Polyethylene Terephthalate Microplastics Generated from Disposable Water Bottles Induce Interferon Signaling Pathways in Mouse Lung Epithelial Cells.

Microplastics (MPs) are present in ambient air in a respirable size fraction; however, their potential impact on human health via inhalation routes is not well documented. In the present study, methods for a lab-scale generation of MPs from regularly used and littered plastic articles were optimized. The toxicity of 11 different types of MPs, both commercially purchased and in-lab prepared MPs, was investigated in lung epithelial cells using cell viability, immune and inflammatory response, and genotoxicity endpoints. The underlying mechanisms were identified by microarray analysis. Although laborious, the laboratory-scale methods generated a sufficient quantity of well characterized MPs for toxicity testing. Of the 11 MPs tested, the small sized polyethylene terephthalate (PETE) MPs prepared from disposable water bottles induced the maximum toxicity. Specifically, the smaller size PETE MPs induced a robust activation of the interferon signaling pathway, implying that PETE MPs are perceived by cells by similar mechanisms as those employed to recognize pathogens. The PETE MPs of heterogenous size and shapes induced cell injury, triggering cell death, inflammatory cascade, and DNA damage, hallmark in vitro events indicative of potential in vivo tissue injury. The study establishes toxicity of specific types of plastic materials in micron and nano size.

Bioinformatics and machine learning to support nanomaterial grouping.

Nanomaterials (NMs) offer plenty of novel functionalities. Moreover, their physicochemical properties can be fine-tuned to meet the needs of specific applications, leading to virtually unlimited numbers of NM variants. Hence, efficient hazard and risk assessment strategies building on New Approach Methodologies (NAMs) become indispensable. Indeed, the design, the development and implementation of NAMs has been a major topic in a substantial number of research projects. One of the promising strategies that can help to deal with the high number of NMs variants is grouping and read-across. Based on demonstrated structural and physicochemical similarity, NMs can be grouped and assessed together. Within an established NM group, read-across may be performed to fill in data gaps for data-poor variants using existing data for NMs within the group. Establishing a group requires a sound justification, usually based on a grouping hypothesis that links specific physicochemical properties to well-defined hazard endpoints. However, for NMs these interrelationships are only beginning to be understood. The aim of this review is to demonstrate the power of bioinformatics with a specific focus on Machine Learning (ML) approaches to unravel the NM Modes-of-Action (MoA) and identify the properties that are relevant to specific hazards, in support of grouping strategies. This review emphasizes the following messages: 1) ML supports identification of the most relevant properties contributing to specific hazards; 2) ML supports analysis of large omics datasets and identification of MoA patterns in support of hypothesis formulation in grouping approaches; 3) omics approaches are useful for shifting away from consideration of single endpoints towards a more mechanistic understanding across multiple endpoints gained from one experiment; and 4) approaches from other fields of Artificial Intelligence (AI) like Natural Language Processing or image analysis may support automated extraction and interlinkage of information related to NM toxicity. Here, existing ML models for predicting NM toxicity and for analyzing omics data in support of NM grouping are reviewed. Various challenges related to building robust models in the field of nanotoxicology exist and are also discussed.

Next Generation Risk Assessment approaches for advanced nanomaterials: Current status and future perspectives.

This manuscript discusses the challenges of applying New Approach Methodologies (NAMs) for safe by design and regulatory risk assessment of advanced nanomaterials (AdNMs). The authors propose a framework for Next Generation Risk Assessment of AdNMs involving NAMs that is aligned to the conventional risk assessment paradigm. This framework is exposure-driven, endpoint-specific, makes best use of pre-existing information, and can be implemented in tiers of increasing specificity and complexity of the adopted NAMs. The tiered structure of the approach, which effectively combines the use of existing data with targeted testing will allow safety to be assessed cost-effectively and as far as possible with even more limited use of vertebrates. The regulatory readiness of state-of-the-art emerging NAMs is assessed in terms of Transparency, Reliability, Accessibility, Applicability, Relevance and Completeness, and their appropriateness for AdNMs is discussed in relation to each step of the risk assessment paradigm along with providing perspectives for future developments in the respective scientific and regulatory areas.

