Critical aspects in dissolution testing of nanomaterials in the oro-gastrointestinal tract: the relevance of juice composition for hazard identification and grouping.

The dissolution of a nanomaterial (NM) in an in vitro simulant of the oro-gastrointestinal (OGI) tract is an important predictor of its biodurability in vivo. The cascade addition of simulated digestive juices (saliva, stomach and intestine), including inorganic/organic biomacromolecules and digestive enzymes (complete composition, referred to as "Type 1 formulation"), strives for realistic representation of chemical composition of the OGI tract. However, the data robustness requires consideration of analytical feasibility, such as the use of simplified media. Here we present a systematic analysis of the effects exerted by different digestive juice formulations on the dissolution% (or half-life values) of benchmark NMs (e.g., zinc oxide, titanium dioxide, barium sulfate, and silicon dioxide). The digestive juices were progressively simplified by removal of components such as organic molecules, enzymes, and inorganic molecules (Type 2, 3 and 4). The results indicate that the "Type 1 formulation" augments the dissolution via sequestration of ions by measurable factors compared to formulations without enzymes (i.e., Type 3 and 4). Type 1 formulation is thus regarded as a preferable option for predicting NM biodurability for hazard assessment. However, for grouping purposes, the relative similarity among diverse nanoforms (NFs) of a NM is decisive. Two similarity algorithms were applied, and additional case studies comprising NFs and non NFs of the same substance were included. The results support the grouping decision by simplified formulation (Type 3) as a robust method for screening and grouping purposes.

How to formulate hypotheses and IATAs to support grouping and read-across of nanoforms.

Manufacturing and functionalizing materials at the nanoscale has led to the generation of a whole array of nanoforms (NFs) of substances varying in size, morphology, and surface characteristics. Due to financial, time, and ethical considerations, testing every unique NF for adverse effects is virtually impossible. Use of hypothesis-driven grouping and read-across approaches, as supported by the GRACIOUS Framework, represents a promising alternative to case-by-case testing that will make the risk assessment process more efficient. Through application of appropriate grouping hypotheses, the Framework facilitates the assessment of similarity between NFs, thereby supporting grouping and read-across of information, minimizing the need for new testing, and aligning with the 3R principles of replacement, reduction, and refinement of animals in toxicology studies. For each grouping hypothesis an integrated approach to testing and assessment (IATA) guides the user in data gathering and acquisition to test the hypothesis, following a structured format to facilitate efficient decision-making. Here we present the template used to generate the GRACIOUS grouping hypotheses encompassing information relevant to "Lifecycle, environmental release, and human exposure", "What they are: physicochemical characteristics", "Where they go: environmental fate, uptake, and toxicokinetics", and "What they do: human and environmental toxicity". A summary of the template-derived hypotheses focusing on human health is provided, along with an overview of the IATAs generated by the GRACIOUS project. We discuss the application and flexibility of the template, providing the opportunity to expand the application of grouping and read-across in a logical, evidence-based manner to a wider range of NFs and substances.

Grouping of orally ingested silica nanomaterials via use of an integrated approach to testing and assessment to streamline risk assessment.

