Roadmap towards safe and sustainable advanced and innovative materials. (Outlook for 2024-2030).

The adoption of innovative advanced materials holds vast potential, contingent upon addressing safety and sustainability concerns. The European Commission advocates the integration of Safe and Sustainable by Design (SSbD) principles early in the innovation process to streamline market introduction and mitigate costs. Within this framework, encompassing ecological, social, and economic factors is paramount. The NanoSafety Cluster (NSC) delineates key safety and sustainability areas, pinpointing unresolved issues and research gaps to steer the development of safe(r) materials. Leveraging FAIR data management and integration, alongside the alignment of regulatory aspects, fosters informed decision-making and innovation. Integrating circularity and sustainability mandates clear guidance, ensuring responsible innovation at every stage. Collaboration among stakeholders, anticipation of regulatory demands, and a commitment to sustainability are pivotal for translating SSbD into tangible advancements. Harmonizing standards and test guidelines, along with regulatory preparedness through an exchange platform, is imperative for governance and market readiness. By adhering to these principles, the effective and sustainable deployment of innovative materials can be realized, propelling positive transformation and societal acceptance.

Toxicological evaluation of primary particulate matter emitted from combustion of aviation fuel.

Recently, Sustainable Aviation Fuel (SAF) blends and novel combustion technologies have been introduced to reduce aircraft engine emissions. However, there is limited knowledge about the impact of combustion technology and fuel composition on toxicity of primary Particulate Matter (PM) emissions, comparable to regulated non-volatile PM (nvPM). In this study, primary PM was collected on filters using a standardised approach, from both a Rich-Quench-Lean (RQL) combustion rig and a bespoke liquid fuelled Combustion Aerosol Standard (CAST) Generator burning 12 aviation fuels including conventional Jet-A, SAFs, and blends thereof. The fuels varied in aromatics (0-25.2%), sulphur (0-3000 ppm) and hydrogen (13.43-15.31%) contents. Toxicity of the collected primary PM was studied in vitro utilising Air-Liquid Interface (ALI) exposure of lung epithelial cells (Calu-3) in monoculture and co-culture with macrophages (differentiated THP-1 cells). Cells were exposed to PM extracted from filters and nebulised from suspensions using a cloud-based ALI exposure system. Toxicity readout parameters were analysed 24h after exposure. RESULTS: showed presence of genotoxicity and changes in gene expression at dose levels which did not induce cytotoxicity. DNA damage was detected through Comet assay in cells exposed to CAST generated samples. Real-Time PCR performed to investigate the expression profile of genes involved in oxidative stress and DNA repair pathways showed different behaviours after exposure to the various PM samples. No differences were found in pro-inflammatory interleukin-8 secretion. This study indicates that primary PM toxicity is driven by wider factors than fuel composition, highlighting that further work is needed to substantiate the full toxicity of aircraft exhaust PM inclusive of secondary PM emanating from numerous engine technologies across the power range burning conventional Jet-A and SAF.

Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials.

To facilitate Safe and Sustainable by Design (SSbD) strategies during the development of nanomaterials (NMs), quick and easy in vitro assays to test for hazard potential at an early stage of NM development are essential. The formation of reactive oxygen species (ROS) and the induction of oxidative stress are considered important mechanisms that can lead to NM toxicity. In vitro assays measuring oxidative stress are therefore commonly included in NM hazard assessment strategies. The fluorescence-based dichloro-dihydro-fluorescein (DCFH) assay for cellular oxidative stress is a simple and cost-effective assay, making it a good candidate assay for SSbD hazard testing strategies. It is however subject to several pitfalls and caveats. Here, we provide further optimizations to the assay using 5-(6)-Chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA-AE, referred to as DCFH probe), known for its improved cell retention. We measured the release of metabolic products of the DCFH probe from cells to supernatant, direct reactions of CM-H2DCFDA-AE with positive controls, and compared the commonly used plate reader-based DCFH assay protocol with fluorescence microscopy and flow cytometry-based protocols. After loading cells with DCFH probe, translocation of several metabolic products of the DCFH probe to the supernatant was observed in multiple cell types. Translocated DCFH products are then able to react with test substances including positive controls. Our results also indicate that intracellularly oxidized fluorescent DCF is able to translocate from cells to the supernatant. In either way, this will lead to a fluorescent supernatant, making it difficult to discriminate between intra- and extra-cellular ROS production, risking misinterpretation of possible oxidative stress when measuring fluorescence on a plate reader. The use of flow cytometry instead of plate reader-based measurements resolved these issues, and also improved assay sensitivity. Several optimizations of the flow cytometry-based DCFH ISO standard (ISO/TS 19006:2016) were suggested, including loading cells with DCFH probe before incubation with the test materials, and applying an appropriate gating strategy including live-death staining, which was not included in the ISO standard. In conclusion, flow cytometry- and fluorescence microscopy-based read-outs are preferred over the classical plate reader-based read-out to assess the level of intracellular oxidative stress using the cellular DCFH assay.

