Hazard identification and safety assessment of the huge variety of nanomaterials (NMs), calls for robust and validated toxicity screening tests in combination with cheminformatics approaches to ...identify factors that can drive toxicity. Cytotoxicity and genotoxicity of seventeen JRC repository NMs, derived from titanium dioxide, zinc oxide, silver and silica, were tested in vitro using human lung alveolar epithelial cells A549. Cytotoxicity was assessed with the AlamarBlue (AB) and colony forming efficiency (CFE) assays, and genotoxicity by the enzyme-linked version of the comet assay. Nanoparticle tracking analysis (NTA) was used to measure size of the NMs in stock and in cell culture medium at different time points. Categorization and ranking of cytotoxic and genotoxic potential were performed (EU-NanoREG2 project approach). Descriptors for prediction of NMs toxicity were identified by quantitative structure-activity relationship (QSAR) analysis. Our results showed that ZnO NMs (NM-110 and NM-111), and Ag NMs (NM-300K and NM-302) were cytotoxic, while the TiO2 and SiO2 NMs were non-cytotoxic. Regarding genotoxicity, TiO2 NM-100, ZnO NM-110, SiO2 NM-203 and Ag NM-300K were categorized as positive. Cheminformatics modeling identified electron properties and overall chemical reactivity as important descriptors for cytotoxic potential, HOMO-LUMO energy parameter, ionization potential, pristine size for the NMs´ genotoxic potential, and presence of surface coating as descriptor for induction of DNA oxidized base lesions.
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•Characterization of physicochemical properties is crucial for hazard identification of nanomaterials (NMs).•Machine learning (2D HCA and PLS) can identify NMs descriptors for hazard prediction.•Distinct descriptors of NMs are related to toxicity of NMs.•In vitro combined with in silico represents a valuable approach to risk assessment of NMs.
The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is ...dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles.
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•NM surfaces result in physical (scattering) & binding interferences with in vitro toxicity assays.•Analysis of literature from 2014 to 2022 shows some improvement in reporting of NMs interferences.•An approach to assess interference and design fit-for purpose controls for NM type / method is presented.•A decision tree to avoid interferences and select mitigation actions / interference controls is included.•Application of the decision tree will improve reliability and reusability of in vitro NM toxicity data..
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•Electrochemical study of interaction of three toxicants with lipid monolayer.•Effect of three toxicants on cell viability and genotoxicity.•Benchmarking of model membrane results ...with live cell assays.•Electrochemical characterisation of membrane interaction mechanism of toxicants.•Identification of biomembrane damage using fluorescent probe assay.
This study compares the performance and output of an electrochemical phospholipid membrane platform against respective in vitro cell-based toxicity testing methods using three toxicants of different biological action (chlorpromazine (CPZ), colchicine (COL) and methyl methanesulphonate (MMS)). Human cell lines from seven different tissues (lung, liver, kidney, placenta, intestine, immune system) were used to validate this physicochemical testing system. For the cell-based systems, the effective concentration at 50 % cell death (EC50) values are calculated. For the membrane sensor, a limit of detection (LoD) value was extracted as a quantitative parameter describing the minimum concentration of toxicant which significantly affects the structure of the phospholipid sensor membrane layer. LoD values were found to align well with the EC50 values when acute cell viability was used as an end-point and showed a similar toxicity ranking of the tested toxicants. Using the colony forming efficiency (CFE) or DNA damage as end-point, a different order of toxicity ranking was observed. The results of this study showed that the electrochemical membrane sensor generates a parameter relating to biomembrane damage, which is the predominant factor in decreasing cell viability when in vitro models are acutely exposed to toxicants. These results lead the way to using electrochemical membrane-based sensors for rapid relevant preliminary toxicity screens.
As part of a large human biomonitoring study, we conducted occupational monitoring in a glass fibre factory in Slovakia. Shopfloor workers (n = 80), with a matched group of administrators in the same ...factory (n = 36), were monitored for exposure to glass fibres and to polycyclic aromatic hydrocarbons (PAHs). The impact of occupational exposure on chromosomal aberrations, DNA damage and DNA repair, immunomodulatory markers, and the role of nutritional and lifestyle factors, as well as the effect of polymorphisms in metabolic and DNA repair genes on genetic stability, were investigated.
The (enzyme-modified) comet assay was employed to measure DNA strand breaks (SBs) and apurinic sites, oxidised and alkylated bases. Antioxidant status was estimated by resistance to H2O2-induced DNA damage. Base excision repair capacity was measured with an in vitro assay (based on the comet assay).
Exposure of workers to fibres was low, but still was associated with higher levels of SBs, and SBs plus oxidised bases, and higher sensitivity to H2O2. Multivariate analysis showed that exposure increased the risk of high levels of SBs by 20%. DNA damage was influenced by antioxidant enzymes catalase and glutathione S-transferase (measured in blood). DNA repair capacity was inversely correlated with DNA damage and positively with antioxidant status. An inverse correlation was found between DNA base oxidation and the percentage of eosinophils (involved in the inflammatory response) in peripheral blood of both exposed and reference groups. Genotypes of XRCC1 variants rs3213245 and rs25487 significantly decreased the risk of high levels of base oxidation, to 0.50 (p = 0.001) and 0.59 (p = 0.001), respectively.
Increases in DNA damage owing to glass fibre exposure were significant but modest, and no increases were seen in chromosome aberrations or micronuclei. However, it is of concern that even low levels of exposure to these fibres can cause significant genetic damage.
•Exposure of workers to glass fibres was associated with increased levels of DNA damage and higher sensitivity to H2O2.•DNA damage was influenced by catalase activity and glutathione S-transferase levels measured in peripheral blood.•XRCC1 variants rs3213245 and rs25487 were associated with a decrease in the risk of high DNA oxidation damage.•Glass fibre exposure did not affect the levels of chromosome aberrations or micronuclei in exposed workers.
Abstract
Genotoxicity testing for nanomaterials remains challenging as standard testing approaches require some adaptation, and further development of nano-specific OECD Test Guidelines (TGs) and ...Guidance Documents (GDs) are needed. However, the field of genotoxicology continues to progress and new approach methodologies (NAMs) are being developed that could provide relevant information on the range of mechanisms of genotoxic action that may be imparted by nanomaterials. There is a recognition of the need for implementation of new and/or adapted OECD TGs, new OECD GDs, and utilization of NAMs within a genotoxicity testing framework for nanomaterials. As such, the requirements to apply new experimental approaches and data for genotoxicity assessment of nanomaterials in a regulatory context is neither clear, nor used in practice. Thus, an international workshop with representatives from regulatory agencies, industry, government, and academic scientists was convened to discuss these issues. The expert discussion highlighted the current deficiencies that exist in standard testing approaches within exposure regimes, insufficient physicochemical characterization, lack of demonstration of cell or tissue uptake and internalization, and limitations in the coverage of genotoxic modes of action. Regarding the latter aspect, a consensus was reached on the importance of using NAMs to support the genotoxicity assessment of nanomaterials. Also highlighted was the need for close engagement between scientists and regulators to (i) provide clarity on the regulatory needs, (ii) improve the acceptance and use of NAM-generated data, and (iii) define how NAMs may be used as part of weight of evidence approaches for use in regulatory risk assessments.
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