A Systematic Genotoxicity Assessment of a Suite of Metal Oxide Nanoparticles Reveals Their DNA Damaging and Clastogenic Potential.

Metal oxide nanoparticles (MONP/s) induce DNA damage, which is influenced by their physicochemical properties. In this study, the high-throughput CometChip and micronucleus (MicroFlow) assays were used to investigate DNA and chromosomal damage in mouse lung epithelial cells induced by nano and bulk sizes of zinc oxide, copper oxide, manganese oxide, nickel oxide, aluminum oxide, cerium oxide, titanium dioxide, and iron oxide. Ionic forms of MONPs were also included. The study evaluated the impact of solubility, surface coating, and particle size on response. Correlation analysis showed that solubility in the cell culture medium was positively associated with response in both assays, with the nano form showing the same or higher response than larger particles. A subtle reduction in DNA damage response was observed post-exposure to some surface-coated MONPs. The observed difference in genotoxicity highlighted the mechanistic differences in the MONP-induced response, possibly influenced by both particle stability and chemical composition. The results highlight that combinations of properties influence response to MONPs and that solubility alone, while playing an important role, is not enough to explain the observed toxicity. The results have implications on the potential application of read-across strategies in support of human health risk assessment of MONPs.

A method for measuring the bioaccessibility of polycyclic aromatic hydrocarbons in cell culture media.

Airborne polycyclic aromatic hydrocarbons (PAHs) and their derivatives are of particular concern for population health due to their abundance and toxicity via inhalation. Lung toxicity testing includes exposing lung epithelial cell lines to PAHs in a culture medium containing inorganic species, lipids, proteins, and other biochemicals where the cell response is influenced among others by the toxic chemical accessibility in the medium. While inhalation bioaccessibility of PAHs and other toxicants was previously studied in surrogate lung fluids, studies measuring bioaccessibility in cell culture media are rare. In this work, a method was developed to characterize PAH bioaccessibility in a culture medium used for mouse lung epithelial (FE1) cells. Further, the optimised method was tested using commercially available standard reference material of urban particulate matter (PM) as well as polyurethane foam passive air samplers (PUF-PAS). The method provided a high precision and recovery of analytes, indicating no losses during sample processing and analysis. PAHs had non-linear concentration-responses, with the culture medium approaching saturation with PM concentration of 500 µg mL-1. The results showed that phenanthrene, a 3-ring PAH, was significantly more bioaccessible than ≥4-ring congeners in the culture medium (up to ∼2.5 folds; p < 0.05). Finally, using pre-deployed PUF-PAS from a residential and an industrial site, five PAHs were found in the culture medium, including naphthalene, phenanthrene, anthracene, fluoranthene, and pyrene. This work provides a proof of concept to enable future studies to assess the inhalation bioaccessibility of polycyclic aromatic compounds and other airborne pollutants collected using PUF-PAS.

Single-Walled vs. Multi-Walled Carbon Nanotubes: Influence of Physico-Chemical Properties on Toxicogenomics Responses in Mouse Lungs.

