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Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production.

Exposure to indoor air pollutants (IAP) has increased recently, with people spending more time indoors. Increased exposures of IAP on a healthy population are poorly understood, and those with allergic respiratory conditions less so. The objective of this study, therefore, was to implement a well-characterised in vitro model of the human alveolar epithelial barrier (A549+PMA differentiated THP-1 incubated with/without IL-13, IL-5 and IL-4) to determine the effects of a standardised indoor particulate (NIST2583) on both a healthy lung model and modelling a type-II inflammatory response. Using concentrations from the literature, and an environmentally appropriate exposure we investigated 232, 464 and 608ng/cm2 of NIST2583 respectively. Membrane integrity (blue dextran), viability (trypan blue), genotoxicity (micronucleus (Mn) assay) and (pro-) and (anti-)inflammatory effects (IL-6, IL-8, IL-33, and IL-10) were then assessed 24hours post exposure to both models. No changes in Mn frequency or membrane integrity in either model was noted when exposed to any concentration of NIST2583 tested. Though, a significant decrease (p

Cell passage number is of high importance in experimental design and can cause biochemical, genetic, and/or morphological changes to a cell. This project aimed to understand the impact of increasing cell passage number on the structure and function of epithelial cells from different organs. Two epithelial cell types were studied, NCI-H441 lung cells (type II alveolar cells), and HepG2 liver cells. Predefined ranges of 11-20, 21-30, 31-40, and 41-50 were studied. Cells were exposed to 1µg/mL tumour necrosis factor α (TNFα) and 15µM hydrogen peroxide (H2O2) via submerged or quasi-air liquid interface, according to GIVIMP guidelines. Endpoints tests included cytotoxicity (lactate dehydrogenase (LDH) release), cell death (BCL2, BAX, FAS), (pro-) inflammatory mediators (IL6 & IL8), oxidative stress (SOD1) and genetic toxicology (DNA damage (p21 & p53)). In addition, confocal microscopy was used to observe any changes to cell morphology. Preliminary data indicates the NCI-H441 epithelial cell passage range has an effect on the secretion of (pro-)inflammatory mediators, with an increase in IL8 and IL6 in higher passage ranges (41-50) compared to lower passage ranges (11-20) when stimulated with TNFα as a positive control. In HepG2 cells there is also an increase in IL8 production across passage range, however, it is considerably less than NCI-H441 cells. In addition, IL6 production in HepG2 cells was negligible across all passage ranges. LDH assay showed minimal variation between cell type and passage number. Data obtained suggests passage range, depending on cell type, has an influence on the effect level of the end point measured.

The development of nanomaterials has seen exponential growth in the last two decades and, more recently, of complex multicomponent nanomaterials (MCNM), which represent challenges for human hazard testing. This study aims to determine exposure-dose-response relationships and the potential human hazard of industrially relevant MCNMs with a tiered testing approach. Tier1 is a simple in vitro assay, while Tier2 is a more complex in vitro model suitable for mimicking inhalation exposure. The intent was to explore the differences between the complexity of the assays and the genotoxicity response. The cytotoxic and genotoxic potential of selected MCNMs were evaluated with an OECD-approved in vitro Tier1 testing approach with nano-specific modifications. Chromosomal breakage (genotoxicity) was measured with the in vitro cytokinesis-blocked micronucleus (CBMN) assay alongside relative population doubling (RPD) to assess cytotoxicity. For Tier1 testing, the CBMN assay was performed on human lymphoblastoid (TK6) cells; a co-culture of lung epithelial cells (A549) and monocytes (d-THP-1) was utilised for Tier2 genotoxicity testing. MCNMs from four industrially-relevant case studies were analysed. Some materials demonstrated differences in the dose-response relationship between the core components and the MCNM when using Tier1 in vitro tests, suggesting a synergistic effect when the materials were combined. These results are compared across the tier testing system to determine if advanced culture models can provide more physiologically relevant data as compared to standard in vitro systems. This research has received funding from the European Union’s Horizon 2020 research and innovation programme for the SUNSHINE project under grant agreement No 952924.

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 utilisation 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 physico-chemical characterisation, lack of demonstration of cell or tissue uptake and internalisation, 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 1) provide clarity on the regulatory needs, 2) improve the acceptance and use of NAMs-generated data, and 3) define how NAMs may be used as part of Weight of Evidence approaches for use in regulatory risk assessments.

