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Nanogo Supplemental Information

Inter-laboratory comparison of in vitro nanotoxicological assays from the NIEHS NanoGo Consortium

Tian Xia, Raymond F. Hamilton Jr, James C. Bonner, Edward D. Crandall, Alison Elder, Farnoosh Fazlollahi, Teri A. Girtsman, Kwang Kim, Somenath Mitra, Susana A. Ntim, Galya Orr, Mami Tagmount, Alexia J. Taylor, Donatello Telesca, Ana Tolic, Christopher Vulpe, Andrea Walker, Xiang Wang, Frank A. Witzmann, Nianqiang Wu, Yumei Xie, Jeffrey Zink, Andre Nel and Andrij Holian

There are conflicting data in the literature regarding engineered nanomaterial (ENM) bioactivity.  Grantees of a NIEHS-funded consortium program performed two phases of in vitro round robin tests using selected ENM to develop in vitro protocols for ENM toxicity screening that would produce reliable and reproducible data with acceptable variability between laboratories.  In addition, the studies were designed to evaluate the relative in vitro bioactivity of a selected panel of well-characterized ENM.  Protocols for ENM dispersion were established and in vitro assay protocols were used to test biological effects, including cell viability assays (MTS, LDH) and an NLRP3 inflammasome activation assay (IL-1β production) using selected ENM (ZnO, TiO2 in various forms, and multiwalled carbon nanotubes (MWCNT) in 3 forms). The tests were performed using selected mammalian cell lines (BEAS-2B, RLE-6TN, and THP-1).  The second phase (phase II) used improved protocols after overcoming several technical issues identified in phase I.  The overall in vitro toxicity profiles of ENM after the round robin tests were: ZnO was cytotoxic to all cell types, but did not induce IL-1β. TiO2 was not cytotoxic except for the nanobelt form, which was cytotoxic and induced significant IL-1β production in THP-1 cells.  MWCNT were generally not very cytotoxic, but activated low levels of IL-1β production. Statistical analysis showed significant improvement in the consistency of measurable outcomes with decreased variability between laboratories.  The protocols provide investigators with the necessary tools to conduct in vitro studies to minimize artifacts in future studies with ENM.

Off-site supplemental section below:  The Protocol section includes detailed protocols for cell growth, particle suspension techniques and assays developed and performed by the NIEHS consortium investigators.  The protocols are divided by cell type and trial number (phase I and phase II).  The Raw Data section provides detailed individual lab data for every assay performed during the consortium.  Table of contents are available at the beginning of the link.  Investigators are encouraged to utilize the information generated by the consortium investigators and to contact us with any questions.