish Institute for Technological Research (VITO), Mol, Belgium.
authors.forename: An
authors.initials: A
authors.lastname: Jacobs
authors.affiliation: Environmental Risk and Health Unit, Flemish Institute for Technological Research (VITO), Mol, Belgium.
authors.forename: Inge
authors.initials: I
authors.lastname: Nelissen
authors.affiliation: Materials Science & Engineering, Indian Institute of Technology Gandhinagar, Gandhinagar, India.
authors.forename: Superb K
authors.initials: SK
authors.lastname: Misra
authors.affiliation: Research Unit in Cellular Biology (URBC), Namur Nanosafety Center (NNC), NAmur Research Institute for Life Sciences (NARILIS), University of Namur (UNamur), Namur, Belgium. Olivier.toussaint@unamur.be.
authors.forename: Olivier
authors.initials: O
authors.lastname: Toussaint
mesh_terms: D001681:Biological Assay / Q000379:methods / Q000592:standards; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003300:Copper / Q000737:chemistry / Q000633:toxicity*; D016207:Cytokines / Q000378:metabolism*; D005060:Europe; D006801:Humans; D007753:Laboratories / Q000592:standards*; D053768:Metal Nanoparticles / Q000737:chemistry / Q000633:toxicity*; D010316:Particle Size; D015203:Reproducibility of Results; D012834:Silver / Q000737:chemistry / Q000633:toxicity*; D013499:Surface Properties; D018675:Toxicity Tests / Q000379:methods / Q000592:standards*
chemical_list: D016207:Cytokines; D012834:Silver; D003300:Copper; C030973:cupric oxide
keywords: In vitro assays; Inter-laboratory studies; Interference; Nanoparticles; Standard operating procedures