title: Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay.
abstract: An important consideration in developing standards and regulations that govern the production and use of commercial nanoscale materials is the development of robust and reliable measurements to monitor the potential adverse biological effects of such products. These measurements typically require cell-based and other biological assays that provide an assessment of the risks associated with the nanomaterial of interest. In this perspective, we describe the use of cause-and-effect (C&E) analysis to design robust, high quality cell-based assays to test nanoparticle-related cytotoxicity. C&E analysis of an assay system identifies the sources of variability that influence the test result. These sources can then be used to design control experiments that aid in establishing the validity of a test result. We demonstrate the application of C&E analysis to the commonly used 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay. This is the first time to our knowledge that C&E analysis has been used to characterize a cell-based toxicity assay. We propose the use of a 96-well plate layout which incorporates a range of control experiments to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance, and demonstrate the performance of the assay using the plate layout in a case study. While the plate layout was formulated specifically for the MTS assay, it is applicable to other cytotoxicity, ecotoxicity (i.e., bacteria toxicity), and nanotoxicity assays after assay-specific modifications.
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: Matthias
authors.initials: M
authors.lastname: Rösslein
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: John T
authors.initials: JT
authors.lastname: Elliott
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: Marc
authors.initials: M
authors.lastname: Salit
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: Elijah J
authors.initials: EJ
authors.lastname: Petersen
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: Cordula
authors.initials: C
authors.lastname: Hirsch
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: Harald F
authors.initials: HF
authors.lastname: Krug
authors.affiliation: Materials-Biology Interactions Laboratory, and ‡International Research Cooperations Manager, Swiss Federal Laboratories for Material Testing and Research (Empa) , CH-9014 St. Gallen, Switzerland.|Cell Systems Science Group, and ∥Genome Scale Measurements Group, National Institute of Standard and Technology , Gaithersburg, Maryland 20899, United States.
authors.forename: Peter
authors.initials: P
authors.lastname: Wick
mesh_terms: D001681:Biological Assay; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D053758:Nanoparticles / Q000633:toxicity*; D011137:Polystyrenes / Q000633:toxicity; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D018675:Toxicity Tests / Q000379:methods*
chemical_list: D011137:Polystyrenes; D013778:Tetrazolium Salts; D013844:Thiazoles; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium