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.

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.

A weight of evidence review of the genotoxicity of titanium dioxide (TiO2).

Titanium dioxide is a ubiquitous white material found in a diverse range of products from foods to sunscreens, as a pigment and thickener, amongst other uses. Titanium dioxide has been considered no longer safe for use in foods (nano and microparticles of E171) by the European Food Safety Authority (EFSA) due to concerns over genotoxicity. There are however, conflicting opinions regarding the safety of Titanium dioxide. In an attempt to clarify the situation, a comprehensive weight of evidence (WoE) assessment of the genotoxicity of titanium dioxide based on the available data was performed. A total of 192 datasets for endpoints and test systems considered the most relevant for identifying mutagenic and carcinogenic potential were reviewed and discussed for both reliability and relevance (by weight of evidence) and in the context of whether the physico-chemical properties of the particles had been characterised. The view of an independent panel of experts was that, of the 192 datasets identified, only 34 met the reliability and quality criteria for being most relevant in the evaluation of genotoxicity. Of these, 10 were positive (i.e. reported evidence that titanium dioxide was genotoxic), all of which were from studies of DNA strand breakage (comet assay) or chromosome damage (micronucleus or chromosome aberration assays). All the positive findings were associated with high cytotoxicity, oxidative stress, inflammation, apoptosis, necrosis, or combinations of these. Considering that DNA and chromosome breakage can be secondary to physiological stress, it is highly likely that the observed genotoxic effects of titanium dioxide, including those with nanoparticles, are secondary to physiological stress. Consistent with this finding, there were no positive results from the in vitro and in vivo gene mutation studies evaluated, although it should be noted that to definitively conclude a lack of mutagenicity, more robust in vitro and in vivo gene mutation studies would be useful. Existing evidence does not therefore support a direct DNA damaging mechanism for titanium dioxide (nano and other forms).

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).

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.

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.

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.

Time dependent impact of copper oxide nanomaterials on the expression of genes associated with oxidative stress, metal binding, inflammation and mucus secretion in single and co-culture intestinal in vitro models.

The potential for ingestion of copper oxide nanomaterials (CuO NMs) is increasing due to their increased exploitation. Investigation of changes in gene expression allows toxicity to be detected at an early stage of NM exposure and can enable investigation of the mechanism of toxicity. Here, undifferentiated Caco-2 cells, differentiated Caco-2 cells, Caco-2/HT29-MTX (mucus secreting) and Caco-2/Raji B (M cell model) co-cultures were exposed to CuO NMs and copper sulphate (CuSO4) in order to determine their impacts. Cellular responses were measured in terms of production of reactive oxygen species (ROS), the gene expression of an antioxidant (haem oxygenase 1 (HMOX1)), the pro-inflammatory cytokine (interleukin 8 (IL8)), the metal binding (metallothionein 1A and 2A (MT1A and MT2A)) and the mucus secreting (mucin 2 (MUC2)), as well as HMOX-1 protein level. While CuSO4 induced ROS production in cells, no such effect was observed for CuO NMs. However, these particles did induce an increase in the level of HMOX-1 protein and upregulation of HMOX1, MT2A, IL8 and MUC2 genes in all cell models. In conclusion, the expression of HMOX1, IL8 and MT2A were responsive to CuO NMs at 4 to 12 h post exposure when investigating the toxicity of NMs using intestinal in vitro models. These findings can inform the selection of endpoints, timepoints and models when investigating NM toxicity to the intestine in vitro in the future.

Acute waterborne and chronic sediment toxicity of silver and titanium dioxide nanomaterials towards the oligochaete, Lumbriculus variegatus.

