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.

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

“…We also evaluated the effect of using serum-free in comparison to serum-containing medium during the incubation of ENM as serum may affect the response of cells to ENM. Figure 1 illustrates the serum and serum-free protocols (Protocols S1 1 , S2 1 ) that were derived from the manufacturer's instructions and from the results of a cause-and-effect analysis of the MTS assay (Rösslein et al, 2014). The protocols include 8 system controls to quantify critical sources of variability in the assay (Fig.…”

A guide to nanosafety testing: Considerations on cytotoxicity testing in different cell models

“…Several consortia have already worked on evaluation and harmonization of cytotoxicity assays for NPs (Elliott Piret et al 2017;Rösslein et al 2015;Xia et al 2013). Moreover, a standard assay specifically dedicated to NPs is currently in preparation using the MTT variant 3-(4,5-…”

Emerging metrology for high-throughput nanomaterial genotoxicology

“…Perhaps more readily available would be ENM reference materials (with well-defined and consistent physicochemical properties) that have been validated for use as positive or negative controls in multiple toxicity assays; such materials are already being pursued by the research community (153). Validation of such materials is not trivial and would require rigorous comparative studies using robust assays that have been examined for ENM interference (10,11). Improved nanotoxicity assays will greatly benefit all realms of nanotechnology, one in particular being precision nanomedicine.…”

“…Unfortunately, this does not work seamlessly and often the resulting toxicity mechanism is obscured or the resulting data are flawed by artefacts due to ENM interference with components in the assay or the assay readout (9,10). However, one excellent example demonstrating the steps required for properly modifying a biological cytotoxicity assay for use with ENMs was recently reported (11). In this work, the authors described a successful approach for optimizing and modifying the control parameters of the highly utilised 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell-viability assay to make it suitable for testing the cytotoxicity of ENMs in a 96-well plate format.…”