Evaluation of sources of variability in a nitric oxide screening assay for engineered nanomaterials

Assays to detect potential biocompatibility issues can play a key role in supporting the development of new technologies such as medical products containing engineered nanomaterials (ENMs). A consensus test method standard on nitric oxide production after cellular ENM exposure was developed and published through ASTM International. In this paper, we describe an evaluation of sources of variability in this method. A significant challenge is ensuring that the protocol contains the necessary control measurements to account for potential issues when testing ENMs. Protocol testing was conducted during draft standard development and post-publication to better understand potential sources of variability such as the impact of insufficient removal of the ENM, the number of cells seeded, the selection of positive control compounds, and the culture techniques of the cells prior to the experiments. Several in-process control measurements were used to monitor the performance of intermediate steps in the assay procedure. Two gold nanoparticles with different surface coatings and nano-sized polystyrene particles were used to demonstrate the applicability of some of the control measurements. This testing revealed which sources of variability were more likely to have a significant impact on the overall assay uncertainty and confirmed the key importance of certain control measurements. These results could also support the standardization of other ENM-related in vitro methods that share similarities in their protocols with the method investigated here. The further development of this method can also support its use to evaluate the potential for substances other than ENMs to induce nitric oxide production.