BACKGROUND: Nanomaterials can exist in different nanoforms (NFs). Their grouping may be supported by the formulation of hypotheses which can be interrogated via integrated approaches to testing and assessment (IATA). IATAs are decision trees that guide the user through tiered testing strategies (TTS) to collect the required evidence needed to accept or reject a grouping hypothesis. In the present paper, we investigated the applicability of IATAs for ingested NFs using a case study that includes different silicon dioxide, SiO2 NFs. Two oral grouping hypotheses addressing local and systemic toxicity were identified relevant for the grouping of these NFs and verified through the application of oral IATAs. Following different Tier 1 and/or Tier 2 in vitro methods of the TTS (i.e., in vitro dissolution, barrier integrity and inflammation assays), we generated the NF datasets. Furthermore, similarity algorithms (e.g., Bayesian method and Cluster analysis) were utilized to identify similarities among the NFs and establish a provisional group(s). The grouping based on Tier 1 and/or Tier 2 testing was analyzed in relation to available Tier 3 in vivo data in order to verify if the read-across was possible and therefore support a grouping decision. RESULTS: The measurement of the dissolution rate of the silica NFs in the oro-gastrointestinal tract and in the lysosome identified them as gradually dissolving and biopersistent NFs. For the local toxicity to intestinal epithelium (e.g. cytotoxicity, membrane integrity and inflammation), the biological results of the gastrointestinal tract models indicate that all of the silica NFs were similar with respect to the lack of local toxicity and, therefore, belong to the same group; in vivo data (although limited) confirmed the lack of local toxicity of NFs. For systemic toxicity, Tier 1 data did not identify similarity across the NFs, with results across different decision nodes being inconsistent in providing homogeneous group(s). Moreover, the available Tier 3 in vivo data were also insufficient to support decisions based upon the obtained in vitro results and relating to the toxicity of the tested NFs. CONCLUSIONS: The information generated by the tested oral IATAs can be effectively used for similarity assessment to support a grouping decision upon the application of a hypothesis related to toxicity in the gastrointestinal tract. The IATAs facilitated a structured data analysis and, by means of the expert's interpretation, supported read-across with the available in vivo data. The IATAs also supported the users in decision making, for example, reducing the testing when the grouping was well supported by the evidence and/or moving forward to advanced testing (e.g., the use of more suitable cellular models or chronic exposure) to improve the confidence level of the data and obtain more focused information.

Comparison of In Vitro Approaches to Assess the Antibacterial Effects of Nanomaterials.

The antibacterial properties of nanomaterials (NMs) can be exploited in a range of consumer products (e.g., wound dressings, food packaging, textiles, medicines). There is also interest in the exploitation of NMs as treatments for infectious diseases to help combat antibiotic resistance. Whilst the antibacterial activity of NMs has been assessed in vitro and in vivo in numerous studies, the methodology used is very varied. Indeed, while numerous approaches are available to assess the antibacterial effect of NMs in vitro, they have not yet been systematically assessed for their suitability and sensitivity for testing NMs. It is therefore timely to consider what assays should be prioritised to screen the antibacterial properties of NMs. The majority of existing in vitro studies have focused on investigating the antibacterial effects exhibited by silver (Ag) NMs and have employed a limited range of assays. We therefore compared the antibacterial effects of copper oxide (CuO) NMs to Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis at various concentrations (12.5-200 µg/mL) using a battery of tests (well and disc diffusion, plate counts-time-kill method, optical density measurement-OD, Alamar Blue and live/dead viability assays, and quantitative polymerase chain reaction). CuO NMs were most toxic to B. subtilis and E. coli, while P. aeruginosa was the least sensitive strain. All assays employed detected the antibacterial activity of CuO NMs; however, they varied in their sensitivity, time, cost, technical difficulty and requirement for specialized equipment. In the future, we suggest that a combination of approaches is used to provide a robust assessment of the antibacterial activity of NMs. In particular, we recommend that the time-kill and OD assays are prioritised due to their greater sensitivity. We also suggest that standard operating protocols are developed so that the antibacterial activity of NMs can be assessed using a harmonised approach.

Assessing the transferability and reproducibility of 3D in vitro liver models from primary human multi-cellular microtissues to cell-line based HepG2 spheroids.

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.

Transgenic zebrafish larvae as a non-rodent alternative model to assess pro-inflammatory (neutrophil) responses to nanomaterials.

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, LTB4, 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. LTB4 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).

Grouping MWCNTs based on their similar potential to cause pulmonary hazard after inhalation: a case-study.

BACKGROUND: The EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing. RESULTS: The case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument. CONCLUSION: This proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of 'similar' MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process.

Integrated approaches to testing and assessment for grouping nanomaterials following dermal exposure.

