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Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy

Zhu, Donghui; Farcal, Lucian; Torres And�n, Fernando; Di Cristo, Luisana; Rotoli, Bianca Maria; Bussolati, Ovidio; Bergamaschi, Enrico; Mech, Agnieszka; Hartmann, Nanna B.; Rasmussen, Kirsten; Riego-Sintes, Juan; Ponti, Jessica; Kinsner-Ovaskainen, Agnieszka; Rossi, Fran�ois; Oomen, Agnes; Bos, Peter; Chen, Rui; Bai, Ru; Chen, Chunying; Rocks, Louise; Fulton, Norma; Ross, Bryony; Hutchison, Gary; Tran, Lang; Mues, Sarah; Ossig, Rainer; Schnekenburger, J�rgen; Campagnolo, Luisa; Vecchione, Lucia; Pietroiusti, Antonio; Fadeel, Bengt


Donghui Zhu

Lucian Farcal

Fernando Torres And�n

Luisana Di Cristo

Bianca Maria Rotoli

Ovidio Bussolati

Enrico Bergamaschi

Agnieszka Mech

Nanna B. Hartmann

Kirsten Rasmussen

Juan Riego-Sintes

Jessica Ponti

Agnieszka Kinsner-Ovaskainen

Fran�ois Rossi

Agnes Oomen

Peter Bos

Rui Chen

Ru Bai

Chunying Chen

Louise Rocks

Norma Fulton

Bryony Ross

Lang Tran

Sarah Mues

Rainer Ossig

J�rgen Schnekenburger

Luisa Campagnolo

Lucia Vecchione

Antonio Pietroiusti

Bengt Fadeel


Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry – hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO – uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques – precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially ‘weak-embryotoxic’ and ZnO and SiO2 NMs as ‘non-embryotoxic’. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.


Zhu, D., Farcal, L., Torres Andón, F., Di Cristo, L., Rotoli, B. M., Bussolati, O., …Fadeel, B. (2015). Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy. PLOS ONE, 10, Article e0127174.

Journal Article Type Article
Acceptance Date Apr 13, 2015
Online Publication Date May 21, 2015
Publication Date May 21, 2015
Deposit Date Jan 14, 2016
Publicly Available Date Aug 26, 2019
Print ISSN 1932-6203
Electronic ISSN 1932-6203
Publisher Public Library of Science
Peer Reviewed Peer Reviewed
Volume 10
Article Number e0127174
Keywords Nanomaterials; in vitro test methods; toxicity assessment;
Public URL
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Comprehensive In Vitro Toxicity Testing of a Panel of Representative Oxide Nanomaterials: First Steps towards an Intelligent Testing Strategy (3.1 Mb)

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Copyright Statement
© 2015 Farcal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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