The use of silver (Ag) and titanium dioxide (TiO2) nanomaterials (NMs) in industrial processes and consumer products has experienced considerable growth since the late 20th century. Throughout their lifecycle, both Ag NM and TiO2NM are released into the environment, with benthic systems anticipated to be the final sink. Their potential toxicity towards benthic species is therefore of major concern. This study investigated the toxicity of silver (Ag; NM-300 K) and titanium dioxide (TiO2; NM-104) NMs to the freshwater oligochaete, Lumbriculus variegatus in acute (0-96-h) waterborne and chronic (28-d) sediment studies. Toxicity was investigated via assessment of mortality, behaviour, and antioxidant enzyme activity. The 96-h LC50 for Ag NMs in water was 0.51 mg/l (95% CI, 0.45-0.56), with L. variegatus displaying inhibited predation-avoidance behaviour compared to controls (6.66 ± 10%) successful response at 24-h), as well as significant increases (p < 0.05) in catalase (CAT) activity at sub-lethal concentrations at 24-h. Behavioural improvement and the return of antioxidant enzymes to control levels was observed after 48 and 72-h. AgNO3 exposure proved more toxic than Ag NM (96-h LC50 = 0.034 mg/l, 95% CI, 0.031-0.037) but resulted in no changes to antioxidant enzymes following sub-lethal exposure. Furthermore, Ag dissolution from Ag NM (~2-4%) could not account for the full extent of toxicity observed, suggesting a nano-specific effect. Increased environmental relevance via the inclusion of Suwannee River Humic Acid (SRHA, 5 mg/l) alleviated sub-lethal Ag NM toxicity despite a comparable 96-h LC50 (0.54 mg/l, 95% CI, 0.51-0.57). Significant effects of Ag NMs in formulated sediments (mortality, biomass) were only recorded according to OECD 225 at the highest test concentration (1333 mg/kg) for Ag NM indicating a potential attenuating effect of sediments towards toxicity. No toxicity was observed for TiO2 NM in aquatic or sediment exposures up to concentrations of 2000 mg/l and 1333 mg/kg, respectively.

Neutrophil activation by nanomaterials in vitro: comparing strengths and limitations of primary human cells with those of an immortalized (HL-60) cell line.

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 (TiO2) 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. TiO2 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.

Is Environmental and Occupational Particulate Air Pollution Exposure Related to Type-2 Diabetes and Dementia? A Cross-Sectional Analysis of the UK Biobank.

Human exposure to particulate air pollution (e.g., PM2.5) can lead to adverse health effects, with compelling evidence that it can increase morbidity and mortality from respiratory and cardiovascular disease. More recently, there has also been evidence that long-term environmental exposure to particulate air pollution is associated with type-2 diabetes mellitus (T2DM) and dementia. There are many occupations that may expose workers to airborne particles and that some exposures in the workplace are very similar to environmental particulate pollution. We conducted a cross-sectional analysis of the UK Biobank cohort to verify the association between environmental particulate air pollution (PM2.5) exposure and T2DM and dementia, and to investigate if occupational exposure to particulates that are similar to those found in environmental air pollution could increase the odds of developing these diseases. The UK Biobank dataset comprises of over 500,000 participants from all over the UK. Environmental exposure variables were used from the UK Biobank. To estimate occupational exposure both the UK Biobank's data and information from a job exposure matrix, specifically developed for UK Biobank (Airborne Chemical Exposure-Job Exposure Matrix (ACE JEM)), were used. The outcome measures were participants with T2DM and dementia. In appropriately adjusted models, environmental exposure to PM2.5 was associated with an odds ratio (OR) of 1.02 (95% CI 1.00 to 1.03) per unit exposure for developing T2DM, while PM2.5 was associated with an odds ratio of 1.06 (95% CI 0.96 to 1.16) per unit exposure for developing dementia. These environmental results align with existing findings in the published literature. Five occupational exposures (dust, fumes, diesel, mineral, and biological dust in the most recent job estimated with the ACE JEM) were investigated and the risks for most exposures for T2DM and for all the exposures for dementia were not significantly increased in the adjusted models. This was confirmed in a subgroup of participants where a full occupational history was available allowed an estimate of workplace exposures. However, when not adjusting for gender, some of the associations become significant, which suggests that there might be a bias between the occupational assessments for men and women. The results of the present study do not provide clear evidence of an association between occupational exposure to particulate matter and T2DM or dementia.

In Utero Exposure to Particulate Air Pollution during Pregnancy: Impact on Birth Weight and Health through the Life Course.

