With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety—preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read‐across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter‐experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM‐cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose‐ and time‐dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label‐free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance‐based monitoring, Multiplex analysis of secreted products, and genotoxicity methods—namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413For further resources related to this article, please visit the WIREs website.

a b s t r a c tThe unique properties of nanomaterials (NMs) are beneficial in numerous industrial and medical applications. However, they could also induce unintended effects. Thus, a proper strategy for toxicity testing is essential in human hazard and risk assessment. Toxicity can be tested in vivo and in vitro; in compliance with the 3Rs, alternative strategies for in vitro testing should be further developed for NMs. Robust, standardized methods are of great importance in nanotoxicology, with comprehensive material characterization and uptake as an integral part of the testing strategy. Oxidative stress has been shown to be an underlying mechanism of possible toxicity of NMs, causing both immunotoxicity and genotoxicity. For testing NMs in vitro, a battery of tests should be performed on cells of human origin, either cell lines or primary cells, in conditions as close as possible to an in vivo situation. Novel toxicity pathways, particularly epigenetic modification, should be assessed along with conventional toxicity testing methods. However, to initiate epigenetic toxicity screens for NM exposure, there is a need to better understand their adverse effects on the epigenome, to identify robust and reproducible causal links between exposure, epigenetic changes and adverse phenotypic endpoints, and to develop improved assays to monitor epigenetic toxicity.

There is serious concern about the potential harmful effects of certain nanomaterials (NMs), on account of their ability to penetrate cell membranes and the increased reactivity that results from their increased surface area compared with bulk chemicals. To assess the safety of NMs, reliable tests are needed. We have investigated the possible genotoxicity of four representative NMs, derived from titanium dioxide, zinc oxide, cerium oxide and silver, in two human cell lines, A549 alveolar epithelial cells and lymphoblastoid TK6 cells. A high-throughput version of the comet assay was used to measure DNA strand beaks (SBs) as well as oxidised purines (converted to breaks with the enzyme formamidopyrimidine DNA glycosylase). In parallel, cytotoxicity was measured with the alamarBlue® assay, and the ability of NM-treated cells to survive was assessed by their colony-forming efficiency. TiO and CeO NMs were only slightly cytotoxic by the alamarBlue® test, and had no long-term effect on colony-forming efficiency. However, both induced DNA damage at non-cytotoxic concentrations; the damage decreased from 3 to 24-h exposure, except in the case of CeO-treated A549 cells. ZnO and Ag NMs affected cell survival, and induced high levels of DNA damage at cytotoxic concentrations. At lower concentrations, there was significant damage, which tended to persist over 24 h. The implication is that all four reference metal NMs tested-whether cytotoxic or not-are genotoxic. A full assessment of NM toxicity should include tests on different cell types, different times of incubation and a wide range of (especially non-cytotoxic) concentrations; a test for cell viability should be performed in parallel. Inclusion of Fpg in the comet assay allows detection of indirect genotoxic effects via oxidative stress.

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Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardizing the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature, and voltage gradient) are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e., cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls) or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay), or photosensitiser plus light to oxidize guanine (positive control for Fpg- or OGG1-sensitive sites). Reference standards are especially valuable when performing a series of experiments over a long period—for example, analysing samples of white blood cells from a large human biomonitoring trial—to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

Changes in the genetic material can lead to serious human health defects, as mutations in somatic cells may cause cancer and can contribute to other chronic diseases. Genotoxic events can appear at both the DNA, chromosomal or (during mitosis) whole genome level. The study of mechanisms leading to genotoxicity is crucially important, as well as the detection of potentially genotoxic compounds. We consider the current state of the art and describe here the main endpoints applied in standard human in vitro models as well as new advanced 3D models that are closer to the in vivo situation. We performed a literature review of in vitro studies published from 2000–2020 (August) dedicated to the genotoxicity of nanomaterials (NMs) in new models. Methods suitable for detection of genotoxicity of NMs will be presented with a focus on advances in miniaturization, organ-on-a-chip and high throughput methods.

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