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This manuscript discusses the challenges of applying New Approach Methodologies (NAMs) for safe by design and regulatory risk assessment of advanced nanomaterials (AdNMs). The authors propose a framework for Next Generation Risk Assessment of AdNMs involving NAMs that is aligned to the conventional risk assessment paradigm. This framework is exposure-driven, endpoint-specific, makes best use of pre-existing information, and can be implemented in tiers of increasing specificity and complexity of the adopted NAMs. The tiered structure of the approach, which effectively combines the use of existing data with targeted testing will allow safety to be assessed cost-effectively and as far as possible with even more limited use of vertebrates. The regulatory readiness of state-of-the-art emerging NAMs is assessed in terms of Transparency, Reliability, Accessibility, Applicability, Relevance and Completeness, and their appropriateness for AdNMs is discussed in relation to each step of the risk assessment paradigm along with providing perspectives for future developments in the respective scientific and regulatory areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Toxicity evaluation of engineered nanomaterials is challenging due to the ever increasing number of materials and because nanomaterials (NMs) frequently interfere with commonly used assays. Hence, there is a need for robust, high-throughput assays with which to assess their hazard potential. The present study aimed at evaluating the applicability of a genotoxicity assay based on the immunostaining and foci counting of the DNA repair protein 53BP1 (p53-binding protein 1), in a high-throughput format, for NM genotoxicity assessment. For benchmarking purposes, we first applied the assay to a set of eight known genotoxic agents, as well as X-ray irradiation (1 Gy). Then, a panel of NMs and nanobiomaterials (NBMs) was evaluated with respect to their impact on cell viability and genotoxicity, and to their potential to induce reactive oxygen species (ROS) production. The genotoxicity recorded using the 53BP1 assay was confirmed using the micronucleus assay, also scored via automated (high-throughput) microscopy. The 53BP1 assay successfully identified genotoxic compounds on the HCT116 human intestinal cell line. None of the tested NMs showed any genotoxicity using the 53BP1 assay, except the positive control consisting in (CoO)(NiO) NMs, while only TiO2 NMs showed positive outcome in the micronucleus assay. Only Fe3O4 NMs caused significant elevation of ROS, not correlated to DNA damage. Therefore, owing to its adequate predictivity of the genotoxicity of most of the tested benchmark substance and its ease of implementation in a high throughput format, the 53BP1 assay could be proposed as a complementary high-throughput screening genotoxicity assay, in the context of the development of New Approach Methodologies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Fontaine et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

There is an urgent need to apply effective, data-driven approaches to reliably predict engineered nanomaterial (ENM) toxicity. Here we introduce a predictive computational framework based on the molecular and phenotypic effects of a large panel of ENMs across multiple in vitro and in vivo models. Our methodology allows for the grouping of ENMs based on multi-omics approaches combined with robust toxicity tests. Importantly, we identify mRNA-based toxicity markers and extensively replicate them in multiple independent datasets. We find that models based on combinations of omics-derived features and material intrinsic properties display significantly improved predictive accuracy as compared to physicochemical properties alone., (© 2022. The Author(s).)

Hazard studies for nanomaterials (NMs) commonly assess whether they activate an inflammatory response. Such assessments often rely on rodents, but alternative models are needed to support the implementation of the 3Rs principles. Zebrafish ( Danio rerio ) offer a viable alternative for screening NM toxicity by investigating inflammatory responses. Here, we used non-protected life stages of transgenic zebrafish (Tg(mpx:GFP) i114 ) with fluorescently-labeled neutrophils to assess inflammatory responses to silver (Ag) and zinc oxide (ZnO) NMs using two approaches. Zebrafish were exposed to NMs via water following a tail fin injury, or NMs were microinjected into the otic vesicle. Zebrafish were exposed to NMs at 3 days post-fertilization (dpf) and neutrophil accumulation at the injury or injection site was quantified at 0, 4, 6, 8, 24, and 48 h post-exposure. Zebrafish larvae were also exposed to fMLF, LTB 4 , CXCL-8, C5a, and LPS to identify a suitable positive control for inflammation induction. Aqueous exposure to Ag and ZnO NMs stimulated an enhanced and sustained neutrophilic inflammatory response in injured zebrafish larvae, with a greater response observed for Ag NMs. Following microinjection, Ag NMs stimulated a time-dependent neutrophil accumulation in the otic vesicle which peaked at 48 h. LTB 4 was identified as a positive control for studies investigating inflammatory responses in injured zebrafish following aqueous exposure, and CXCL-8 for microinjection studies that assess responses in the otic vesicle. Our findings support the use of transgenic zebrafish to rapidly screen the pro-inflammatory effects of NMs, with potential for wider application in assessing chemical safety (e.g. pharmaceuticals).