In high-income countries, and increasingly in lower- and middle-income countries, chronic non-communicable diseases (NCDs) have become the primary health burden. It is possible that in utero exposure to environmental pollutants such as particulate matter (PM) may have an impact on health later in life, including the development of NCDs. Due to a lack of data on foetal growth, birth weight is often used in epidemiologic studies as a proxy to assess impacts on foetal development and adverse birth outcomes since it is commonly recorded at birth. There are no research studies with humans that directly link PM exposure in utero to birth weight (BW) and subsequently, the effects of lower BW on health outcomes in old age. It is, however, plausible that such associations exist, and it is thus important to assess the potential public health impacts of PM across the life course, and it is plausible to use birth weight as an indicator of risk. We therefore split this narrative review into two parts. In the first part, we evaluated the strength of the evidence on the impact of PM exposure during the entire pregnancy on birth weight outcomes in ten meta-analyses. In the second part, we reviewed the literature linking lower birth weight to childhood and adult chronic cardiovascular disease to explore the potential implications of PM exposure in utero on health later in life. Within the reviewed meta-studies on birth weight, there is sufficient evidence that PM pollution is associated with lower birth weight, i.e., the majority of meta-studies found statistically significant reductions in birth weight. From the second part of the review, it is evident that there is good evidence of associations between lower birth weight and subsequent cardiovascular disease risk. It is thus plausible that in utero exposure to PM is associated with lower birth weight and persisting biological changes that could be associated with adverse health effects in adulthood. Based on the reviewed evidence, however, the magnitude of later life cardiovascular health impacts from in utero exposure and its impact on BW are likely to be small compared to health effects from exposure to particulate air pollution over a whole lifetime.

Evaluation of the Suitability of an Existing Job-Exposure Matrix for the Assessment of Exposure of UK Biobank Participants to Dust, Fumes, and Diesel Exhaust Particulates.

Many epidemiological studies have shown an association between outdoor particulate air pollutants and increased morbidity and mortality. Inhalation of ambient aerosols can exacerbate or promote the development of cardiovascular and pulmonary diseases as well as other diseases, such as type 2 diabetes mellitus (T2DM) and neurodegenerative diseases. Occupational exposure to dust, fumes and diesel exhaust particulates can also cause adverse health outcomes and there are numerous occupations where workers are exposed to airborne particles that are similar to ambient air pollution. An individual's job title has normally been identified as a major determinant of workplace exposure in epidemiological studies. This has led to the development of Job-Exposure Matrices (JEMs) as a way of characterising specific workplace exposures. One JEM for airborne chemical exposures is the Airborne Chemical Exposure Job-Exposure Matrix (ACE JEM), developed specifically for the UK Biobank cohort. The objective of this paper is to evaluate the suitability of the ACE JEM in assessing occupational aerosol exposure of participants in the UK Biobank. We searched the scientific literature to identify exposure data linked to selected jobs in the ACE JEM and compared these data with the JEM assessments. Additionally, we carried out an independent expert-based assessment of exposure to compare with the JEM estimates. There is good published evidence to substantiate the high dust and biological dust assignments in the JEM and more limited evidence for diesel exhaust particulates. There is limited evidence in the published literature to substantiate moderate or low exposure assignments in the JEM. The independent expert-based assessment found good agreement at the two extremes of exposure in the JEM (high and no exposure), with uncertainty in all other classifications. The ACE JEM assignments are probably reliable for highly exposed jobs and for jobs assigned as unexposed. However, the assignments for medium and low exposures are less reliable. The ACE JEM is likely to be a good tool to examine associations between occupational exposures to particulates and chronic disease, although it should be used with caution. Further efforts should be made to improve the reliability of the ACE JEM.

Using 3D gastrointestinal tract in vitro models with microfold cells and mucus secreting ability to assess the hazard of copper oxide nanomaterials.