Abstract Scientific, financial, and ethical drivers have led to unprecedented interest in implementing human-relevant, mechanistic in vitro and in silico testing approaches. Further, as non-animal approaches are being developed and validated, researchers are interested in strategies that can immediately reduce the use of animals in toxicology testing. Here, we aim to outline a testing strategy for assessing genotoxicity beginning with standard in vitro methods, such as the bacterial reverse mutation test and the in vitro micronucleus test, followed by a second tier of in vitro assays including those using advanced 3D tissue models. Where regulatory agencies require in vivo testing, one demonstrated strategy is to combine genotoxicity studies traditionally conducted separately into a single test or to integrate genotoxicity studies into other toxicity studies. Standard setting organisations and regulatory agencies have encouraged such strategies, and examples of their use can be found in the scientific literature. Employing approaches outlined here will reduce animal use as well as study time and costs.

Nanopesticides are not only in an advanced state of research and development but have started to appear on the market. Industry and regulatory agencies need a consolidated and comprehensive framework and guidance for human health risk assessments. In this perspective we develop such a comprehensive framework by exploring two case studies from relevant product types: an active ingredient delivered with a nanocarrier system, and a nanoparticle as an active ingredient. For a nanocarrier system, three entities are tracked during the assessment: the nanocarrier–active ingredient complex, the empty nanocarrier remaining after the complete release of the active ingredient, and the released active ingredient. For the nanoparticle of pure active ingredient, only two entities are relevant: the nanoparticle and the released ions. We suggest important adaptations of the existing pesticide framework to determine the relevant nanopesticide entities and their concentrations for toxicity testing. Depending on the nature of the nanopesticides, additional data requirements, such as those pertaining to durability in biological media and potential for crossing biological barriers, have also been identified. Overall, our framework suggests a tiered approach for human health risk assessment, which is applicable for a range of nanopesticide products to support regulators and industry in making informed decisions on nanopesticide submissions. Brief summaries of suitable methods including references to existing standards (if available) have been included together with an analysis of current knowledge gaps. Our study is an important step towards a harmonized approach accepted by regulatory agencies for assessing nanopesticides. This perspective presents a framework with a tiered approach for human health risk assessment of nanopesticides that will facilitate informed decisions by regulators and industry.

Background With the continued integration of engineered nanomaterials (ENMs) into everyday applications, it is important to understand their potential for inducing adverse human health effects. However, standard in vitro hazard characterisation approaches suffer limitations for evaluating ENM and so it is imperative to determine these potential hazards under more physiologically relevant and realistic exposure scenarios in target organ systems, to minimise the necessity for in vivo testing. The aim of this study was to determine if acute (24 h) and prolonged (120 h) exposures to five ENMs (TiO2, ZnO, Ag, BaSO4 and CeO2) would have a significantly different toxicological outcome (cytotoxicity, (pro-)inflammatory and genotoxic response) upon 3D human HepG2 liver spheroids. In addition, this study evaluated whether a more realistic, prolonged fractionated and repeated ENM dosing regime induces a significantly different toxicity outcome in liver spheroids as compared to a single, bolus prolonged exposure. Results Whilst it was found that the five ENMs did not impede liver functionality (e.g. albumin and urea production), induce cytotoxicity or an IL-8 (pro-)inflammatory response, all were found to cause significant genotoxicity following acute exposure. Most statistically significant genotoxic responses were not dose-dependent, with the exception of TiO2. Interestingly, the DNA damage effects observed following acute exposures, were not mirrored in the prolonged exposures, where only 0.2–5.0 µg/mL of ZnO ENMs were found to elicit significant (p ≤ 0.05) genotoxicity. When fractionated, repeated exposure regimes were performed with the test ENMs, no significant (p ≥ 0.05) difference was observed when compared to the single, bolus exposure regime. There was Conclusion In conclusion, whilst there was no difference between a single, bolus or fractionated, repeated ENM prolonged exposure regimes upon the toxicological output of 3D HepG2 liver spheroids, there was a difference between acute and prolonged exposures. This study highlights the importance of evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