The European Green Deal outlines ambitions to build a more sustainable, climate neutral, and circular economy by 2050. To achieve this, the European Commission has published the Chemicals Strategy for Sustainability: Towards a Toxic-Free Environment, which provides targets for innovation to better protect human and environmental health, including challenges posed by hazardous chemicals and animal testing. The European project PATROLS (Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment) has addressed multiple aspects of the Chemicals Strategy for Sustainability by establishing a battery of new approach methodologies, including physiologically anchored human and environmental hazard assessment tools to evaluate the safety of engineered nanomaterials. PATROLS has delivered and improved innovative tools to support regulatory decision-making processes. These tools also support the need for reducing regulated vertebrate animal testing; when used at an early stage of the innovation pipeline, the PATROLS tools facilitate the safe and sustainable development of new nano-enabled products before they reach the market., (© 2022 The Authors. Small published by Wiley-VCH GmbH.)

Exposure to engineered nanomaterials (ENM) poses a potential health risk to humans through long-term, repetitive low-dose exposures. Currently, this is not commonplace within in vitro lung cell cultures. Therefore, the purpose of this study was to consider the optimal exposure approach toward determining the stability, sensitivity and validity of using in vitro lung cell mono- and co-cultures to determine ENM hazard. A range of exposure scenarios were conducted with DQ 12 (previously established as a positive particle control) (historic and re-activated), TiO 2 (JRC NM-105) and BaSO 4 (JRC NM-220) on both monocultures of A549 cells as well as co-cultures of A549 cells and differentiated THP-1 cells. Cell cultures were exposed to either a single, or a repeated exposure over 24, 48- or 72-hours at in vivo extrapolated concentrations of 0-5.2 µg/cm 2 , 0-6 µg/cm 2 and 0-1µg/cm 2 . The focus of this study was the pro-inflammatory, cytotoxic and genotoxic response elicited by these ENMs. Exposure to DQ 12 caused pro-inflammatory responses after 48 hours repeat exposures, as well as increases in micronucleus frequency. Neither TiO 2 nor BaSO 4 elicited a pro-inflammatory response at this time point. However, there was induction of IL-6 after 24 hours TiO 2 exposure. In conclusion, it is important to consider the appropriateness of the positive control implemented, the cell culture model, the time of exposure as well as the type of exposure (bolus or fractionated) before establishing if an in vitro model is appropriate to determine the level of response to the specific ENM of interest.

Assessment of nanomaterial (NM) induced inflammatory responses has largely relied on rodent testing via measurement of leukocyte accumulation in target organs. Despite observations that NMs activate neutrophil driven inflammatory responses in vivo , a limited number of studies have investigated neutrophil responses to NMs in vitro . We compared responses between the human neutrophil-like HL-60 cell line and human primary neutrophils following exposure to silver (Ag), zinc oxide (ZnO), copper oxide (CuO) and titanium dioxide (TiO 2 ) NMs. NM cytotoxicity and neutrophil activation were assessed by measuring cellular metabolic activity, cytokine production, respiratory burst, and release of neutrophil extracellular traps. We observed a similar pattern of response between HL-60 cells and primary neutrophils, however we report that some neutrophil functions are compromised in the cell line. Ag NMs were consistently observed to stimulate neutrophil activation, with CuO NMs inducing similar though weaker responses. TiO 2 NMs did not induce a neutrophil response in either cell type. Interestingly, ZnO NMs readily induced activation of HL-60 cells but did not appear to activate primary cells. Our findings are relevant to the development of a tiered testing strategy for NM hazard assessment which promotes the use of non-rodent models. Whilst we acknowledge that HL-60 cells may not be a perfect substitute for primary cells and require further investigation regarding their ability to predict neutrophil activation, we recommend their use for initial screening of NM-induced inflammation. Primary human neutrophils can then be used for more focused assessments of neutrophil activation before progressing to in vivo models where necessary.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