Comparing the quality of care in physician networks to usual care for elderly patients in three German regions: a quasi-experimental cohort study.

OBJECTIVES: Patients in Germany have free choice of physicians in the ambulatory care sector and can consult them as often as they wish without a referral. This can lead to inefficiencies in treatment pathways. In response, some physicians have formed networks to improve the coordination and quality of care. This study aims to investigate whether the care provided by these networks results in better health and process outcomes than usual care. STUDY DESIGN: This was a quasi-experimental cohort study. METHODS: We analysed claims data from 2017 to 2018 in Bavaria, Brandenburg, and Westphalia-Lippe. Our study population includes patients aged 65 years or older with heart failure (n = 267,256), back pain (n = 931,672), or depression (n = 483,068). We compared condition-specific and generic quality indicators between patients treated in physician networks and usual care. Ambulatory care-sensitive emergency department cases were used as a primary outcome measure. Imbalances between the groups were minimized using propensity score matching. RESULTS: Rates of ambulatory care-sensitive emergency department cases yielded insignificant differences between networks and usual care in the depression and heart failure subgroups. For back pain patients, rates were 0.17 percentage points higher (P < 0.01) in network patients compared with usual care. Among network patients, generic indicators for prevention and coordination showed significantly better performance. For instance, the rate of completed vaccination against influenza is 3.03 percentage points higher (P < 0.01), and the rate of specialist visits after referral is 1.6 percentage points higher (P < 0.01) in heart failure patients, who are treated in physician networks. This is accompanied by higher rates of polypharmacy. Furthermore, the results for condition-specific indicators suggest that for most indicators, a greater proportion of the care provided by physician networks adhered to national treatment guidelines. CONCLUSIONS: Our findings suggest that physician networks in Germany do not reduce rates of ambulatory care-sensitive emergency department cases but perform better than usual care in terms of care coordination and prevention. Further research is needed to confirm our findings and explore the implications of the potentially higher rates of polypharmacy seen in physician networks.

Micro- and nanoplastics concepts for particle and fibre toxicologists.

Micro- and nanoplastic particles (MNP) are omnipresent as either pollution or intentionally used in consumer products, released from packaging or even food. There is an exponential increase in the production of plastics. With the realization of bioaccumulation in humans, toxicity research is quickly expanding. There is a rapid increase in the number of papers published on the potential implications of exposure to MNP which necessitates a call for quality criteria to be applied when doing the research. At present, most papers on MNP describe the effects of commercially available polymer (mostly polystyrene) beads that are typically not the MNP of greatest concern. This is not a fault of the research community, necessarily, as the MNPs to which humans are exposed are usually not available in the quantities needed for toxicological research and innovations are needed to supply environmentally-relevant MNP models. In addition, like we have learned from decades of research with particulate matter and engineered nanomaterials, sample physicochemical characteristics and preparation can have major impacts on the biological responses and interpretation of the research findings. Lastly, MNP dosimetry may pose challenges as (1) we are seeing early evidence that plastics are already in the human body at quite high levels that may be difficult to achieve in acute in vitro studies and (2) plastics are already in the diets fed to preclinical models. This commentary highlights the pitfalls and recommendations for particle and fibre toxicologists that should be considered when performing and disseminating the research.

In vitro neurotoxicity of particles from diesel and biodiesel fueled engines following direct and simulated inhalation exposure.