Single-walled carbon nanotubes (SWCNTs) and multi-walled carbon nanotubes (MWCNTs) are nanomaterials with one or multiple layers of carbon sheets. While it is suggested that various properties influence their toxicity, the specific mechanisms are not completely known. This study was aimed to determine if single or multi-walled structures and surface functionalization influence pulmonary toxicity and to identify the underlying mechanisms of toxicity. Female C57BL/6J BomTac mice were exposed to a single dose of 6, 18, or 54 µg/mouse of twelve SWCNTs or MWCNTs of different properties. Neutrophil influx and DNA damage were assessed on days 1 and 28 post-exposure. Genome microarrays and various bioinformatics and statistical methods were used to identify the biological processes, pathways and functions altered post-exposure to CNTs. All CNTs were ranked for their potency to induce transcriptional perturbation using benchmark dose modelling. All CNTs induced tissue inflammation. MWCNTs were more genotoxic than SWCNTs. Transcriptomics analysis showed similar responses across CNTs at the pathway level at the high dose, which included the perturbation of inflammatory, cellular stress, metabolism, and DNA damage responses. Of all CNTs, one pristine SWCNT was found to be the most potent and potentially fibrogenic, so it should be prioritized for further toxicity testing.

Toxicity of Metal Oxide Nanoparticles: Looking through the Lens of Toxicogenomics.

The impact of solubility on the toxicity of metal oxide nanoparticles (MONPs) requires further exploration to ascertain the impact of the dissolved and particulate species on response. In this study, FE1 mouse lung epithelial cells were exposed for 2-48 h to 4 MONPs of varying solubility: zinc oxide, nickel oxide, aluminum oxide, and titanium dioxide, in addition to microparticle analogues and metal chloride equivalents. Previously published data from FE1 cells exposed for 2-48 h to copper oxide and copper chloride were examined in the context of exposures in the present study. Viability was assessed using Trypan Blue staining and transcriptomic responses via microarray analysis. Results indicate material solubility is not the sole property governing MONP toxicity. Transcriptional signaling through the 'HIF-1α Signaling' pathway describes the response to hypoxia, which also includes genes associated with processes such as oxidative stress and unfolded protein responses and represents a conserved response across all MONPs tested. The number of differentially expressed genes (DEGs) in this pathway correlated with apical toxicity, and a panel of the top ten ranked DEGs was constructed (Hmox1, Hspa1a, Hspa1b, Mmp10, Adm, Serpine1, Slc2a1, Egln1, Rasd1, Hk2), highlighting mechanistic differences among tested MONPs. The HIF-1α pathway is proposed as a biomarker of MONP exposure and toxicity that can help prioritize MONPs for further evaluation and guide specific testing strategies.

Unchanged pulmonary toxicity of ZnO nanoparticles formulated in a liquid matrix for glass coating.

The inclusion of nanoparticles can increase the quality of certain products. One application is the inclusion of Zinc oxide (ZnO) nanoparticles in a glass coating matrix to produce a UV-absorbing coating for glass sheets. Yet, the question is whether the inclusion of ZnO in the matrix induces toxicity at low exposure levels. To test this, mice were given single intratracheal instillation of 1) ZnO powder (ZnO), 2) ZnO in a glass matrix coating in its liquid phase (ZnO-Matrix), and 3) the matrix with no ZnO (Matrix). Doses of ZnO were 0.23, 0.67, and 2 µg ZnO/mouse. ZnO Matrix doses had equal amounts of ZnO, while Matrix was adjusted to have an equal volume of matrix as ZnO Matrix. Post-exposure periods were 1, 3, or 28 d. Endpoints were pulmonary inflammation as bronchoalveolar lavage (BAL) fluid cellularity, genotoxicity in lung and liver, measured by comet assay, histopathology of lung and liver, and global gene expression in lung using microarrays. Neutrophil numbers were increased to a similar extent with ZnO and ZnO-Matrix at 1 and 3 d. Only weak genotoxicity without dose-response effects was observed in the lung. Lung histology showed an earlier onset of inflammation in material-exposed groups as compared to controls. Microarray analysis showed a stronger response in terms of the number of differentially regulated genes in ZnO-Matrix exposed mice as compared to Matrix only. Activated canonical pathways included inflammatory and cardiovascular ones. In conclusion, the pulmonary toxicity of ZnO was not changed by formulation in a liquid matrix for glass coating.

The High-Throughput In Vitro CometChip Assay for the Analysis of Metal Oxide Nanomaterial Induced DNA Damage.

