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WIREs Nanomed Nanobiotechnol
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Alternative in vitro assays in nanomaterial toxicology

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Abstract Nanomaterials are acclaimed for their novel properties, for which broad new uses are being discovered with increasing frequency. It is obvious that, as the properties change, unwanted properties (toxicity) are to be expected as well. Current toxicology, however, is already overwhelmed with the challenge of addressing new chemicals, not to mention the enormous number of old chemicals never properly assessed. Limitations of traditional approaches range from animal welfare issues, which were a strong driving force for alternative approaches (the 3Rs concept) over the last two decades, to aspects of throughput and accuracy of the predicted toxicities. The latter has prompted discussion about a revolutionary change in chemical safety assessment, now known as Toxicology for the 21st Century (Tox‐21c). The multitude of possible formulations of nanomaterials to be assessed for novel toxic properties makes these alternative approaches especially attractive, given the well recognized limitations of traditional animal‐based approaches—limitations that might be even more pronounced for nanomaterials, which have notably altered biokinetics. WIREs Nanomed Nanobiotechnol 2011 3 545–573 DOI: 10.1002/wnan.153 This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials

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Strategy for in vitro nanotoxicology research.124

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The mechanistic approach to in vitro nanotoxicology.115

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SEM pictures of Balb/3T3 cells exposed to 100 µM Co‐nano for 4 h. Preparation by Critical Point Dryer technique (with kind permission by Dr. T. Sasaki, Unit BMS, Joint Research Centre, Ispra, Italy). We observe an agglomeration of NP between two cells and the cell membrane ‘engulfing’ the particles.115

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SEM pictures of Balb/3T3 cells exposed to 100 µM Co‐nano for 4 h. Preparation by Critical Point Dryer technique (with kind permission by Dr. T. Sasaki, Unit BMS, Joint Research Centre, Ispra, Italy). The observed degradation of the light suggests an effective penetration of NP into cells.115

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Comet assay of Balb/3T3 cells exposed for 2 h to 1 µM of cobalt NP or equimolar concentrations of Co2+ ions. (a) Positive control (300 µM of H2O2, relevant DNA damage). (b) ModerateDNA damage induced by the two cobalt compounds (left: Co NP; right: Co2+ ions).115

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The radiolabelling of solid cobalt NP by neutron activation in the nuclear reactor. Exposure of NP to a neutron flux of 1013 neutrons cm−2 s−1 (Triga Mark II reactor, University of Pavia, Italy) or of 2 × 1014 neutrons cm−2 s−1 (HFR reactor, petten, the Netherlands) led to Co [60Co]‐radiolabeled NP with different specific radioactivity to be used for uptake and intracellular distribution studies in Balb/3T3 cells.135

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Morphology of Balb/3T3 cells unexposed (control) or exposed to increasing concentrations of cobalt NP (Alexa‐488‐phalloidine and propidium iodide staining).129

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Foci type III induced by Co nanopaticles into two clones of Balb/3T3 cell line. Concurrent cytotoxicity and neoplastic morphological transformation assays on two clones of Balb/3T3 cell line exposed to cobalt NP.128 Left: clone HRI (Hatano Research Institute, Ochiai, Hadano, Kanagawa 257, Japan). Right: clone ECVAM (supplied by Laboratorio centro Substrati, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia (IZS), Brescia, Italy).

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