Following advancements in the field of genotoxicology, it has become widely accepted that 3D models are not only more physiologically relevant but also have the capacity to elucidate more complex biological processes that standard 2D monocultures are unable to. Whilst 3D liver models have been developed to evaluate the short-term genotoxicity of chemicals, the aim of this study was to develop a 3D model that could be used with the regulatory accepted in vitro micronucleus (MN) following low-dose, longer-term (5 days) exposure to engineered nanomaterials (ENMs). A comparison study was carried out between advanced models generated from two commonly used liver cell lines, namely HepaRG and HepG2, in spheroid format. While both spheroid systems displayed good liver functionality and viability over 14 days, the HepaRG spheroids lacked the capacity to actively proliferate and, therefore, were considered unsuitable for use with the MN assay. This study further demonstrated the efficacy of the in vitro 3D HepG2 model to be used for short-term (24 h) exposures to genotoxic chemicals, aflatoxin B1 (AFB1) and methyl-methanesulfonate (MMS). The 3D HepG2 liver spheroids were shown to be more sensitive to DNA damage induced by AFB1 and MMS when compared to the HepG2 2D monoculture. This 3D model was further developed to allow for longer-term (5 day) ENM exposure. Four days after seeding, HepG2 spheroids were exposed to Zinc Oxide ENM (0-2 µg/ml) for 5 days and assessed using both the cytokinesis-block MN (CBMN) version of the MN assay and the mononuclear MN assay. Following a 5-day exposure, differences in MN frequency were observed between the CBMN and mononuclear MN assay, demonstrating that DNA damage induced within the first few cell cycles is distributed across the mononucleated cell population. Together, this study demonstrates the necessity to adapt the MN assay accordingly, to allow for the accurate assessment of genotoxicity following longer-term, low-dose ENM exposure.

When considering air pollution particles (AP), they are often assessed in isolation, when this is not how the population is exposed to them. Within the indoor environment, there are bioaerosols present, which contain various allergens including house dust mite (HDM) and lipopolysaccharides (LPS). This RESPIRE study aims to apply a well-characterised in vitro model of the alveolar barrier (type I and II epithelial cells (NCI-H441 and hAELVi at relevant in vivo ratios) and macrophage-like cells) to determine the effects of these bioaerosols on the toxicological and immunological responses to AP. To achieve this, we will investigate cytotoxicity, inflammatory responses, morphology, oxidative stress, membrane integrity and genetic toxicology of these models with specific focus on responses from human allergic responses and airway diseases (i.e., bronchiolitis-interstitial lung disease and sarcoidosis) to determine the additional health considerations the addition of bioaerosols to AP has on our models. We will utilise a physiologically relevant aerosolization exposure method) and a standardised indoor (NIST2583) particulate at an environmentally relevant concentration to mimic air pollution along with HDM and LPS to determine if the addition of these environmentally appropriate bioaerosols influence the responses to IAP. This project is funded by the recently initiated RESPIRE project (NE/W002264/1), as part of the SPF Clean Air Programme.

In vitro lung models are a valuable tool to assess the inhalation hazard of air pollutants, bioaerosols, chemicals and nanomaterials. Though it is essential that these models are characterised and anatomically relevant to the area of the human lung they seek to represent. Previously within inhalation toxicology, focus has been placed on the use of monocultures of alveolar epithelial cells, however use of alveolar co-culture models have been presented as more realistic and sensitive models. Here, we present the development of a novel triple cell co-culture system, cultured at the air-liquid interface that is sensitive to LPS and particulate matter. This co-culture contains alveolar type 1 cells (hAELVi), alveolar type 2 cells (NCI-H441) and alveolar macrophages (differentiated THP-1 (dTHP-1) using PMA (20 nM)). Using CellTrackers™ with confocal microscopy, the ratio of hAELVi cells to NCI-H441 cells was confirmed to be a histologically relevant 16:1 ratio forming a tight monolayer shown through blue dextran membrane permeability assay. Macrophages were seeded on top of the epithelial co-culture at an anatomically relevant density of 1 macrophage per 18x103 μm2 (confirmed via confocal microscopy) to form the triple cell co-culture. The model, when challenged, released more interleukin-8 and TNF-α than the epithelial mono- and bi-culture systems when exposed to LPS (1 μg/mL for 24 hrs) (p

