Toward achieving harmonization in a nanocytotoxicity assay measurement through an interlaboratory comparison study

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John T. Elliott
Matthias Rösslein
Nam Woong Song
Blaza Toman
Agnieszka Kinsner- Ovaskainen
Rawiwan Maniratanachote
Marc L. Salit
Elijah J. Petersen
Fatima Sequeira
Erica Romsos
Soo Jin Kim
Jieun Lee
Nadia R. von Moos
François Rossi
Cordula Hirsch
Harald F. Krug
Wongsakorn Suchaoin
Peter Wick


Development of reliable cell-based nanotoxicology assays is important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nanocytotoxicity MTS cell viability assay based on the A549 cell line. The consensus EC50 values were 22.1 mg/L (95% confidence intervals 16.9 mg/L to 27.2 mg/L) and 52.6 mg/L (44.1 mg/L to 62.6 mg/L) for positively charged polystyrene nanoparticles for the serum-free and serum conditions, respectively, and 49.7 μmol/L (47.5 μmol/L to 51.5 μmol/L) and 77.0 μmol/L (54.3 μmol/L to 99.4 μmol/L) for positive chemical control cadmium sulfate for the serum-free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nanocytotoxicity assay results. The combi­nation of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

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Elliott, J. T., Rösslein, M., Song, N. W., Toman, B., Kinsner- Ovaskainen, A., Maniratanachote, R., Salit, M. L., Petersen, E. J., Sequeira, F., Romsos, E., Kim, S. J., Lee, J., von Moos, N. R., Rossi, F., Hirsch, C., Krug, H. F., Suchaoin, W. and Wick, P. (2017) “Toward achieving harmonization in a nanocytotoxicity assay measurement through an interlaboratory comparison study”, ALTEX - Alternatives to animal experimentation, 34(2), pp. 201–218. doi: 10.14573/altex.1605021.

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