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Abstract(s)
The toxicity of NMs in submerged conditions with the alveolar cell line A549 was finalized, concerning the four selected nanomaterials (NMs): TiO2 (NM-100 and NM-1001), CeO2 (NM-2102) and BaSO4 (NM-220). Cytotoxicity was analysed using the MTT assay and the colony forming efficiency assay (CFE or clonogenic assay). The alkaline comet plus the FPG-modified assays and the cytokynesis-blocked micronucleus assay were used to address the genotoxic effects. The tests were performed with A549 cells cultured in conventional submerged cultures. Following the recommendation of the guidance document, the quality of the NM batch dispersion, as well as that of the NM dilution in the complete cells exposure medium, were analyzed using dynamic light scattering (DLS) performed shortly after dispersion and after incubation during 24h, 72h and 7 days. Most of the batch dispersions were in similar size range of the NANoREG benchmark data. In general, the dilution of the batch in cell culture medium showed no major impact on NM sizes and, after 24h incubation, sedimentation of the NMs probably contributed to the observation of lower size dispersed NMs when analyzing the suspension by DLS.
The CFE assay with A549 cells was used to assess the cytotoxicity of a set of NMs that were agreed upon with NILU to allow data comparison between the two laboratories. Two alternative methods were used to perform the assay – exposure of cells at a high desnsity to the NM (24h) or exposure of a small number of adherent cells to the NM during 7 days – and the following NMs were tested: silver NMs (NM-300k and NM-302) as well as zinc oxide (NM-110), amorphous silicon dioxide (NM-2000 and NM-203), a multiwalled carbon nanotube (NM-401) and titanium dioxide (NM-103).
Also, within a collaborative work with NILU aimed at characterizing the genotoxicity of three metallic NMs using regulatory tests (the hprt mutation assay, the comet assay and the micronucleus assay) results from the micronucleus assay in V79 cells were obtained for NM-100, NM-302 and NM-2102. All results were analyzed using adequate statistical tools.
All data have been uploaded into the NanoReg Database entry tool with the exception of CFE for which no model database has been produced. The later data will be sent to the task leaders in order to be uploaded at CIRCABC.
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Keywords
Genotoxicidade Ambiental Nanotoxicologia