The impact of micro-, nanoplastics (MNPs) and particulate matter (PM) upon human health are escalating concerns worldwide. MNPs released from surgical face masks, and PM from ambient air pollution, can both be inhaled and consequently have potentially significant negative impacts upon human health, particularly on our lung epithelial cell structure and function. Currently, the impact of the realistic co-exposure of these samples is unknown. Therefore, the purpose of this study was to determine the potential inhaled hazardous health effects of the co-exposure of MNPs and ambient (indoor and outdoor) air PM. Using an advanced in vitro testing model combined with a specific mechanistic toxicology approach, it is hypothesised that the co-exposure approach will elucidate important information regarding the differential toxicology of ambient (indoor and outdoor) air PM. The in vitro system will consist of alveolar epithelial type-II cells (-NCI-H441) combined with differentiated monocytes (dTHP-1), cultured at the relevant in vivo ratio and at the air-liquid interface. Cell cultures will be exposed for both 4 and 24hrs to MNP (commercially available polypropylene (PP) and blue face mask extracted PP), PM (NIST 2584 and NIST 1649a respectively) and then subsequent MNP+PM (both NIST 2584 and NIST 1649a respectively) via aerosol (VITROCELL Cloud12), using alternative dosimetry patterns. The biochemical endpoints of cytotoxicity, oxidative stress, (pro-)inflammatory response and genotoxicity, will then be assessed to provide the basis for future global analytical techniques to examine the currently unknown human hazard of inhaled MNPs (+/- PM).

A primary toxicological focus has been the development of cellular models and exposure systems representative of nanomaterial inhalation due to the inevitable risk of environmental or occupational exposure. This current study compared the genotoxic and immunogenic potential of two nanomaterials: copper oxide (CuO) and zinc oxide (ZnO) and a positive-particle control tungsten-carbide cobalt (WC-Co), in type-II alveolar epithelial cells (A549) utilising cell culture and exposure systems of increasing complexity. Firstly, treatment was undertaken in submerged-culture at a dose range of 1–100 μg/cm2 for all materials. This was repeated with A549s established at the air-liquid interface using a quasi-ALI exposure technique. Finally, complexity of the quasi-ALI exposure was increased with the addition of a dynamic-flow system (Kirkstall Quasi-Vivo,600) allowing media circulation on the basal side of A549 cultures. For all exposures, cytotoxicity, chromosomal damage, and immunogenicity were assessed by relative population doubling, cytokinesis blocked micronucleus assay and interleukin-8 quantification respectively. Compared to the untreated control, significant cytotoxicity (~50%;p

Many in vitro studies that focus upon lung toxicology use models that assume, or are specifically based upon adult lung structure, function, and physiology. Yet, the most vulnerable groups of the human population (i.e., young, elderly, and diseased) have an altered lung structure and function. Notably, the developing lung (in utero) and infant lungs exhibit notable differences in term of cell type, number, ratio, organ size and surface area. Recent work has identified the detrimental implications of being exposed to AP while the lung is developing. To elucidate the mechanisms associated with AP exposure to the developing lung, the aim of this study will create and characterise an in vitro model of the foetal human bronchial region composed of cells physiologically appropriate to various stages of development, as well as an older child’s airway, notably the developing alveoli. This will allow us to determine the developmental effects of air pollution particles at an air-liquid interface (ALI) utilising a physiologically relevant aerosol exposure and standardised particulate aiming to mimic indoor and outdoor air pollution. For this work, a global approach will be utilised to determine up and down regulation of various pathways associated with both development and exposure to AP. A further aim will include comparison with an “adult” lung to analysis the potential developmental implications exposure to AP may illicit. This project is funded by the recently initiated RESPIRE project (NE/W002264/1), as part of the SPF Clean Air Programme.