Exposure to different nanoforms (NFs) via the dermal route is expected in occupational and consumer settings and thus it is important to assess their dermal toxicity and the contribution of dermal exposure to systemic bioavailability. We have formulated four grouping hypotheses for dermal toxicity endpoints which allow NFs to be grouped to streamline and facilitate risk assessment. The grouping hypotheses are developed based on insight into how physicochemical properties of NFs (i.e. composition, dissolution kinetics, size, and flexibility) influence their fate and hazard following dermal exposure. Each hypothesis is accompanied by a tailored Integrated Approach to Testing and Assessment (IATA) that is structured as a decision tree and tiered testing strategies (TTS) for each relevant question (at decision nodes) that indicate what information is needed to guide the user to accept or reject the grouping hypothesis. To develop these hypotheses and IATAs, we gathered and analyzed existing information on skin irritation, skin sensitization, and dermal penetration of NFs from the published literature and performed experimental work to generate data on NF dissolution in sweat simulant fluids. We investigated the dissolution of zinc oxide and silicon dioxide NFs in different artificial sweat fluids, demonstrating the importance of using physiologically relevant conditions for dermal exposure. All existing and generated data informed the formulation of the grouping hypotheses, the IATAs, and the design of the TTS. It is expected that the presented IATAs will accelerate the NF risk assessment for dermal toxicity via the application of read-across.

Determining nanoform similarity via assessment of surface reactivity by abiotic and in vitro assays.

Grouping of substances is a method used to streamline hazard and risk assessment. Assessment of similarity provides the scientific evidence needed for formation of groups. This work reports on justification of grouping of nanoforms (NFs) via similarity of their surface reactivity. Four reactivity assays were used for concentration dependent detection of reactive oxygen species (ROS) generated by NFs: abiotic assays FRAS, EPR and DCFH2-DA, as well as the in vitro assay of NRF2/ARE responsive luciferase reporter activation in the HEK293 cell line. Representative materials (CuO, Mn2O3, BaSO4, CeO2 and ZnO) and three case studies of each several NFs of iron oxides, Diketopyrrolopyrroles (DPP)-based organic pigments and silicas were assessed. A novel similarity assessment algorithm was applied to quantify similarities between pairs of NFs, in a four-step workflow on concentration-response curves, individual concentration and response ranges, and finally the representative materials. We found this algorithm to be applicable to all abiotic and in vitro assays that were tested. Justification of grouping must include the increased potency of smaller particles via the scaling of effects with specific surface, and hence quantitative similarity analysis was performed on concentration-response in mass-metrics. CuO and BaSO4 were the most and least reactive representative materials respectively, and all assays found BaSO4/CuO not similar, as confirmed by their different NOAECs of in vivo studies. However, similarity outcomes from different reactivity assays were not always in agreement, highlighting the need to generate data by one assay for the representative materials and the candidate group of NFs. Despite low similarity scores in vitro some pairs of case study NFs can be accepted as sufficiently similar because the in vivo NOAECs are similar, highlighting the conservative assessment by the abiotic assays.

The application of existing genotoxicity methodologies for grouping of nanomaterials: towards an integrated approach to testing and assessment.

The incorporation of nanomaterials (NMs) in consumer products has proven to be highly valuable in many sectors. Unfortunately, however, the same nano specific physicochemical properties, which make these material attractive, might also contribute to hazards for people exposed to these materials. The physicochemical properties of NMs will impact their interaction with biological surroundings and influence their fate and their potential adverse effects such as genotoxicity. Due to the large and expanding number of NMs produced, their availability in different nanoforms (NFs) and their utilization in various formats, it is impossible for risk assessment to be conducted on an individual NF basis. Alternative methods, such as grouping are needed for streamlining hazard assessment. The GRACIOUS Framework provides a logical and science evidenced approach to group similar NFs, allowing read-across of hazard information from source NFs (or non-NFs) with adequate hazard data to target NFs that lack such data. Here, we propose a simple three-tiered testing strategy to gather evidence to determine whether different NFs are sufficiently similar with respect to their potential to induce genotoxicity, in order to be grouped. The tiered testing strategy includes simple in vitro models as well as a number of alternative more complex multi-cellular in vitro models to allow for a better understanding of secondary NM-induced DNA damage, something that has been more appropriate in vivo until recently.

How can we justify grouping of nanoforms for hazard assessment? Concepts and tools to quantify similarity.