Metal oxide nanomaterials (MONMs) are among the most highly utilized classes of nanomaterials worldwide, though their potential to induce DNA damage in living organisms is known. High-throughput in vitro assays have the potential to greatly expedite analysis and understanding of MONM induced toxicity while minimizing the overall use of animals. In this study, the high-throughput CometChip assay was used to assess the in vitro genotoxic potential of pristine copper oxide (CuO), zinc oxide (ZnO), and titanium dioxide (TiO2) MONMs and microparticles (MPs), as well as five coated/surface-modified TiO2 NPs and zinc (II) chloride (ZnCl2) and copper (II) chloride (CuCl2) after 2-4 h of exposure. The CuO NPs, ZnO NPs and MPs, and ZnCl2 exposures induced dose- and time-dependent increases in DNA damage at both timepoints. TiO2 NPs surface coated with silica or silica-alumina and one pristine TiO2 NP of rutile crystal structure also induced subtle dose-dependent DNA damage. Concentration modelling at both post-exposure timepoints highlighted the contribution of the dissolved species to the response of ZnO, and the role of the nanoparticle fraction for CuO mediated genotoxicity, showing the differential impact that particle and dissolved fractions can have on genotoxicity induced by MONMs. The results imply that solubility alone may be insufficient to explain the biological behaviour of MONMs.

Characterization of ENM Dynamic Dose-Dependent MOA in Lung with Respect to Immune Cells Infiltration.

The molecular effects of exposures to engineered nanomaterials (ENMs) are still largely unknown. In classical inhalation toxicology, cell composition of bronchoalveolar lavage (BAL) is a toxicity indicator at the lung tissue level that can aid in interpreting pulmonary histological changes. Toxicogenomic approaches help characterize the mechanism of action (MOA) of ENMs by investigating the differentially expressed genes (DEG). However, dissecting which molecular mechanisms and events are directly induced by the exposure is not straightforward. It is now generally accepted that direct effects follow a monotonic dose-dependent pattern. Here, we applied an integrated modeling approach to study the MOA of four ENMs by retrieving the DEGs that also show a dynamic dose-dependent profile (dddtMOA). We further combined the information of the dddtMOA with the dose dependency of four immune cell populations derived from BAL counts. The dddtMOA analysis highlighted the specific adaptation pattern to each ENM. Furthermore, it revealed the distinct effect of the ENM physicochemical properties on the induced immune response. Finally, we report three genes dose-dependent in all the exposures and correlated with immune deregulation in the lung. The characterization of dddtMOA for ENM exposures, both for apical endpoints and molecular responses, can further promote toxicogenomic approaches in a regulatory context.

AOP173 key event associated pathway predictor - online application for the prediction of benchmark dose lower bound (BMDLs) of a transcriptomic pathway involved in MWCNTs-induced lung fibrosis.

Nano-QSAR model allows for prediction of the toxicity of materials that have not been experimentally tested before by linking the nano-related structural properties with the biological responses induced by nanomaterials. Prediction of adverse effects caused by substances without having to perform time- and cost-consuming experiments makes QSAR models promising tools for supporting risk assessment. However, very often, newly developed nano-QSAR models are not used in practice due to the complexity of their algorithms, the necessity to have experience in chemoinformatics, and their poor accessibility. In this perspective, the aim of this paper is to encourage developers of the QSAR models to take the effort to prepare user-friendly applications based on predictive models. This would make the developed models accessible to a wider community, and, in effect, promote their further application by regulators and decision-makers. Here, we describe a web-based application that enables to predict the transcriptomic pathway-level response perturbated in the lungs of mice exposed to multiwalled carbon nanotubes. The developed application is freely available at http://aop173-event1.nanoqsar-aop.com/apps/aop_app. It requires only two types of input information related to analyzed nanotubes (their length and diameter) to assess the doses that initiate the inflammation process that may lead to lung fibrosis.

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.