Combustion-derived particulate matter (PM) is a major source of air pollution. Efforts to reduce diesel engine emission include the application of biodiesel. However, while urban PM exposure has been linked to adverse brain effects, little is known about the direct effects of PM from regular fossil diesel (PMDEP) and biodiesel (PMBIO) on neuronal function. Furthermore, it is unknown to what extent the PM-induced effects in the lung (e.g., inflammation) affect the brain. This in vitro study investigates direct and indirect toxicity of PMDEP and PMBIO on the lung and brain and compared it with effects of clean carbon particles (CP). PM were generated using a common rail diesel engine. CP was sampled from a spark generator. First, effects of 48 h exposure to PM and CP (1.2-3.9 µg/cm2) were assessed in an in vitro lung model (air-liquid interface co-culture of Calu-3 and THP1 cells) by measuring cell viability, cytotoxicity, barrier function, inflammation, and oxidative and cell stress. None of the exposures caused clear adverse effects and only minor changes in gene expression were observed. Next, the basal medium was collected for subsequent simulated inhalation exposure of rat primary cortical cells. Neuronal activity, recorded using microelectrode arrays (MEA), was increased after acute (0.5 h) simulated inhalation exposure. In contrast, direct exposure to PMDEP and PMBIO (1-100 µg/mL; 1.2-119 µg/cm2) reduced neuronal activity after 24 h with lowest observed effect levels of respectively 10 µg/mL and 30 µg/mL, indicating higher neurotoxic potency of PMDEP, whereas neuronal activity remained unaffected following CP exposure. These findings indicate that combustion-derived PM potently inhibit neuronal function following direct exposure, while the lung serves as a protective barrier. Furthermore, PMDEP exhibit a higher direct neurotoxic potency than PMBIO, and the data suggest that the neurotoxic effects is caused by adsorbed chemicals rather than the pure carbon core.

First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

Spatial distribution of selenium-mercury in Arctic seabirds.

Mercury (Hg) is a metallic trace element toxic for humans and wildlife that can originate from natural and anthropic sources. Hg spatial gradients have been found in seabirds from the Arctic and other oceans, suggesting contrasting toxicity risks across regions. Selenium (Se) plays a protective role against Hg toxicity, but its spatial distribution has been much less investigated than that of Hg. From 2015 to 2017, we measured spatial co-exposure of Hg and Se in blood samples of two seabird species, the Brünnich's guillemot (Uria lomvia) and the black-legged kittiwake (Rissa tridactyla) from 17 colonies in the Arctic and subarctic regions, and we calculated their molar ratios (Se:Hg), as a measure of Hg sequestration by Se and, therefore, of Hg exposure risk. We also evaluated concentration differences between species and ocean basins (Pacific-Arctic and Atlantic-Arctic), and examined the influence of trophic ecology on Hg and Se concentrations using nitrogen and carbon stable isotopes. In the Atlantic-Arctic ocean, we found a negative west-to-east gradient of Hg and Se for guillemots, and a positive west-to-east gradient of Se for kittiwakes, suggesting that these species are better protected from Hg toxicity in the European Arctic. Differences in Se gradients between species suggest that they do not follow environmental Se spatial variations. This, together with the absence of a general pattern for isotopes influence on trace element concentrations, could be due to foraging ecology differences between species. In both oceans, the two species showed similar Hg concentrations, but guillemots showed lower Se concentrations and Se:Hg than kittiwakes, suggesting a higher Hg toxicity risk in guillemots. Within species, neither Hg, nor Se or Se:Hg differed between both oceans. Our study highlights the importance of considering Se together with Hg, along with different species and regions, when evaluating Hg toxic effects on marine predators in international monitoring programs.

Airway contraction and cytokine release in isolated rat lungs induced by wear particles from the road and tire interface and road vehicle brakes.

The dominant road traffic particle sources are wear particles from the road and tire interface, and from vehicle brake pads. The aim of this work was to investigate the effect of road and brake wear particles on pulmonary function and biomarkers in isolated perfused rat lungs. Particles were sampled from the studded tire wear of three road pavements containing different rock materials in a road simulator; and from the wear of two brake pad materials using a pin-on-disk machine. Isolated rat lungs inhaled the coarse and fine fractions of the sampled particles resulting in an estimated total particle lung dose of 50 µg. The tidal volume (TV) was measured during the particle exposure and the following 50 min. Perfusate and BALF were analyzed for the cytokines TNF, CXCL1 and CCL3. The TV of lungs exposed to rock materials was significantly reduced after 25 min of exposure compared to the controls, for quartzite already after 4 min. The particles of the heavy-duty brake pads had no effect on the TV. Brake particles resulted in a significant elevation of CXCL1 in the perfusate. Brake particles showed significant elevations of all three measured cytokines, and quartzite showed a significant elevation of TNF in BALF. The study shows that the toxic effect on lungs exposed to airborne particles can be investigated using measurements of tidal volume. Furthermore, the study shows that the choice of rock material in road pavements has the potential to affect the toxicity of road wear PM10.

Placental-fetal distribution of carbon particles in a pregnant rabbit model after repeated exposure to diluted diesel engine exhaust.