An Environmental Risk Assessment (ERA) for nanomaterials (NMs) is outlined in this paper. Contrary to other recent papers on the subject, the main data requirements, models and advancement within each of the four risk assessment domains are described, i.e., in the: (i) materials, (ii) release, fate and exposure, (iii) hazard and (iv) risk characterisation domains. The material, which is obviously the foundation for any risk assessment, should be described according to the legislatively required characterisation data. Characterisation data will also be used at various levels within the ERA, e.g., exposure modelling. The release, fate and exposure data and models cover the input for environmental distribution models in order to identify the potential (PES) and relevant exposure scenarios (RES) and, subsequently, the possible release routes, both with regard to which compartment(s) NMs are distributed in line with the factors determining the fate within environmental compartment. The initial outcome in the risk characterisation will be a generic Predicted Environmental Concentration (PEC), but a refined PEC can be obtained by applying specific exposure models for relevant media. The hazard information covers a variety of representative, relevant and reliable organisms and/or functions, relevant for the RES and enabling a hazard characterisation. The initial outcome will be hazard characterisation in test systems allowing estimating a Predicted No-Effect concentration (PNEC), either based on uncertainty factors or on a NM adapted version of the Species Sensitivity Distributions approach. The risk characterisation will either be based on a deterministic risk ratio approach (i.e., PEC/PNEC) or an overlay of probability distributions, i.e., exposure and hazard distributions, using the nano relevant models., Competing Interests: The authors declare no conflict of interest.

A first European Conference on Computational Nanotoxicology, CompNanoTox, was held in November 2015 in Benahavís, Spain with the objectives to disseminate and integrate results from the European modeling and database projects (NanoPUZZLES, ModENPTox, PreNanoTox, MembraneNanoPart, MODERN, eNanoMapper and EU COST TD1204 MODENA) as well as to create synergies within the European NanoSafety Cluster. This conference was supported by the COST Action TD1204 MODENA on developing computational methods for toxicological risk assessment of engineered nanoparticles and provided a unique opportunity for cross fertilization among complementary disciplines. The efforts to develop and validate computational models crucially depend on high quality experimental data and relevant assays which will be the basis to identify relevant descriptors. The ambitious overarching goal of this conference was to promote predictive nanotoxicology, which can only be achieved by a close collaboration between the computational scientists (e.g. database experts, modeling experts for structure, (eco) toxicological effects, performance and interaction of nanomaterials) and experimentalists from different areas (in particular toxicologists, biologists, chemists and material scientists, among others). The main outcome and new perspectives of this conference are summarized here.

Nanotechnology is one of the most important technological developments of the 21st century. In silico methods to predict toxicity, such as quantitative structure-activity relationships (QSARs), promote the safe-by-design approach for the development of new materials, including nanomaterials. In this study, a set of cytotoxicity experimental data corresponding to 19 data points for silica nanomaterials were investigated, to compare the widely employed CORAL and Random Forest approaches in terms of their usefulness for developing so-called 'nano-QSAR' models. 'External' leave-one-out cross-validation (LOO) analysis was performed, to validate the two different approaches. An analysis of variable importance measures and signed feature contributions for both algorithms was undertaken, in order to interpret the models developed. CORAL showed a more pronounced difference between the average coefficient of determination (R²) for training and for LOO (0.83 and 0.65 for training and LOO, respectively), compared to Random Forest (0.87 and 0.78 without bootstrap sampling, 0.90 and 0.78 with bootstrap sampling), which may be due to overfitting. With regard to the physicochemical properties of the nanomaterials, the aspect ratio and zeta potential were found to be the two most important variables for Random Forest, and the average feature contributions calculated for the corresponding descriptors were consistent with the clear trends observed in the data set: less negative zeta potential values and lower aspect ratio values were associated with higher cytotoxicity. In contrast, CORAL failed to capture these trends., (2016 FRAME.)

Commercialization of nanotechnologies entails a regulatory requirement for understanding their environmental, health and safety (EHS) risks. Today we face challenges to assess these risks, which emerge from uncertainties around the interactions of manufactured nanomaterials (MNs) with humans and the environment. In order to reduce these uncertainties, it is necessary to generate sound scientific data on hazard and exposure by means of relevant frameworks and tools. The development of such approaches to facilitate the risk assessment (RA) of MNs has become a dynamic area of research. The aim of this paper was to review and critically analyse these approaches against a set of relevant criteria. The analysis concluded that none of the reviewed frameworks were able to fulfill all evaluation criteria. Many of the existing modelling tools are designed to provide screening-level assessments rather than to support regulatory RA and risk management. Nevertheless, there is a tendency towards developing more quantitative, higher-tier models, capable of incorporating uncertainty into their analyses. There is also a trend towards developing validated experimental protocols for material identification and hazard testing, reproducible across laboratories. These tools could enable a shift from a costly case-by-case RA of MNs towards a targeted, flexible and efficient process, based on grouping and read-across strategies and compliant with the 3R (Replacement, Reduction, Refinement) principles. In order to facilitate this process, it is important to transform the current efforts on developing databases and computational models into creating an integrated data and tools infrastructure to support the risk assessment and management of MNs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of "nanotoxicology research" based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