COVID-19 through Adverse Outcome Pathways: Building networks to better understand the disease - 3rd CIAO AOP Design Workshop.

On April 28-29, 2021, 50 scientists from different fields of expertise met for the 3rd online CIAO workshop. The CIAO project "Modelling the Pathogenesis of COVID-19 using the Adverse Outcome Pathway (AOP) framework" aims at building a holistic assembly of the available scientific knowledge on COVID-19 using the AOP framework. An individual AOP depicts the disease progression from the initial contact with the SARS-CoV-2 virus through biological key events (KE) toward an adverse outcome such as respiratory distress, anosmia or multiorgan failure. Assembling the individual AOPs into a network highlights shared KEs as central biological nodes involved in multiple outcomes observed in COVID-19 patients. During the workshop, the KEs and AOPs established so far by the CIAO members were presented and posi­tioned on a timeline of the disease course. Modulating factors influencing the progression and severity of the disease were also addressed as well as factors beyond purely biological phenomena. CIAO relies on an interdisciplinary crowd­sourcing effort, therefore, approaches to expand the CIAO network by widening the crowd and reaching stakeholders were also discussed. To conclude the workshop, it was decided that the AOPs/KEs will be further consolidated, inte­grating virus variants and long COVID when relevant, while an outreach campaign will be launched to broaden the CIAO scientific crowd.

Non-Animal Strategies for Toxicity Assessment of Nanoscale Materials: Role of Adverse Outcome Pathways in the Selection of Endpoints.

Faster, cheaper, sensitive, and mechanisms-based animal alternatives are needed to address the safety assessment needs of the growing number of nanomaterials (NM) and their sophisticated property variants. Specifically, strategies that help identify and prioritize alternative schemes involving individual test models, toxicity endpoints, and assays for the assessment of adverse outcomes, as well as strategies that enable validation and refinement of these schemes for the regulatory acceptance are needed. In this review, two strategies 1) the current nanotoxicology literature review and 2) the adverse outcome pathways (AOPs) framework, a systematic process that allows the assembly of available mechanistic information concerning a toxicological response in a simple modular format, are presented. The review highlights 1) the most frequently assessed and reported ad hoc in vivo and in vitro toxicity measurements in the literature, 2) various AOPs of relevance to inhalation toxicity of NM that are presently under development, and 3) their applicability in identifying key events of toxicity for targeted in vitro assay development. Finally, using an existing AOP for lung fibrosis, the specific combinations of cell types, exposure and test systems, and assays that are experimentally supported and thus, can be used for assessing NM-induced lung fibrosis, are proposed.

Bringing together scientific disciplines for collaborative undertakings: a vision for advancing the adverse outcome pathway framework.

BACKGROUND: Decades of research to understand the impacts of various types of environmental occupational and medical stressors on human health have produced a vast amount of data across many scientific disciplines. Organizing these data in a meaningful way to support risk assessment has been a significant challenge. To address this and other challenges in modernizing chemical health risk assessment, the Organisation for Economic Cooperation and Development (OECD) formalized the adverse outcome pathway (AOP) framework, an approach to consolidate knowledge into measurable key events (KEs) at various levels of biological organisation causally linked to disease based on the weight of scientific evidence (http://oe.cd/aops). Currently, AOPs have been considered predominantly in chemical safety but are relevant to radiation. In this context, the Nuclear Energy Agency's (NEA's) High-Level Group on Low Dose Research (HLG-LDR) is working to improve research co-ordination, including radiological research with chemical research, identify synergies between the fields and to avoid duplication of efforts and resource investments. To this end, a virtual workshop was held on 7 and 8 October 2020 with experts from the OECD AOP Programme together with the radiation and chemical research/regulation communities. The workshop was a coordinated effort of Health Canada, the Electric Power Research Institute (EPRI), and the Nuclear Energy Agency (NEA). The AOP approach was discussed including key issues to fully embrace its value and catalyze implementation in areas of radiation risk assessment. CONCLUSIONS: A joint chemical and radiological expert group was proposed as a means to encourage cooperation between risk assessors and an initial vision was discussed on a path forward. A global survey was suggested as a way to identify priority health outcomes of regulatory interest for AOP development. Multidisciplinary teams are needed to address the challenge of producing the appropriate data for risk assessments. Data management and machine learning tools were highlighted as a way to progress from weight of evidence to computational causal inference.