Ambient nitrogen dioxide (NO2) and particulate matter (PM) concentrations are often found to be proportional, such that inhalation of one pollutant is expected to be accompanied by the inhalation of the other. Epidemiological evidence consistently demonstrates the health burdens associated with the inhalation of NO2 and PM, though has difficulty deciphering the inhalation hazard of either pollutant individually. Airway models can be exposed to NO2 and PM separately or as a co-exposure in vitro, allowing mechanisms of toxicity to be elucidated. Herein, NCI-H441 cells were exposed to either 780 ng/cm2 Printex 90 carbon black (CB) (a surrogate model of PM) using a VitroCell Cloud12, 5 ppm NO2 (within a modified Coy hypoxic cabinet) or a combination of NO2+CB over 24 hours. Cytotoxicity was not altered by NO2 or CB (trypan blue exclusion assay), however CB, and to a lesser extent CB+NO2 exposure resulted in a non-significant increase in membrane permeability (p>0.05). qPCR revealed no alterations in expression of superoxide dismutase 1 (SOD1) for any exposure condition. Both CB and NO2 exposures resulted in significantly increased IL6 and IL8 release (ELISA) (p

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2′,7′-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

In vitro genotoxicity studies are a quick and high throughput approach to assess the genotoxic potential of chemicals; however, the reliability of these tests and their relevance to in vivo effects depends on the choice of representative cell line and optimisation of assay conditions. For chemicals like urethane that require specific metabolic activation to cause genotoxicity, it is important that in vitro tests are conducted using cell lines exhibiting the activity and induction of CYP450 enzymes, including CYP2E1 enzyme that is important in the metabolism of urethane, at a concentration representing actual or perceived chemical exposure. We compared 2D MCL-5 cells and HepG2 cells with 3D HepG2 hanging drop spheroids to determine the genotoxicity of urethane using the micronucleus assay. Our 2D studies with MCL-5 did not show any statistically significant genotoxicity [99% relative population doubling (RPD)] compared to controls for concentrations and time point tested in vitro. HepG2 cells grown as 2D indicated that exposure to urethane of up to 30 mM for 23 h did not cause any genotoxic effect (102% RPD) but, at higher concentrations, genotoxicity was produced with only 89–85% RPD. Furthermore, an exposure of 20–50 mM for 23 h using 3D hanging drop spheroid assays revealed a higher MN frequency, thus exhibiting in vitro genotoxicity of urethane in metabolically active cell models. In comparison with previous studies, this study indicated that urethane genotoxicity is dose, sensitivity of cell model (2D vs. 3D) and exposure dependent.

It is well understood that poor diet and lifestyle choices can increase the risk of cancer. It is also well documented that cancer is a disease of DNA mutations, with mutations in key genes driving carcinogenesis. Measuring these mutations in a minimally invasive way may be informative as to which exposures are harmful and thus allow us to introduce primary preventative measures, in a bid to reduce cancer incidences. Here, we have measured mutations in the phosphatidylinositol glycan class A (PIG-A) gene in erythrocytes from healthy volunteers (n = 156) and from non-cancer patients attending the local endoscopy department (n = 144). The X-linked PIG-A gene encodes an enzyme involved in glycosylphosphatidylinositol (GPI) anchor synthesis. A silencing mutation in which leads to the absence of GPI anchors on the extracellular surface which can be rapidly assessed using flow cytometry. The background level of PIG-A mutant erythrocytes was 2.95 (95% CI: 2.59–3.67) mutant cells (10−6). Older age increased mutant cell frequency (P < 0.001). There was no difference in mutant cell levels between males and females (P = 0.463) or smokers and non-smokers (P = 0.186). In the endoscopy group, aspirin users had lower mutant frequencies (P = 0.001). Further information on diet and exercise was available for the endoscopy patient group alone, where those with a higher health promotion index score had lower mutant frequencies (P = 0.011). Higher dietary intake of vegetables reduced mutant cell levels (P = 0.022). Participants who exercised for at least 1 h a week appeared to have reduced mutant frequencies than those who did not exercise, although this was not statistically significant (P = 0.099). This low background level of mutant erythrocytes in a population makes this assay an attractive tool to monitor exposures such as those associated with lifestyles and diet, as demonstrated here.

Human ENP exposure is inevitable and the novel, size-dependent physicochemical properties that enable ENPs to be beneficial in innovative technologies are concomitantly causing heightened public concerns as to their potential adverse effects upon human health. This study aims to deduce the mechanisms associated with potential ENP mediated (geno)toxicity and impact upon telomere integrity, if any, of varying concentrations of both ∼16 nm (4.34 × 10

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 μg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.