BACKGROUND: Airborne pollution particles have been shown to translocate from the mother's lung to the fetal circulation, but their distribution and internal placental-fetal tissue load remain poorly explored. Here, we investigated the placental-fetal load and distribution of diesel engine exhaust particles during gestation under controlled exposure conditions using a pregnant rabbit model. Pregnant dams were exposed by nose-only inhalation to either clean air (controls) or diluted and filtered diesel engine exhaust (1 mg/m3) for 2 h/day, 5 days/week, from gestational day (GD) 3 to GD27. At GD28, placental and fetal tissues (i.e., heart, kidney, liver, lung and gonads) were collected for biometry and to study the presence of carbon particles (CPs) using white light generation by carbonaceous particles under femtosecond pulsed laser illumination. RESULTS: CPs were detected in the placenta, fetal heart, kidney, liver, lung and gonads in significantly higher amounts in exposed rabbits compared with controls. Through multiple factor analysis, we were able to discriminate the diesel engine exposed pregnant rabbits from the control group taking all variables related to fetoplacental biometry and CP load into consideration. Our findings did not reveal a sex effect, yet a potential interaction effect might be present between exposure and fetal sex. CONCLUSIONS: The results confirmed the translocation of maternally inhaled CPs from diesel engine exhaust to the placenta which could be detected in fetal organs during late-stage pregnancy. The exposed can be clearly discriminated from the control group with respect to fetoplacental biometry and CP load. The differential particle load in the fetal organs may contribute to the effects on fetoplacental biometry and to the malprogramming of the fetal phenotype with long-term effects later in life.

In vitro neurotoxicity screening of engine oil- and hydraulic fluid-derived aircraft cabin bleed-air contamination.

In most airplanes, cabin air is extracted from the turbine compressors, so-called bleed air. Bleed air can become contaminated by leakage of engine oil or hydraulic fluid and possible neurotoxic constituents, like triphenyl phosphate (TPhP) and tributyl phosphate (TBP). The aim of this study was to characterize the neurotoxic hazard of TBP and TPhP, and to compare this with the possible hazard of fumes originating from engine oils and hydraulic fluids in vitro. Effects on spontaneous neuronal activity were recorded in rat primary cortical cultures grown on microelectrode arrays following exposure for 0.5 h (acute), and 24 h and 48 h (prolonged) to TBP and TPhP (0.01-100 µM) or fume extracts (1-100 µg/mL) prepared from four selected engine oils and two hydraulic fluids by a laboratory bleed air simulator. TPhP and TBP concentration-dependently reduced neuronal activity with equal potency, particularly during acute exposure (TPhP IC50: 10-12 µM; TBP IC50: 15-18 µM). Engine oil-derived fume extracts persistently reduced neuronal activity. Hydraulic fluid-derived fume extracts showed a stronger inhibition during 0.5 h exposure, but the degree of inhibition attenuates during 48 h. Overall, fume extracts from hydraulic fluids were more potent than those from engine oils, in particular during 0.5 h exposure, although the higher toxicity is unlikely to be due only to higher levels of TBP and TPhP in hydraulic fluids. Our combined data show that bleed air contaminants originating from selected engine oils or hydraulic fluids exhibit neurotoxic hazard in vitro, with fumes derived from the selected hydraulic fluids being most potent.

The State of the Art and Challenges of In Vitro Methods for Human Hazard Assessment of Nanomaterials in the Context of Safe-by-Design.

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.

Establishing relationships between particle-induced in vitro and in vivo inflammation endpoints to better extrapolate between in vitro markers and in vivo fibrosis.