Workers involved in producing nanomaterials or using nanomaterials in manufacturing plants are likely to have earlier and higher exposure to manufactured/engineered nanomaterials (ENM) than the general population. This is because both the volume handled and the probability of the effluence of 'free' nanoparticles from the handled volume are much higher during a production process than at any other stage in the lifecycle of nanomaterials and nanotechnology-enabled products. Risk assessment (RA) techniques using control banding (CB) as a framework for risk transfer represents a robust theory but further progress on implementing the model is required so that risk can be transferred to insurance companies. Following a review of RA in general and hazard measurement in particular, we subject a Structural Alert Scheme methodology to three industrial case studies using ZrO2 , TiO2 , and multi-walled carbon nanotubes (MWCNT). The materials are tested in a pristine state and in a remediated (coated) state, and the respective emission and hazard rates are tested alongside the material performance as originally designed. To our knowledge, this is the first such implementation of a CB RA in conjunction with an ENM performance test and offers both manufacturers and underwriters an insight into future applications., (© 2015 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals, Inc.)

Nanomaterials (NMs) display many unique and useful physico-chemical properties. However, reliable approaches are needed for risk assessment of NMs. The present study was performed in the FP7-MARINA project, with the objective to identify and evaluate in vitro test methods for toxicity assessment in order to facilitate the development of an intelligent testing strategy (ITS). Six representative oxide NMs provided by the EC-JRC Nanomaterials Repository were tested in nine laboratories. The in vitro toxicity of NMs was evaluated in 12 cellular models representing 6 different target organs/systems (immune system, respiratory system, gastrointestinal system, reproductive organs, kidney and embryonic tissues). The toxicity assessment was conducted using 10 different assays for cytotoxicity, embryotoxicity, epithelial integrity, cytokine secretion and oxidative stress. Thorough physico-chemical characterization was performed for all tested NMs. Commercially relevant NMs with different physico-chemical properties were selected: two TiO2 NMs with different surface chemistry - hydrophilic (NM-103) and hydrophobic (NM-104), two forms of ZnO - uncoated (NM-110) and coated with triethoxycapryl silane (NM-111) and two SiO2 NMs produced by two different manufacturing techniques - precipitated (NM-200) and pyrogenic (NM-203). Cell specific toxicity effects of all NMs were observed; macrophages were the most sensitive cell type after short-term exposures (24-72h) (ZnO>SiO2>TiO2). Longer term exposure (7 to 21 days) significantly affected the cell barrier integrity in the presence of ZnO, but not TiO2 and SiO2, while the embryonic stem cell test (EST) classified the TiO2 NMs as potentially 'weak-embryotoxic' and ZnO and SiO2 NMs as 'non-embryotoxic'. A hazard ranking could be established for the representative NMs tested (ZnO NM-110 > ZnO NM-111 > SiO2 NM-203 > SiO2 NM-200 > TiO2 NM-104 > TiO2 NM-103). This ranking was different in the case of embryonic tissues, for which TiO2 displayed higher toxicity compared with ZnO and SiO2. Importantly, the in vitro methodology applied could identify cell- and NM-specific responses, with a low variability observed between different test assays. Overall, this testing approach, based on a battery of cellular systems and test assays, complemented by an exhaustive physico-chemical characterization of NMs, could be deployed for the development of an ITS suitable for risk assessment of NMs. This study also provides a rich source of data for modeling of NM effects.

