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Paget V.,CEA Fontenay-aux-roses | Moche H.,Institute Pasteur Of Lille | Moche H.,Servier Group | Moche H.,Lille 2 University of Health and Law | And 6 more authors.
Toxicological Sciences | Year: 2015

Although tungsten carbide-cobalt (WC-Co) nanoparticles (NPs) have been widely used because of their robustness, their risk to human health remains poorly studied, despite the International Agency for Research on Cancer (IARC) classifying them as "probably carcinogenic" for humans (Group 2A) in 2006. Our current study aimed at defining the cytotoxicity and genotoxicity of one set of commercially available 60-nm diameter WC-Co NPs on three human cell lines representative of potential target organs: A549 (lung), Hep3B (liver), and Caki-1 (kidney). The cytotoxicity of WC-Co NPs was determined by evaluating cell impedance (xCELLigence), cell survival/death, and cell cycle checkpoints. Flow cytometry was used to not only evaluate cell cycle checkpoints, but to also estimate reactive oxygen species (ROS) generation. In addition, γ-H2Ax foci detection (confocal microscopy), considered to be the most sensitive technique for studying DNA double-strand breaks, was utilized to evaluate genotoxicity. As a final part of this study, we assessed the cellular incorporation of WC-Co NPs, first byflow cytometry (side scatter), and then by confocal microscopy (light reflection) to ensure that the NPs had entered cells. Overall, our current findings demonstrate that WC-Co NPs induce cell mortality, DNA double-strand breaks, and cell cycle arrest in human renal (Caki-1) and liver (Hep3B) cell lines, but do not induce significant cytotoxic effects in A549 lung cells. Interestingly, although WC-Co NPs effectively entered the cells in all 3 lines tested, ROS were detected in Caki-1 and Hep3B, but not in A549. This may explain the great differences in the cytotoxic and genotoxic effects we observed between these lines. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

Dearfield K.L.,U.S. Department of Agriculture | Thybaud V.,Sanofi S.A. | Cimino M.C.,U.S. Environmental Protection Agency | Custer L.,Bristol Myers Squibb | And 16 more authors.
Environmental and Molecular Mutagenesis | Year: 2011

Appropriate follow-up actions and decisions are needed when evaluating and interpreting clear positive results obtained in the in vitro assays used in the initial genotoxicity screening battery (i.e., the battery of tests generally required by regulatory authorities) to assist in overall risk-based decision making concerning the potential effects of human exposure to the agent under test. Over the past few years, the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Project Committee on the Relevance and Follow-up of Positive Results in In Vitro Genetic Toxicity (IVGT) Testing developed a decision process flow chart to be applied in case of clear positive results in vitro. It provides for a variety of different possibilities and allows flexibility in choosing follow-up action(s), depending on the results obtained in the initial battery of assays and available information. The intent of the Review Subgroup was not to provide a prescriptive testing strategy, but rather to reinforce the concept of weighing the totality of the evidence. The Review Subgroup of the IVGT committee highlighted the importance of properly analyzing the existing data, and considering potential confounding factors (e.g., possible interactions with the test systems, presence of impurities, irrelevant metabolism), and chemical modes of action when analyzing and interpreting positive results in the in vitro genotoxicity assays and determining appropriate follow-up testing. The Review Subgroup also examined the characteristics, strengths, and limitations of each of the existing in vitro and in vivo genotoxicity assays to determine their usefulness in any follow-up testing. © 2010 Wiley-Liss, Inc.

Lorge E.,Servier Group
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2010

The reference genotoxic agents mitomycin C, cadmium chloride, 2-aminoanthracene, vinblastine sulphate and 5-fluorouracil were tested in the in vitro micronucleus assay, in mouse lymphoma L5178Y cells and in human lymphoblastoid cells TK6, without cytokinesis block. This was done in support of the toxicity measures recommended in the late 2007 version of the draft OECD Test Guideline 487 for the testing of chemicals.Relative Population Doubling and Relative Increase in Cell Counts, used for the selection of the highest concentrations to be evaluated for genotoxicity assessment, based on a 50 ± 5% cytotoxicity, both allowed to equally detect positive mitomycin C, cadmium chloride, 2-aminoanthracene, vinblastine sulphate and 5-fluorouracil on L5178Y and/or TK6 cells. Therefore, these parameters, recommended in the draft Test Guideline 487, are suitable to select the concentrations at the cytotoxicity required for genotoxicity assessment in the in vitro micronucleus assay without cytokinesis block. © 2010 Elsevier B.V.

Lorge E.,Servier Group | Hayashi M.,NIHS | Albertini S.,F.Hoffmann LaRoche AG | Kirkland D.,Covance
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2010

A decrease in the cytokinesis-block proliferation index (CBPI) or replication index (RI) is routinely used to determine cytotoxicity of a test compound and therefore the choice of its appropriate test concentration for the . in vitro micronucleus (MN) test conducted in the presence of cytochalasin B. As a number of laboratories prefer to conduct the . in vitro MN test in the absence of cytochalasin B, it is important that selected test concentrations, based on cytotoxicity, should be similar to what they would have been if cytochalasin B had been used, and should be relevant of a true cytotoxicity. By using models to analyse the dynamics of the cell cultures with and without cytochalasin B we have compared different methods for evaluation of cytotoxicity, and demonstrate that relative decrease in population doubling or relative increase in cell counts are the most appropriate measures of cytotoxicity to compare with reduction in CBPI or RI. © 2008 Elsevier B.V..

Galloway S.,Merck And Co. | Lorge E.,Servier Group | Aardema M.J.,Procter and Gamble | Aardema M.J.,Marilyn Aardema Consulting LLC | And 10 more authors.
Mutation Research - Genetic Toxicology and Environmental Mutagenesis | Year: 2011

The selection of maximum concentrations for in vitro mammalian cell genotoxicity assays was reviewed at the 5th International Workshop on Genotoxicity Testing (IWGT), 2009. Currently, the top concentration recommended when toxicity is not limiting is 10. mM or 5. mg/ml, whichever is lower. The discussion was whether to reduce the limit, and if so whether the 1. mM limit proposed for human pharmaceuticals was appropriate for testing other chemicals. The consensus was that there was reason to consider reducing the 10. mM limit, and many, but not all, attendees favored a reduction to 1. mM. Several proposals are described here for the concentration limit. The in vitro cytogenetics expert working group also discussed appropriate measures and level of cytotoxicity. Data were reviewed from a multi-laboratory trial of the in vitro micronucleus (MN) assay with multiple cell types and several types of toxicity measurements. The group agreed on a preference for toxicity measures that take cell proliferation after the beginning of treatment into account (relative increase in cell counts, relative population doubling, cytokinesis block proliferation index or replicative index), and that this applies both to in vitro MN assays and to in vitro chromosome aberration assays. Since relative cell counts (RCC) underestimate toxicity, many group members favored making a recommendation against the use of RCC as a toxicity measure for concentration selection. All 14 chemicals assayed for MN induction in the multi-laboratory trial were detected without exceeding 50% toxicity by any measure, but some were positive only at concentrations with toxicity quite close to 50%. The expert working group agreed to accept the cytotoxicity range recommended by OECD guideline 487 (55 ± 5% toxicity at the top concentration scored). This also reinforces the original intent of the guidance for the in vitro chromosome aberration assay, where ">50%" was intended to target the range close to 50% toxicity. © 2011 Elsevier B.V.

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