BACKGROUND: Toxicity assessment for regulatory purposes is starting to move away from traditional in vivo methods and towards new approach methodologies (NAM) such as high-throughput in vitro models and computational tools. For materials with limited hazard information, utilising quantitative Adverse Outcome Pathways (AOPs) in a testing strategy involving NAM can produce information relevant for risk assessment. The aim of this work was to determine the feasibility of linking in vitro endpoints to in vivo events, and moreover to key events associated with the onset of a chosen adverse outcome to aid in the development of NAM testing strategies. To do this, we focussed on the adverse outcome pathway (AOP) relating to the onset of pulmonary fibrosis. RESULTS: We extracted in vivo and in vitro dose-response information for particles known to induce this pulmonary fibrosis (crystalline silica, specifically α-quartz). To test the in vivo-in vitro extrapolation (IVIVE) determined for crystalline silica, cerium dioxide nanoparticles (nano-CeO2) were used as a case study allowing us to evaluate our findings with a less studied substance. The IVIVE methodology outlined in this paper is formed of five steps, which can be more generally summarised into two categories (i) aligning the in vivo and in vitro dosimetry, (ii) comparing the dose-response curves and derivation of conversion factors. CONCLUSION: Our analysis shows promising results with regards to correlation of in vitro cytokine secretion to in vivo acute pulmonary inflammation assessed by polymorphonuclear leukocyte influx, most notable is the potential of using IL-6 and IL-1ß cytokine secretion from simple in vitro submerged models as a screening tool to assess the likelihood of lung inflammation at an early stage in product development, hence allowing a more targeted investigation using either a smaller, more targeted in vivo study or in the future a more complex in vitro protocol. This paper also highlights the strengths and limitations as well as the current difficulties in performing IVIVE assessment and suggestions for overcoming these issues.

Neuromodulatory and neurotoxic effects of e-cigarette vapor using a realistic exposure method.

The most direct effects of inhaled harmful constituents are the effects on the airways. However, inhaled compounds can be rapidly absorbed and subsequently result in systemic effects. For example, e-cigarette vapor has been shown to evoke local effects in the lung, although little is known about subsequent effects in secondary target organs such as the brain. Traditionally, such effects are tested using in vivo models. As an alternative, we have combined two in vitro systems, which are Air-Liquid-Interface (ALI) cultured alveolar cells (A549) and rat primary cortical cultures grown on multi-well microelectrode arrays. This allows us to assess the neurological effects of inhaled compounds. We have used exposure to e-cigarette vapor, containing nicotine, menthol, or vanillin to test the model. Our results show that ALI cultured A549 cells respond to the exposure with the production of cytokines (IL8 and GROalpha). Furthermore, nicotine, menthol, and vanillin were found on the basolateral side of the cell culture, which indicates their translocation. Upon transfer of the basolateral medium to the primary cortical culture, exposure-related changes in spontaneous electrical activity were observed correlating with the presence of e-liquid components in the medium. These clear neuromodulatory effects demonstrate the feasibility of combining continuous exposure of ALI cultured cells with subsequent exposure of neuronal cells to assess neurotoxicity. Although further optimization steps are needed, such a combination of methods is important to assess the neurotoxic effects of inhaled compounds realistically. As such, an approach like this could play a role in future mechanism-based risk assessment strategies.

An inter-laboratory effort to harmonize the cell-delivered in vitro dose of aerosolized materials.

Air-liquid interface (ALI) lung cell models cultured on permeable transwell inserts are increasingly used for respiratory hazard assessment requiring controlled aerosolization and deposition of any material on ALI cells. The approach presented herein aimed to assess the transwell insert-delivered dose of aerosolized materials using the VITROCELL® Cloud12 system, a commercially available aerosol-cell exposure system. An inter-laboratory comparison study was conducted with seven European partners having different levels of experience with the VITROCELL® Cloud12. A standard operating procedure (SOP) was developed and applied by all partners for aerosolized delivery of materials, i.e., a water-soluble molecular substance (fluorescence-spiked salt) and two poorly soluble particles, crystalline silica quartz (DQ12) and titanium dioxide nanoparticles (TiO2 NM-105). The material dose delivered to transwell inserts was quantified with spectrofluorometry (fluorescein) and with the quartz crystal microbalance (QCM) integrated in the VITROCELL® Cloud12 system. The shape and agglomeration state of the deposited particles were confirmed with transmission electron microscopy (TEM). Inter-laboratory comparison of the device-specific performance was conducted in two steps, first for molecular substances (fluorescein-spiked salt), and then for particles. Device- and/or handling-specific differences in aerosol deposition of VITROCELL® Cloud12 systems were characterized in terms of the so-called deposition factor (DF), which allows for prediction of the transwell insert-deposited particle dose from the particle concentration in the aerosolized suspension. Albeit DF varied between the different labs from 0.39 to 0.87 (mean (coefficient of variation (CV)): 0.64 (28%)), the QCM of each VITROCELL® Cloud 12 system accurately measured the respective transwell insert-deposited dose. Aerosolized delivery of DQ12 and TiO2 NM-105 particles showed good linearity (R2 > 0.95) between particle concentration of the aerosolized suspension and QCM-determined insert-delivered particle dose. The VITROCELL® Cloud 12 performance for DQ12 particles was identical to that for fluorescein-spiked salt, i.e., the ratio of measured and salt-predicted dose was 1.0 (29%). On the other hand, a ca. 2-fold reduced dose was observed for TiO2 NM-105 (0.54 (41%)), which was likely due to partial retention of TiO2 NM-105 agglomerates in the vibrating mesh nebulizer of the VITROCELL® Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL® Cloud 12 is a reliable tool for dosimetry, which accounts for potential variations of the transwell insert-delivered dose due to device-, handling- and/or material-specific effects. With the detailed protocol presented herein, all seven partner laboratories were able to demonstrate dose-controlled aerosolization of material suspensions using the VITROCELL® Cloud12 exposure system at dose levels relevant for observing in vitro hazard responses. This is an important step towards regulatory approved implementation of ALI lung cell cultures for in vitro hazard assessment of aerosolized materials.

