Challenges with toxicity testing nanomaterials: in vitro assay interference
In vitro toxicology is an essential tool to screen and study the toxicity of nanomaterials. However, some properties of nanomaterials can hinder the outcome of an experiment through assay interference, leading to a risk of false positive or false negative results. The potential for assay interference needs to be understood and addressed in toxicology studies.
Recent research has shown that…
- a good knowledge of the physico-chemical characteristics of the nanomaterials being tested is essential;
- interactions between the nanomaterials and the assay systems should be systematically analysed;
- multiple assays need to be used for each end-point (e.g. cytotoxicity, genotoxicity, inflammation) studied.
Kroll et al. (2009) published a review about the challenges associated with toxicological in vitro testing of nanomaterials. The authors described many assays classically used for in vitro testing and reported cases of interference related to nanomaterials properties such as a high surface area, reactivity, and optical properties. More recently, Kroll et al. (2011) illustrated the importance of testing for interference, by checking for adsorption of analyte, optical interference with the reading system used, and reactivity with assay reagents. They also demonstrated that by adapting some protocols for nanomaterials testing, for example by adding washing steps, it was possible to reduce interference issues.
A specific example can be seen in the work of Han et al. (2011) who published a study about the use of the lactate dehydrogenase assay (LDH) for cytotoxicity, and its validity with nanomaterials. LDH is a protein located in the cells that is released in the cell culture medium when the cell membrane is damaged, for example through cell death. In order to limit effects of interference, a kinetic assay was used to measure LDH activity. Moreover, the ability of nanomaterials to adsorb LDH, or to inhibit its activity or to chemically react with some of the components of the assay were all rigorously tested. It was shown that metallic copper nanoparticles could inhibit LDH activity, thought likely due to the release of copper ions, and that TiO2 nanoparticles at high concentration could adsorb LDH. Therefore, despite this reduced LDH activity measured, the authors could show that in some cases it could be unrelated to cytotoxicity and therefore lead to valid interpretation of results.
What does this mean?
Whilst assay interference has been known for many years, some specific properties of nanomaterials (e.g. high surface area, optical properties and reactivity) are more prone to interact with assay systems leading to interference. Confidence in the results from toxicity testing can be maintained by selecting and using appropriate controls and using multiple assays for a common endpoint (e.g. cytotoxicity) to identify possible interferences to avoid misinterpretation of results.
SAFENANO’s in vitro
toxicity testing services use controls for interference in parallel with the nanomaterial under test, and a battery of assays to validate the accuracy of the results.
For further information on SAFENANO’s Toxicity and Product Safety Services, click here
Han X, Gelein R, Corson N, Wade-Mercer P, Jiang J, Biswas P, Finkelstein JN, Elder A, Oberdörster G. 2011, “Validation of an LDH assay for assessing nanoparticles toxicity.” Toxicology. vol. 287. no. 1-3, pp. 99-104. Kroll A, Pillukat MH, Hahn D, Schnekenburger J. 2009, “Current in vitro methods in nanoparticle risk assessment: limitations and challenges.” Eur J Pharm Biopharm. vol. 72, no. 2, pp. 370-7. Kroll A, Dierker C, Rommel C, Hahn D, Wohlleben W, Schulze-Isfort C, Göbbert C, Voetz M, Hardinghaus F, Schnekenburger J. 2011, “Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays.” Part Fibre Toxicol. 8:9.