The risk of each nanoform (NF) of the same substance cannot be assumed to be the same, as they may vary in their physicochemical characteristics, exposure and hazard. However, neither can we justify a need for more animal testing and resources to test every NF individually. To reduce the need to test all NFs, (regulatory) information requirements may be fulfilled by grouping approaches. For such grouping to be acceptable, it is important to demonstrate similarities in physicochemical properties, toxicokinetic behaviour, and (eco)toxicological behaviour. The GRACIOUS Framework supports the grouping of NFs, by identifying suitable grouping hypotheses that describe the key similarities between different NFs. The Framework then supports the user to gather the evidence required to test these hypotheses and to subsequently assess the similarity of the NFs within the proposed group. The evidence needed to support a hypothesis is gathered by an Integrated Approach to Testing and Assessment (IATA), designed as decision trees constructed of decision nodes. Each decision node asks the questions and provides the methods needed to obtain the most relevant information. This White paper outlines existing and novel methods to assess similarity of the data generated for each decision node, either via a pairwise analysis conducted property-by-property, or by assessing multiple decision nodes simultaneously via a multidimensional analysis. For the pairwise comparison conducted property-by-property we included in this White paper: The x-fold, Bayesian and Arsinh-OWA distance algorithms performed comparably in the scoring of similarity between NF pairs. The Euclidean distance was also useful, but only with proper data transformation. The x-fold method does not standardize data, and thus produces skewed histograms, but has the advantage that it can be implemented without programming knowhow. A range of multidimensional evaluations, using for example dendrogram clustering approaches, were also investigated. Multidimensional distance metrics were demonstrated to be difficult to use in a regulatory context, but from a scientific perspective were found to offer unexpected insights into the overall similarity of very different materials. In conclusion, for regulatory purposes, a property-by-property evaluation of the data matrix is recommended to substantiate grouping, while the multidimensional approaches are considered to be tools of discovery rather than regulatory methods.

Bayesian based similarity assessment of nanomaterials to inform grouping.

Nanoforms can be manufactured in plenty of variants by differing their physicochemical properties and toxicokinetic behaviour which can affect their hazard potential. To avoid testing of each single nanomaterial and nanoform variation and subsequently save resources, grouping and read-across strategies are used to estimate groups of substances, based on carefully selected evidence, that could potentially have similar human health and environmental hazard impact. A novel computational similarity method is presented aiming to compare dose-response curves and identify sets of similar nanoforms. The suggested method estimates the statistical model that best fits the data by leveraging pairwise Bayes Factor analysis to compare pairs of curves and evaluate whether each of the nanoforms is sufficiently similar to all other nanoforms. Pairwise comparisons to benchmark materials are used to define threshold similarity values and set the criteria for identifying groups of nanoforms with comparatively similar toxicity. Applications to use case data are shown to demonstrate that the method can support grouping hypotheses linked to a certain hazard endpoint and route of exposure.

An in vitro investigation of the hepatic toxicity of PEGylated polymeric redox responsive nanoparticles.

It can be challenging to deliver drugs to cancer cells in a targeted manner at an effective dose. Polymeric nanoparticles (NPs) are promising drug delivery systems that can be targeted to cancer cells using redox responsive elements. More specifically, intracellular and extracellular levels of the antioxidant glutathione (GSH) are elevated in cancer cells and therefore the use of NPs with a cleavable GSH-responsive element allowing these NPs to target cancer cells and trigger the release of their cargo (e.g. anticancer drugs). The aim of this study was to assess the hepatotoxicity of polymeric NP delivery systems with and without a redox sensitive element. Copolymer poly (lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) NPs with (RR-NPs) and without (nRR-NPs) a redox responsive dithiylethanoate ester linker were synthesised and their toxicity assessed in vitro. As the liver is a primary site of NP accumulation, the C3A hepatocyte cell line was used to assess NP toxicity in vitro via investigation of cytotoxicity, cytokine production, genotoxicity, intracellular reactive oxygen species (ROS) production, intracellular calcium concentration, and hepatocyte function (albumin and urea production). The cellular uptake of NPs was also assessed as this may influence the cellular dose and, therefore, the cellular response. Both NPs had no detrimental impact on cell viability. However, both NPs stimulated an increase in cytokine (IL-1ra) and ROS production and decreased hepatocyte function, with the greatest effect observed for nRR-NPs. Only nRR-NPs caused DNA damage. Cells internalised both NPs and caused a (sub-lethal) increase in intracellular calcium levels. Therefore, whilst the NPs did not have a negative impact on cell viability, the NPs were able to elicit sub-lethal toxicity. By using a battery of tests we were able to demonstrate that RR-NPs may be less toxic than nRR-NPs. Our findings can therefore feed into the development of safer and more effective nanomedicines and into the design of testing strategies to assess polymeric NP safety based on knowledge of their mechanism of toxicity.