Transcriptomics-Based and AOP-Informed Structure-Activity Relationships to Predict Pulmonary Pathology Induced by Multiwalled Carbon Nanotubes.

This study presents a novel strategy that employs quantitative structure-activity relationship models for nanomaterials (Nano-QSAR) for predicting transcriptomic pathway level response using lung tissue inflammation, an essential key event (KEs) in the existing adverse outcome pathway (AOP) for lung fibrosis, as a model response. Transcriptomic profiles of mouse lungs exposed to ten different multiwalled carbon nanotubes (MWCNTs) are analyzed using statistical and bioinformatics tools. Three pathways "agranulocyte adhesion and diapedesis," "granulocyte adhesion and diapedesis," and "acute phase signaling," that (1) are commonly perturbed across the MWCNTs panel, (2) show dose response (Benchmark dose, BMDs), and (3) are anchored to the KEs identified in the lung fibrosis AOP, are considered in modelling. The three pathways are associated with tissue inflammation. The results show that the aspect ratio (κ) of MWCNTs is directly correlated with the pathway BMDs. The study establishes a methodology for QSAR construction based on canonical pathways and proposes a MWCNTs grouping strategy based on the κ-values of the specific pathway associated genes. Finally, the study shows how the AOP framework can help guide QSAR modelling efforts; conversely, the outcome of the QSAR modelling can aid in refining certain aspects of the AOP in question (here, lung fibrosis).

Impact of copper oxide particle dissolution on lung epithelial cell toxicity: response characterization using global transcriptional analysis.

The in vitro and in vivo toxicity of copper oxide nanoparticles (CuO NPs) is attributed to both particle and dissolved copper ion species. However, a clear understanding of (1) the specific cellular responses that are modulated by the two species and (2) the temporal dynamics in toxicity, as the proportional amount of particulate and ionic forms change over time, is lacking. In the current study, in vitro responses to microparticulate CuO (CuO MPs), CuO NPs, and dissolved Cu2+ were characterized in order to elucidate particle and ion-induced kinetic effects. Particle dissolution experiments were carried out in a relevant cell culture medium, using CuO NPs and MPs. Mouse lung epithelial cells were exposed for 2-48 h with 1-25 µg/mL CuO MPs, CuO NPs, or 7 and 54 µg/mL CuCl2. Cellular viability and genome-wide transcriptional responses were assessed. Dose and time-dependent cytotoxicity were observed in CuO NP exposed cells, which was delayed and subtle in CuCl2 and not observed in CuO MPs treated cells. Analyses of differentially expressed genes and associated pathway perturbations showed that dissolved ions released by CuO NPs in the extracellular medium are insufficient to account for the observed potency and cytotoxicity. Further organization of gene expression results in an Adverse Outcome Pathway (AOP) framework revealed a series of key events potentially involved in CuO NPs toxicity. The AOP is applicable to toxicity induced by metal oxide nanoparticles of varying solubility, and thus, can facilitate the development of in vitro alternative strategies to screen their toxicity.

Pulmonary toxicity and gene expression changes after short-term inhalation exposure to surface-modified copper oxide nanoparticles.