Road tunnel-derived coarse, fine and ultrafine particulate matter: physical and chemical characterization and pro-inflammatory responses in human bronchial epithelial cells.

BACKGROUND: Traffic particulate matter (PM) comprises a mixture of particles from fuel combustion and wear of road pavement, tires and brakes. In countries with low winter temperatures the relative contribution of mineral-rich PM from road abrasion may be especially high due to use of studded tires during winter season. The aim of the present study was to sample and characterize size-fractioned PM from two road tunnels paved with different stone materials in the asphalt, and to compare the pro-inflammatory potential of these fractions in human bronchial epithelial cells (HBEC3-KT) in relation to physicochemical characteristics. METHODS: The road tunnel PM was collected with a vacuum pump and a high-volume cascade impactor sampler. PM was sampled during winter, both during humid and dry road surface conditions, and before and after cleaning the tunnels. Samples were analysed for hydrodynamic size distribution, content of elemental carbon (EC), organic carbon (OC) and endotoxin, and the capacity for acellular generation of reactive oxygen species. Cytotoxicity and pro-inflammatory responses were assessed in HBEC3-KT cells after exposure to coarse (2.5-10 µm), fine (0.18-2.5 µm) and ultrafine PM (≤ 0.18 µm), as well as particles from the respective stone materials used in the pavement. RESULTS: The pro-inflammatory potency of the PM samples varied between road tunnels and size fractions, but showed more marked responses than for the stone materials used in asphalt of the respective tunnels. In particular, fine samples showed significant increases as low as 25 µg/mL (2.6 µg/cm2) and were more potent than coarse samples, while ultrafine samples showed more variable responses between tunnels, sampling conditions and endpoints. The most marked responses were observed for fine PM sampled during humid road surface conditions. Linear correlation analysis showed that particle-induced cytokine responses were correlated to OC levels, while no correlations were observed for other PM characteristics. CONCLUSIONS: The pro-inflammatory potential of fine road tunnel PM sampled during winter season was high compared to coarse PM. The differences between the PM-induced cytokine responses were not related to stone materials in the asphalt. However, the ratio of OC to total PM mass was associated with the pro-inflammatory potential.

Unveiling the Toxicity of Fine and Nano-Sized Airborne Particles Generated from Industrial Thermal Spraying Processes in Human Alveolar Epithelial Cells.

High-energy industrial processes have been associated with particle release into workplace air that can adversely affect workers' health. The present study assessed the toxicity of incidental fine (PGFP) and nanoparticles (PGNP) emitted from atmospheric plasma (APS) and high-velocity oxy-fuel (HVOF) thermal spraying. Lactate dehydrogenase (LDH) release, 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) metabolisation, intracellular reactive oxygen species (ROS) levels, cell cycle changes, histone H2AX phosphorylation (γ-H2AX) and DNA damage were evaluated in human alveolar epithelial cells at 24 h after exposure. Overall, HVOF particles were the most cytotoxic to human alveolar cells, with cell viability half-maximal inhibitory concentration (IC50) values of 20.18 µg/cm2 and 1.79 µg/cm2 for PGFP and PGNP, respectively. Only the highest tested concentration of APS-PGFP caused a slight decrease in cell viability. Particle uptake, cell cycle arrest at S + G2/M and γ-H2AX augmentation were observed after exposure to all tested particles. However, higher levels of γ-H2AX were found in cells exposed to APS-derived particles (~16%), while cells exposed to HVOF particles exhibited increased levels of oxidative damage (~17% tail intensity) and ROS (~184%). Accordingly, APS and HVOF particles seem to exert their genotoxic effects by different mechanisms, highlighting that the health risks of these process-generated particles at industrial settings should not be underestimated.

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.

In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures.

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air-liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.