BACKGROUND: Copper oxide nanomaterials (CuO NMs) are exploited in many products including inks, cosmetics, textiles, wood preservatives and food contact materials. Their incorporation into these products may enhance oral exposure in consumer, environmental and occupational settings. Undifferentiated and differentiated monocultures of Caco-2 cells are commonly used to assess NM toxicity to the intestine in vitro. However, the integration of other cell types into Caco-2 in vitro models increases their physiological relevance. Therefore, the aim of this study is to evaluate the toxicity of CuO NMs and copper sulphate (CuSO4) to intestinal microfold (M) cell (Caco-2/Raji B) and mucus secreting (Caco-2/HT29-MTX) co-culture in vitro models via assessment of their impact on barrier integrity, viability and interleukin (IL)-8 secretion. The translocation of CuO NMs and CuSO4 across the intestinal barrier was also investigated in vitro. RESULTS: CuO NMs and CuSO4 impaired the function of the intestinal barrier in the co-culture models [as indicated by a reduction in transepithelial electrical resistance (TEER) and Zonular occludens (ZO-1) staining intensity]. Cu translocation was observed in both models but was greatest in the Caco-2/Raji B co-culture. CuO NMs and CuSO4 stimulated an increase in IL-8 secretion, which was greatest in the Caco-2/HT29-MTX co-culture model. CuO NMs and CuSO4 did not stimulate a loss of cell viability, when assessed using light microscopy, nuclei counts and scanning electron microscopy. CuO NMs demonstrated a relatively similar level of toxicity to CuO4 in both Caco-2/Raji B and Caco-2/HT29-MTX co- culture models. CONCLUSIONS: The Caco-2/Raji B co-culture model was more sensitive to CuO NM and CuSO4 toxicity than the Caco-2/HT29-MTX co-culture model. However, both co-culture models were less sensitive to CuO NM and CuSO4 toxicity than simple monocultures of undifferentiated and differentiated Caco-2 cells, which are more routinely used to investigate NM toxicity to the intestine. Obtained data can therefore feed into the design of future studies which assess the toxicity of substances (e.g. NMs) and pathogens to the intestine (e.g. by informing model and endpoint selection). However, more testing with a wider panel of NMs would be beneficial in order to help select which in vitro models and endpoints to prioritise when screening the safety of ingested NMs. Comparisons with in vivo findings will also be essential to identify the most suitable in vitro model to screen the safety of ingested NMs.

The influence of organic modification on the cytotoxicity of clay particles to keratinocytes, hepatocytes and macrophages; an investigation towards the safe use of polymer-clay nanocomposite packaging.

Organically modified clays can be used as nanofillers in polymer-clay nanocomposites to create bio-based packaging with improved strength and barrier properties. The impact of organic modification on the physico-chemical properties and toxicity of clays has yet to be fully investigated but is essential to ensure their safe use. Two organoclays, named N116_HDTA and N116_TMSA, were prepared using a commercially available sodium bentonite clay and the organic modifiers hexadecyl trimethyl ammonium bromide (HDTA) and octadecyl trimethyl ammonium chloride (TMSA). An in vitro hazard assessment was performed using HaCaT skin cells, C3A liver cells, and J774.1 macrophage-like cells. Organic modification with HDTA and TMSA increased the hazard potential of the organoclays in all cell models, as evidenced by the higher levels of cytotoxicity measured. N116_TMSA caused the greatest loss in viability with IC50 values of 3.2, 3.6 and 6.1 µg/cm2 calculated using J774.1, HaCaT and C3A cell lines, respectively. Cytotoxic effects were dictated by the amount of free or displaced organic modifier present in the exposure suspensions. The parent bentonite clay also caused distinct cytotoxic effects in J774.1 macrophage-like cells with associated TNF-α release. Such information on the hazard profile of organoclays, can feed into risk assessments for these materials.

Assessing the bioactivity of crystalline silica in heated high-temperature insulation wools.