Copper oxide nanoparticles (CuO NPs) have previously been shown to cause dose-dependent pulmonary toxicity following inhalation. Here, CuO NPs (10 nm), coated with polyethylenimine (PEI) or ascorbate (ASC) resulting in positively or negatively charged NPs, respectively, were evaluated. Rats were exposed nose-only to similar exposure dose levels of ASC or PEI coated CuO NPs for 5 consecutive days. On day 6 and day 27 post-exposure, pulmonary toxicity markers in bronchoalveolar lavage fluid (BALF), lung histopathology and genome-wide transcriptomic changes in lungs, were assessed. BALF analyses showed a dose-dependent pulmonary inflammation and cell damage, which was supported by the lung histopathological findings of hypertrophy/hyperplasia of bronchiolar and alveolar epithelium, interstitial and alveolar inflammation, and paracortical histiocytosis in mediastinal lymph nodes for both types of CuO NPs. Transcriptomics analysis showed that pathways related to inflammation and cell proliferation were significantly activated. Additionally, we found evidence for the dysregulation of drug metabolism-related genes, especially in rats exposed to ASC-coated CuO NPs. Overall, no differences in the type of toxic effects and potency between the two surface coatings could be established, except with respect to the (regional) dose that initiates bronchiolar and alveolar hypertrophy. This disproves our hypothesis that differences in surface coatings affect the pulmonary toxicity of CuO NPs.

Adverse Outcome Pathway Development for Assessment of Lung Carcinogenicity by Nanoparticles.

Lung cancer, one of the most common and deadly forms of cancer, is in some cases associated with exposure to certain types of particles. With the rise of nanotechnology, there is concern that some engineered nanoparticles may be among such particles. In the absence of epidemiological evidence, assessment of nanoparticle carcinogenicity is currently performed on a time-consuming case-by-case basis, relying mainly on animal experiments. Non-animal alternatives exist, including a few validated cell-based methods accepted for regulatory risk assessment of nanoparticles. Furthermore, new approach methodologies (NAMs), focused on carcinogenic mechanisms and capable of handling the increasing numbers of nanoparticles, have been developed. However, such alternative methods are mainly applied as weight-of-evidence linked to generally required animal data, since challenges remain regarding interpretation of the results. These challenges may be more easily overcome by the novel Adverse Outcome Pathway (AOP) framework, which provides a basis for validation and uptake of alternative mechanism-focused methods in risk assessment. Here, we propose an AOP for lung cancer induced by nanosized foreign matter, anchored to a selection of 18 standardized methods and NAMs for in silico- and in vitro-based integrated assessment of lung carcinogenicity. The potential for further refinement of the AOP and its components is discussed in relation to available nanosafety knowledge and data. Overall, this perspective provides a basis for development of AOP-aligned alternative methods-based integrated testing strategies for assessment of nanoparticle-induced lung cancer.

Microplastics and nanoplastics science: collecting and characterizing airborne microplastics in fine particulate matter.

Microplastic (MP) pollution in the environment is increasing, leading to growing concerns about human exposures and the subsequent impact on health. Although marine MP research has received significant attention in recent years, only a few studies have attempted characterization of MP in air and examined the MP uptake and influence via inhalation on human health. Moreover, the methods used for MP characterization in the marine environment require further optimization to be applicable to MP in the air. This paper details method for collecting and characterizing MP < 2.5 µm in air samples for the purposes of toxicological assessment. The first phase of the study evaluated (a) the suitability of various filter types to collect respirable airborne MP <2.5 µm, and; (b) the ability of Raman and enhanced darkfield-hyperspectral spectroscopy methods to identify MP reference standards collected from spiked filters and in cells after exposure to reference MP. In the second phase, these methods were employed to characterize MP <2.5 µm in personal, indoor and outdoor filter air samples and in cells following exposure to filter extracted material. The results showed the presence of a variety of MP in the respirable size fraction (0.1-1 µm aerodynamic diameter). Silver membrane filters were found not suitable for collecting and analyzing MP <2.5 µm. While it was easy to detect reference MP in cells post-exposure, the identity of only two types of air-borne MP was confirmed in cells. The study highlighted possible sources of artifacts and inconsistencies in analyzing airborne MP.