There is an ongoing demand to develop options to reduce hazard testing of substances and materials on a case-by-case basis. Grouping approaches offer a way to share or re-use safety-related information between similar substances, providing insights that can inform the Safe and Sustainable by-Design (SSbD)2 of new materials. Here, an existing grouping hypothesis template for single-component nanomaterials (NMs)3 is expanded to facilitate systematic consideration of grouping for multicomponent nanomaterials (MCNMs)4 relevant to SSbD. Modifications to the template include additional information on a) the complexity of physical and chemical composition; b) the emerging properties driving the MCNM functionality; c) the potential for MCNM components to transform with different rates, leading to complex exposure scenarios; d) prioritisation and simplification of grouping decisions related to material properties (what they are), fate/toxicokinetics (where they go) and the hazard mechanisms (what they do). Existing information and data are used to formulate a matrix of sub-hypotheses that individually relate one (or more) indicators of 'what they are' to a single indicator of either 'where they go' or 'what they do'. The resultant sub-hypotheses are easier to assess than the all-encompassing over-arching hypothesis required for regulatory application of grouping. The estimated level of impact of each indicator is used to prioritise the sub-hypothesis assessment. Accepting or rejecting each prioritised sub-hypothesis is facilitated by the application of tiered testing strategies promoting the use of relevant existing data, new approach methodologies and machine learning-based models. A case study of SiO2@ZnO MCNM is provided to demonstrate the template's usefulness in an SSbD context.

In response to the significant global crop losses caused by insect pests, which affect up to 40% of crops annually, there is an urgent need for safer food protection methods. This study addresses this need by proactively developing a safe and sustainable by design (SSbD) alternative to synthetic pesticides. Guided by the EC-JRC SSbD framework, the research focuses on an advanced low density polyethylene (LDPE) film embedding a multicomponent nanomaterial (MCNM), consisting of bentonite nanoclays and clove essential oil (BNT-CEO), designed to repel beetles. In detail, a three-step premarket safe-by-design assessment was performed. The first step was the safety assessment of the BNT-CEO material through i) physicochemical characterization, ii) screening for potential hazards of chemical precursors, and iii) preliminary in vitro toxicity tests. Afterwards, worker safety during both BNT-CEO synthesis and LDPE(BNT-CEO) production was assessed, analyzing dust generation and workers' potential exposure through an industrial hygiene survey followed by occupational monitoring. Lastly, consumers' safety was covered assessing the LDPE(BNT-CEO) film degradation and potential for migration of chemicals, by comparing pristine and accelerated-aged samples. Compliance with EU Regulation 10/2011 was verified by analyzing the migration of substances into food simulants. The integration of these safety evaluations early in the design process of BNT-CEO and LDPE(BNT-CEO) allowed confirmation of the material's compliance with regulatory limits and contributed to the validation of the assessment procedure as proposed by the SSbD framework. The approach here applied demonstrates how to successfully balance effective pest protection with minimal impact on consumers and workers, paving the way for the development of safer and sustainable food packaging solutions.

The movement towards an animal-free testing approach for risk assessment represents a key paradigm shift in toxicology. Risk assessment of engineered and anthropogenic nanoscale materials (NM) is dependent on reliable hazard characterization, which requires validated test methods and models, and increasingly on mechanistic insights into the mode of action. The properties that make NMs so advantageous for a wide range of commercial and industrial applications also pose a challenge when it comes to safety testing under in vitro and in chemico experimental settings. Their large reactive surface area makes NMs prone to interactions with assay reagents, readout signals, or intermediate steps of many test assays, leading to the potential for biased results and data inconsistencies, collectively referred to as interferences. Therefore, methods and protocols developed and validated for conventional chemicals often require adaptation and checking for reliability in NMs' toxicity assessment. This review presents the collected scientific knowledge on NMs-induced interferences for the most common in vitro toxicity assays and methods related to cytotoxicity, oxidative stress and inflammatory response evaluation. Our analysis of existing scientific literature showed that the challenge of NMs-induced interference was not explicitly addressed in more than 90% of the papers published up to 2014 reporting the safety and toxicity of NMs. In later years, increasing number of studies tackled the interference challenge in toxicity testing of NMs, which initiated exhaustive work on standardization and validation of existing regulatory-relevant in vitro test protocols and guidelines. Due to the specificity of the different NMs and the range of ways they can potentially interfere with in vitro assays, interference and fit-for purpose controls should be included for each NM type and method applied, unless label-free assays are selected. Here, we provide a decision tree to guide researchers on how to design experiments to avoid interferences during in vitro testing by taking appropriate mitigation actions and how to include proper interference controls in their experimental design where complete avoidance is not possible. The application of this decision tree will improve the reliability, comparability and reusability of in vitro toxicity data on engineered NMs or ENMs, increasing the relevance of in silico hazard data for use in risk assessment and in science-based risk governance of NMs. The approach is applicable more broadly also, to advanced materials and to hazard assessment of anthropogenic nanoscale materials such as microplastic and tyre-wear particles.

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.