Due to the expansive application of TiO2 and its variance in physico-chemical characteristics, the toxicological profile of TiO2, in all its various forms, requires evaluation. This study aimed to assess the hazard of five TiO2 particle-types in relation to their cytotoxic profile correlated to their cellular interaction, specifically in human lymphoblast (TK6) and type-II alveolar epithelial (A549) cells. Treatment with the test materials was undertaken at a concentration range of 1–100 μg/cm2 over 24 and 72 h exposure. TiO2 interaction with both cell types was visualised by transmission electron microscopy, supported by energy-dispersive X-ray. None of the TiO2 materials tested promoted cytotoxicity in either cell type over the concentration and time range studied. All materials were observed to interact with the A549 cells and were further noted to be internalised following 24 h exposure. In contrast, only the pigmentary rutile was internalised by TK6 lymphoblasts after 24 h exposure. Where uptake was observed there was no evidence, as determined by 2D microscopy techniques, of particle localisation within the nucleus of either cell type. This study indicates that industrially relevant TiO2 particles demonstrate cell interactions that are cell-type dependent and do not induce cytotoxicity at the applied dose range.

Due to the expansive application of TiO

Human exposure to carbon black (CB) is inevitable due to its widespread applications in the medical, industrial and consumer sectors. With an ageing population, it is imperative that the effects of (nano)particle exposure in individuals with compromised immunity or infection are considered. Since barrier immunity provides the first line of defence against CB and the human skin and lung pathogen, Staphylococcus aureus, this work focuses on studying the impact of CB exposure upon compromised immunity during infection on human skin and lung epithelial cells in vitro. The principal aim of the work was to develop an epithelial cell model to characterise (co-)exposure to CB and S. aureus. The work used two human epithelial cell lines, HaCaT (skin) and A549 (lung), ELISA technology to assess the (pro-)inflammatory response, aseptic microbiology techniques to grow S. aureus and a Zetasizer, EDX spectroscopy, and both scanning and transmission electron microscopy (SEM and TEM) to characterise the CB under the conditions used in the study. Physicochemical characterisation of CB confirmed its shape, dramatic polydispersity and potential to aggregate. CB significantly inhibited S. aureus growth, but in a biological media dependent manner. CB did not induce cytokines or antimicrobial peptides from skin and lung epithelial cells, when given alone. In contrast, S. aureus induced a robust interleukin (IL)-8 response in both skin and lung epithelial cells. IL-6 and human beta defensin (hβD)-2 could only be detected when cells were stimulated with S. aureus. However, co-exposure to CB (100µg/ml) and S. aureus resulted in significant inhibition of IL-8 (compared to S. aureus only induced levels). Furthermore, the same co-exposure induced significantly more hβD-2 (compared to S. aureus alone). The ability to detect pathogen responses to particle, in addition to epithelial responses to particle and pathogen is an advance on assessing cell responses under ‘healthy’ conditions and supports the need for developing exposure models under stressed or immunosuppressed conditions. This model will be useful for studying mechanisms of exposure in at-risk groups, including factory workers, the elderly and immunocompromised. Advanced models, that better represent human pathophysiology are essential for understanding cellular mechanisms of toxicity in the 21st Century.

Background Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (p p Conclusions Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound’s subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001–770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the “misleading” in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing. Electronic supplementary material The online version of this article (10.1007/s00204-020-02902-3) contains supplementary material, which is available to authorized users.

Background: Toxicological evaluation of engineered nanomaterials (ENMs) is essential for occupational health and safety, particularly where bulk manufactured ENMs such as few-layer graphene (FLG) are concerned. Additionally, there is a necessity to develop advanced in vitro models when testing ENMs to provide a physiologically relevant alternative to invasive animal experimentation. The aim of this study was to determine the genotoxicity of non-functionalised (neutral), amine- and carboxyl-functionalised FLG upon both human-transformed type-I (TT1) alveolar epithelial cell monocultures, as well as co-cultures of TT1 and differentiated THP-1 monocytes (d.THP-1 (macrophages)). Results: In monocultures, TT1 and d.THP-1 macrophages showed a statistically significant (pin vitro cytokinesis blocked micronucleus (CBMN) assay revealed significant (pConclusions: Therefore, FLG genotoxicity when examined in monocultures, results in primary-indirect DNA damage; whereas co-cultured cells reveal secondary mechanisms of DNA damage.

