Assay conditions can influence the outcome of cytotoxicity tests of nanomaterials: Better assay characterization is needed to compare studies

Background: Reliable in vitro studies that generate consistent toxicity data on nanomaterials on a high-throughput scale will be of invaluable significance in the next few years. Aim: In this study, we checked the influence of several steps of the experimental design on the outcome: we investigated the role of cell density, viability assay and particle dispersion method, including the influence of serum and effect of a surfactant (Tween 80). Methods: The dose-response curve was assessed for ground multiwalled carbon nanotubes (CNT) and the silica benchmark Min-U-Sil, on lung epithelial cells (A549 cells) and macrophages (stimulated THP-1 cells). Results: The cell density used in the cytotoxicity study has an impact on the outcome: for the same concentration of Min-U-Sil, the viability of A549 cells varied from 10% to 55% with increasing cell density. Whereas foetal calf serum attenuated the cytotoxicity of Min-U-Sil, this effect was not seen for CNT. The results show how cell culture conditions can modify the outcome of a toxicological experiment, as shown in this study for Tween 80 to disperse the test agent. Conclusions: These experiments illustrates that results reported in literature can only be compared when, in addition to the use of a benchmark particle, a detailed method description is available. Therefore, more emphasis is needed on a standardized design for cytotoxicity studies. (C) 2009 Elsevier Ltd. All rights reserved.