The Road to Achieving the European Commission's Chemicals Strategy for Nanomaterial Sustainability-A PATROLS Perspective on New Approach Methodologies.

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.

Development of a standard operating procedure for the DCFH2-DA acellular assessment of reactive oxygen species produced by nanomaterials.

Improved strategies are required for testing nanomaterials (NMs) to make hazard and risk assessment more efficient and sustainable. Including reduced reliance on animal models, without decreasing the level of human health protection. Acellular detection of reactive oxygen species (ROS) may be useful as a screening assay to prioritize NMs of high concern. To improve reliability and reproducibility, and minimize uncertainty, a standard operating procedure (SOP) has been developed for the detection of ROS using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH2-DA) assay. The SOP has undergone an inter- and intra-laboratory comparison, to evaluate robustness, reliability, and reproducibility, using representative materials (ZnO, CuO, Mn2O3, and BaSO4 NMs), and a number of calibration tools to normalize data. The SOP includes an NM positive control (nanoparticle carbon black (NPCB)), a chemical positive control (SIN-1), and a standard curve of fluorescein fluorescence. The interlaboratory comparison demonstrated that arbitrary fluorescence units show high levels of partner variability; however, data normalization improved variability. With statistical analysis, it was shown that the SIN-1 positive control provided an extremely high level of reliability and reproducibility as a positive control and as a normalization tool. The NPCB positive control can be used with a relatively high level of reproducibility, and in terms of the representative materials, the reproducibility CuO induced-effects was better than for Mn2O3. Using this DCFH2-DA acellular assay SOP resulted in a robust intra-laboratory reproduction of ROS measurements from all NMs tested, while effective reproduction across different laboratories was also demonstrated; the effectiveness of attaining reproducibility within the interlaboratory assessment was particle-type-specific.

Particulate and drug-induced toxicity assessed in novel quadruple cell human primary hepatic disease models of steatosis and pre-fibrotic NASH.

In an effort to replace, reduce and refine animal experimentation, there is an unmet need to advance current in vitro models that offer features with physiological relevance and enhanced predictivity of in vivo toxicological output. Hepatic toxicology is key following chemical, drug and nanomaterials (NMs) exposure, as the liver is vital in metabolic detoxification of chemicals as well as being a major site of xenobiotic accumulation (i.e., low solubility particulates). With the ever-increasing production of NMs, there is a necessity to evaluate the probability of consequential adverse effects, not only in health but also in clinically asymptomatic liver, as part of risk stratification strategies. In this study, two unique disease initiation and maintenance protocols were developed and utilised to mimic steatosis and pre-fibrotic NASH in scaffold-free 3D liver microtissues (MT) composed of primary human hepatocytes, hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. The characterized diseased MT were utilized for the toxicological assessment of a panel of xenobiotics. Highlights from the study included: 1. Clear experimental evidence for the pre-existing liver disease is important in the augmentation of xenobiotic-induced hepatotoxicity and 2. NMs are able to activate stellate cells. The data demonstrated that pre-existing disease is vital in the intensification of xenobiotic-induced liver damage. Therefore, it is imperative that all stages of the wide spectrum of liver disease are incorporated in risk assessment strategies. This is of significant consequence, as a substantial number of the general population suffer from sub-clinical liver injury without any apparent or diagnosed manifestations.

An in vitro assessment of the toxicity of two-dimensional synthetic and natural layered silicates.