High-Temperature Insulation Wools (HTIW), such as alumino silicate wools (Refractory Ceramic Fibers) and Alkaline Earth Silicate wools, are used in high-temperature industries for thermal insulation. These materials have an amorphous glass-like structure. In some applications, exposure to high temperatures causes devitrification resulting in the formation of crystalline species including crystalline silica. The formation of this potentially carcinogenic material raises safety concerns regarding after-use handling and disposal. This study aims to determine whether cristobalite formed in HTIW is bioactive in vitro. Mouse macrophage (J774A.1) and human alveolar epithelial (A549) cell lines were exposed to pristine HTIW of different compositions, and corresponding heat-treated samples. Cell death, cytokine release, and reactive oxygen species (ROS) formation were assessed in both cell types. Cell responses to aluminum lactate-coated fibers were assessed to determine if responses were caused by crystalline silica. DQ12 α-quartz was used as positive control, and TiO2 as negative control. HTIW did not induce cell death or intracellular ROS, and their ability to induce pro-inflammatory mediator release was low. In contrast, DQ12 induced cytotoxicity, a strong pro-inflammatory response and ROS generation. The modest pro-inflammatory mediator responses of HTIW did not always coincide with the formation of cristobalite in heated fibers; therefore, we cannot confirm that devitrification of HTIW results in bioactive cristobalite in vitro. In conclusion, the biological responses to HTIW observed were not attributable to a single physicochemical characteristic; instead, a combination of physicochemical characteristics (cristobalite content, fiber chemistry, dimensions and material solubility) appear to contribute to induction of cellular responses.

Exposure to Environmental and Occupational Particulate Air Pollution as a Potential Contributor to Neurodegeneration and Diabetes: A Systematic Review of Epidemiological Research.

It has been hypothesised that environmental air pollution, especially airborne particles, is a risk factor for type 2 diabetes mellitus (T2DM) and neurodegenerative conditions. However, epidemiological evidence is inconsistent and has not been previously evaluated as part of a systematic review. Our objectives were to carry out a systematic review of the epidemiological evidence on the association between long-term exposure to ambient air pollution and T2DM and neurodegenerative diseases in adults and to identify if workplace exposures to particles are associated with an increased risk of T2DM and neurodegenerative diseases. Assessment of the quality of the evidence was carried out using the GRADE system, which considers the quality of the studies, consistency, directness, effect size, and publication bias. Available evidence indicates a consistent positive association between ambient air pollution and both T2DM and neurodegeneration risk, such as dementia and a general decline in cognition. However, corresponding evidence for workplace exposures are lacking. Further research is required to identify the link and mechanisms associated with particulate exposure and disease pathogenesis and to investigate the risks in occupational populations. Additional steps are needed to reduce air pollution levels and possibly also in the workplace environment to decrease the incidence of T2DM and cognitive decline.

The Essential Elements of a Risk Governance Framework for Current and Future Nanotechnologies.

Societies worldwide are investing considerable resources into the safe development and use of nanomaterials. Although each of these protective efforts is crucial for governing the risks of nanomaterials, they are insufficient in isolation. What is missing is a more integrative governance approach that goes beyond legislation. Development of this approach must be evidence based and involve key stakeholders to ensure acceptance by end users. The challenge is to develop a framework that coordinates the variety of actors involved in nanotechnology and civil society to facilitate consideration of the complex issues that occur in this rapidly evolving research and development area. Here, we propose three sets of essential elements required to generate an effective risk governance framework for nanomaterials. (1) Advanced tools to facilitate risk-based decision making, including an assessment of the needs of users regarding risk assessment, mitigation, and transfer. (2) An integrated model of predicted human behavior and decision making concerning nanomaterial risks. (3) Legal and other (nano-specific and general) regulatory requirements to ensure compliance and to stimulate proactive approaches to safety. The implementation of such an approach should facilitate and motivate good practice for the various stakeholders to allow the safe and sustainable future development of nanotechnology.

Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.

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.

Impact of copper oxide nanomaterials on differentiated and undifferentiated Caco-2 intestinal epithelial cells; assessment of cytotoxicity, barrier integrity, cytokine production and nanomaterial penetration.

BACKGROUND: Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. METHODS: Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 µg/cm2 Cu (equivalent to 1.95 to 250 µg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 µg/cm2 for CuO NMs, and 4.25 µg/cm2 for copper sulphate (CuSO4), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO4 on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO4 on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO4 was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (Papp); a measure of barrier permeability to CuO NMs. For all experiments, CuSO4 was used as an ionic control. RESULTS: CuO NMs and CuSO4 caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO4 translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO4 decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted. CONCLUSIONS: CuO NMs and CuSO4 stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the Papp and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO4. Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.