Nanotechnologies have reached maturity and market penetration that require nano‐specific changes in legislation and harmonization among legislation domains, such as the amendments to REACH for nanomaterials (NMs) which came into force in 2020. Thus, an assessment of the components and regulatory boundaries of NMs risk governance is timely, alongside related methods and tools, as part of the global efforts to optimise nanosafety and integrate it into product design processes, via Safe(r)‐by‐Design (SbD) concepts. This paper provides an overview of the state‐of‐the‐art regarding risk governance of NMs and lays out the theoretical basis for the development and implementation of an effective, trustworthy and transparent risk governance framework for NMs. The proposed framework enables continuous integration of the evolving state of the science, leverages best practice from contiguous disciplines and facilitates responsive re‐thinking of nanosafety governance to meet future needs. To achieve and operationalise such framework, a science‐based Risk Governance Council (RGC) for NMs is being developed. The framework will provide a toolkit for independent NMs' risk governance and integrates needs and views of stakeholders. An extension of this framework to relevant advanced materials and emerging technologies is also envisaged, in view of future foundations of risk research in Europe and globally., This study received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 814425 (RiskGONE) and No 814572 (NanoSolveIT).

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (≤14 days). These spheroid models (≥500 µm in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the 'gold standard' micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. The liver's role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.

Six-transmembrane epithelial antigen of the prostate-2 (STEAP2) expression is increased in prostate cancer when compared to normal prostate, suggesting STEAP2 may drive prostate cancer progression. This study aimed to establish the functional role of STEAP2 in prostate tumourigenesis and evaluate if its knockdown resulted in reduced invasive potential of prostate cancer cells. PC3 and LNCaP cells were transfected with STEAP2 siRNA and proliferation, migration, invasion and gene expression analyses were performed. STEAP2 immunohistochemistry was applied to assess the protein expression and localisation according to Gleason score in 164 prostate cancer patients. Invasion significantly decreased in both cell lines following STEAP2 knockdown. PC3 proliferation and migration capacity significantly reduced, while LNCaP cell morphology and growth characteristics were altered. Additionally, STEAP2 downstream targets associated with driving invasion were identified as MMP3, MMP10, MMP13, FGFR4, IL1β, KiSS1 and SERPINE1 in PC3 cells and, MMP7 in LNCaP cells, with CD82 altered in both. In patient tissues, STEAP2 expression was significantly increased in prostate cancer samples and this significantly correlated with Gleason score. These data demonstrate that STEAP2 drives aggressive prostate cancer traits by promoting proliferation, migration and invasion and significantly influencing the transcriptional profile of ten genes underlying the metastatic cascade.

The liver’s role in metabolism of chemicals makes it an appropriate tissue for toxicity testing. Current testing protocols, such as animal testing and two-dimensional liver cell systems, offer limited resemblance to in vivo liver cell behaviour, in terms of gene expression profiles and metabolic competence; thus, they do not always accurately predict human toxicology. In vitro three-dimensional liver cell models offer an attractive alternative. This study reports on the development of a 3D liver model, using HepG2 cells, by a hanging-drop technique, with a focus on evaluating spheroid growth characteristics and suitability for genotoxicity testing. The cytokinesis-blocked micronucleus assay protocol was adapted to enable micronucleus (MN) detection in the 3D spheroid models. This involved evaluating the difference between hanging vs non-hanging drop positions for dosing of the test agents and comparison of automated Metafer scoring with manual scoring for MN detection in HepG2 spheroids. The initial seeding density, used for all experiments, was 5000 cells/20 μl drop hanging spheroids, harvested on day 4, with >75% cell viability. Albumin secretion (7.8 g/l) and both CYP1A1 and CYP1A2 gene expression were highest in the 3D environment at day 4. Exposure to metabolically activated genotoxicants for 24 h resulted in a 6-fold increase in CYP1A1 enzyme activity (3 μM B[a]P) and a 30-fold increase in CYP1A2 enzyme activity (5 μM PhIP) in 3D hanging spheroids. MN inductions in response to B[a]P or PhIP were 2-fold and 3-fold, respectively, and were greater in 3D hanging spheroids than in 2D format, showing that hanging spheroids are more sensitive to genotoxic agents. HepG2 hanging-drop spheroids are an exciting new alternative system for genotoxicity studies, due to their improved structural and physiological properties, relative to 2D cultures.