Natural Layered Silicates (NLS) and Synthetic Layered Silicates (SLS) are a diverse group of clay minerals that have attracted great interest in various branches of industry. However, despite growing demand for this class of material, their impact on human health has not been fully investigated. Therefore, the aim of this study was to evaluate and compare the potential toxic effects of a wide range of commercially available SLS and NLS of varying physicochemical properties (lithium (Li) or fluoride (F) content and size). Mouse BALB/c monocyte macrophage (J774A.1) and human monocyte-derived macrophages (MDMs) were chosen as in vitro models of alveolar macrophages. Montmorillonite, hectorite, Medium (med) F/High Li and Low F/Med Li particles, were cytotoxic to cells and induced potent pro-inflammatory responses. The remaining particles (No F/Very (V)Low Li, No F/Med Li, No F/Low Li, High F/Med Li and High F/Med Li washed) were non- to relatively low- cytotoxic and inflammogenic, in both type of cells. In an acellular condition none of the tested samples increased reactive oxygen species (ROS), while ROS generation was observed following exposure to sublethal concentrations of Med F/High Li, Low F/Med Li, montmorillonite and hectorite samples, in J774A.1 cells. Based on the results obtained in this study the toxic potency of tested samples was not associated with lithium or fluoride content, but appeared to be dependent on particle size, with the platelets of larger dimension and lower surface area being more potent than the smaller platelet particles with higher surface area. In addition, the increased bioactivity of Med F/High Li and Low F/Med Li was associated with endotoxin contamination. Obtained results demonstrated that layered silicate materials have different toxicological profiles and suggest that toxicological properties of a specific layered silicate should be investigated on an individual basis.

An Integrated Approach to Testing and Assessment to Support Grouping and Read-Across of Nanomaterials After Inhalation Exposure.

Introduction: Here, we describe the generation of hypotheses for grouping nanoforms (NFs) after inhalation exposure and the tailored Integrated Approaches to Testing and Assessment (IATA) with which each specific hypothesis can be tested. This is part of a state-of-the-art framework to support the hypothesis-driven grouping and read-across of NFs, as developed by the EU-funded Horizon 2020 project GRACIOUS. Development of Grouping Hypotheses and IATA: Respirable NFs, depending on their physicochemical properties, may dissolve either in lung lining fluid or in acidic lysosomal fluid after uptake by cells. Alternatively, NFs may also persist in particulate form. Dissolution in the lung is, therefore, a decisive factor for the toxicokinetics of NFs. This has led to the development of four hypotheses, broadly grouping NFs as instantaneous, quickly, gradually, and very slowly dissolving NFs. For instantaneously dissolving NFs, hazard information can be derived by read-across from the ions. For quickly dissolving particles, as accumulation of particles is not expected, ion toxicity will drive the toxic profile. However, the particle aspect influences the location of the ion release. For gradually dissolving and very slowly dissolving NFs, particle-driven toxicity is of concern. These NFs may be grouped by their reactivity and inflammation potency. The hypotheses are substantiated by a tailored IATA, which describes the minimum information and laboratory assessments of NFs under investigation required to justify grouping. Conclusion: The GRACIOUS hypotheses and tailored IATA for respiratory toxicity of inhaled NFs can be used to support decision making regarding Safe(r)-by-Design product development or adoption of precautionary measures to mitigate potential risks. It can also be used to support read-across of adverse effects such as pulmonary inflammation and subsequent downstream effects such as lung fibrosis and lung tumor formation after long-term exposure.

Grouping Hypotheses and an Integrated Approach to Testing and Assessment of Nanomaterials Following Oral Ingestion.

The risk assessment of ingested nanomaterials (NMs) is an important issue. Here we present nine integrated approaches to testing and assessment (IATAs) to group ingested NMs following predefined hypotheses. The IATAs are structured as decision trees and tiered testing strategies for each decision node to support a grouping decision. Implications (e.g., regulatory or precautionary) per group are indicated. IATAs integrate information on durability and biopersistence (dissolution kinetics) to specific hazard endpoints, e.g., inflammation and genotoxicity, which are possibly indicative of toxicity. Based on IATAs, groups of similar nanoforms (NFs) of a NM can be formed, such as very slow dissolving, highly biopersistent and systemically toxic NFs. Reference NMs (ZnO, SiO2 and TiO2) along with related NFs are applied as case studies to testing the oral IATAs. Results based on the Tier 1 level suggest a hierarchy of biodurability and biopersistence of TiO2 > SiO2 > ZnO, and are confirmed by in vivo data (Tier 3 level). Interestingly, our analysis suggests that TiO2 and SiO2 NFs are able to induce both local and systemic toxicity along with microbiota dysbiosis and can be grouped according to the tested fate and hazard descriptors. This supports that the decision nodes of the oral IATAs are suitable for classification and assessment of the toxicity of NFs.