Comparative hazard identification of nanomaterials (NMs) can aid in the prioritisation for further toxicity testing. Here, we assessed the acute lung, systemic and liver responses in C57BL/6N mice for three NMs to provide a hazard ranking. A silver (Ag), non-functionalised zinc oxide (ZnO) and a triethoxycaprylylsilane functionalised ZnO NM suspended in water with 2% mouse serum were examined 24 hours following a single intratracheal instillation (I.T.). An acute pulmonary inflammation was noted (marked by a polymorphonuclear neutrophil influx) with cell damage (LDH and total protein) in broncho-alveolar lavage fluid (BALF) after administration of both non-functionalised and functionalised ZnO. The latter also induced systemic inflammation measured as an increase in blood neutrophils and a decrease in blood lymphocytes. Exposure to Ag NM was not accompanied by pulmonary inflammation or cytotoxicity, or by systemic inflammation. A decrease in glutathione levels was demonstrated in the liver following exposure to high doses of all three nanomaterials irrespective of any noticeable inflammatory or cytotoxic effects in the lung. By applying benchmark dose (BMD) modeling statistics to compare potencies of the NMs, we rank functionalised ZnO ranked the highest based on the largest number of affected endpoints, as well as the strongest responses observed after 24 hours. The non-functionalised ZnO NM gave an almost similar response, whereas Ag NM did not cause an acute response at similar doses.

Substantial limitations and uncertainties hinder the exposure assessment of engineered nanomaterials (ENMs). The present deficit of reliable measurements and models will inevitably lead in the near term to qualitative and uncertain exposure estimations, which may fail to support adequate risk assessment and management. Therefore it is necessary to complement the current toolset with user-friendly methods for near-term nanosafety evaluation. This paper proposes an approach for relative exposure screening of ENMs. For the first time, an exposure model explicitly implements quantitative weight of evidence (WoE) methods and utilises expert judgement for filling data gaps in the available evidence-base. Application of the framework is illustrated for screening of exposure scenarios for nanoscale titanium dioxide, carbon nanotubes and fullerenes, but it is applicable to other nanomaterials as well. The results show that the WoE-based model overestimates exposure for scenarios where expert judgement was substantially used to fill data gaps, which suggests its conservative nature. In order to test how variations in input data influence the obtained results, probabilistic Monte Carlo sensitivity analysis was applied to demonstrate that the model performs in stable manner.

PARTICLE_RISK was one of the first multidisciplinary projects funded by the European Commission's Framework Programme that was responsible for evaluating the implications of nanomaterial (NM) exposure on human health. This project was the basis for this review which identifies the challenges that exist within the assessment of NM risk. We have retrospectively reflected on the findings of completed nanotoxicology studies to consider what progress and advances have been made within the risk assessment of NMs, as well as discussing the direction that nanotoxicology research is taking and identifying the limitations and failings of existing research. We have reflected on what commonly encountered challenges exist and explored how these issues may be resolved. In particular, the following is discussed (i) NM selection (ii) NM physico-chemical characterisation; (iii) NM dispersion; (iv) selection of relevant doses and concentrations; (v) identification of relevant models, target sites and endpoints; (vi) development of alternatives to animal testing; and (vii) NM risk assessment. These knowledge gaps are relatively well recognised by the scientific community and recommendations as to how they may be overcome in the future are provided. It is hoped that this will help develop better defined hypothesis driven research in the future that will enable comprehensive risk assessments to be conducted for NMs. Importantly, the nanotoxicology community has responded and adapted to advances in knowledge over recent years to improve the approaches used to assess NM hazard, exposure and risk. It is vital to learn from existing information provided by ongoing or completed studies to avoid unnecessary duplication of effort, and to offer guidance on aspects of the experimental design that should be carefully considered prior to the start of a new study.

Nanotechnology has become the focus of a large amount of scientific, political, and financial interest. Limited information on the exposure to nanomaterials is available, with only a few occupational exposure studies having been performed. While laboratory animal studies on the biological effects of some nanomaterials have been published, no epidemiological studies have been reported to date. This lack of data on exposure and human health effects hinders risk assessment of these materials. As the use of nanomaterials increases rapidly, it is of vital importance that the risk assessment community understands the complexities of the issues surrounding the manufacture, use and disposal of nanomaterials, the potential of environmental and occupational exposure to human populations, as well as adverse health outcomes. For this to happen, it is in many ways necessary for the scientific community to also understand what questions risk assessors need to ask, and what research will best answer them. Risk management of nanomaterials requires more information as to the human and ecological effects of exposure to various nanomaterials. At this time, there are no specific regulations for nanomaterials, but a few efforts to include nanomaterials under existing environmental regulations have begun. The purpose of this article is to describe the potential regulations for nanomaterials, and the current issues related to the risk assessment of nanomaterials.