I was about to call this blog post From spreadsheets to RDF , after the post last week. But then I decided to just use the pattern I typically use. Why I wanted to use that shorter term in the first place was that one of the thing I like about the AMBIT software (of OpenTox and eNanoMapper fame) is its RDF support (see doi:10.1186/1756-0500-4-487). But RDF, ontologies, those are hard things.

The rapid development of engineered nanomaterials (ENMs) causes humans to become increasingly exposed to them. Therefore, a better understanding of the health impact of ENMs is highly demanded. Considering the 3Rs (Replacement, Reduction, and Refinement) principle, in vitro and computational methods are excellent alternatives for testing on animals. Among computational methods, nano-quantitative structure-activity relationship (nano-QSAR), which links the physicochemical and structural properties of EMNs with biological activities, is one of the leading method. The nature of toxicological experiments has evolved over the last decades; currently, one experiment can provide thousands of measurements of the organism's functioning at the molecular level. At the same time, the capacity of the in vitro systems to mimic the human organism is also improving significantly. Hence, the authors would like to discuss whether the nano-QSAR approach follows modern toxicological studies and takes full advantage of the opportunities offered by modern toxicological platforms. Challenges and possibilities for improving data integration are underlined narratively, including the need for a consensus built between the in vitro and the QSAR domains.

BACKGROUND:The establishment of reliable and robust in vitro models for hazard assessment, a prerequisite for moving away from animal testing, requires the evaluation of model transferability and reproducibility. Lung models that can be exposed via the air, by means of an air-liquid interface (ALI) are promising in vitro models for evaluating the safety of nanomaterials (NMs) after inhalation exposure. We performed an inter-laboratory comparison study to evaluate the transferability and reproducibility of a lung model consisting of the human bronchial cell line Calu-3 as a monoculture and, to increase the physiologic relevance of the model, also as a co-culture with macrophages (either derived from the THP-1 monocyte cell line or from human blood monocytes). The lung model was exposed to NMs using the VITROCELL® Cloud12 system at physiologically relevant dose levels. RESULTS:Overall, the results of the 7 participating laboratories are quite similar. After exposing Calu-3 alone and Calu-3 co-cultures with macrophages, no effects of lipopolysaccharide (LPS), quartz (DQ12) or titanium dioxide (TiO2) NM-105 particles on the cell viability and barrier integrity were detected. LPS exposure induced moderate cytokine release in the Calu-3 monoculture, albeit not statistically significant in most labs. In the co-culture models, most laboratories showed that LPS can significantly induce cytokine release (IL-6, IL-8 and TNF-α). The exposure to quartz and TiO2 particles did not induce a statistically significant increase in cytokine release in both cell models probably due to our relatively low deposited doses, which were inspired by in vivo dose levels. The intra- and inter-laboratory comparison study indicated acceptable interlaboratory variation for cell viability/toxicity (WST-1, LDH) and transepithelial electrical resistance, and relatively high inter-laboratory variation for cytokine production. CONCLUSION:The transferability and reproducibility of a lung co-culture model and its exposure to aerosolized particles at the ALI were evaluated and recommendations were provided for performing inter-laboratory comparison studies. Although the results are promising, optimizations of the lung model (including more sensitive read-outs) and/or selection of higher deposited doses are needed to enhance its predictive value before it may be taken further towards a possible OECD guideline.

Allergic diseases represent an increasing global health problem, among which food and respiratory allergies play an important part. Inhalation of sensitizers from food sources primes the immune system for an exacerbated response upon subsequent exposure via the ingestion route. With the increasing emergence of novel food additives and proteins and growing morbidity in both food and respiratory allergies, the hazard assessment of the allergenicity of food components is of paramount importance. A three-dimensional (3D) alveolar in vitro model for respiratory sensitization prediction was downscaled from the 6-to the 24-well plate insert format, to meet the industry's high-throughput (HTS) screening requirements. The resemblance of the cellular architecture of the downscaled model to the alveolar region of the lung was evaluated through microscopy techniques and the functionality of the in vitro model was assessed following air-liquid interface (ALI) exposure to ammonium persulfate (APS). The model represents a promising in vitro tool to screen for the respiratory sensitization potential of food components in a fast and reliable approach.

It is well known that copper oxide nanoparticles (CuO NPs) are heavily toxic on in vitro systems. In human alveolar epithelial cells, the mechanism of toxicity is mostly related to oxidative insults, coming from intracellularly dissolved copper ions, finally leading to apoptotic or autophagic cell death. Our hypothesis is based on possible early oxidative events coming from specific NP surface reactivity able to undermine the cell integrity and to drive cell to death, independently from Lysosomal-Enhanced Trojan Horse mechanism. Two types of CuO NPs, with different oxidative potential, were selected and tested on A549 cells for 1 h and 3 h at 10, 25, 50 and 100 µg/ml. Cells were then analyzed for viability and oxidative change of the proteome. Oxidative by-products were localized by immunocytochemistry and cell-NP interactions characterized by confocal and electron microscopy techniques. The results show that CuO NPs induced oxidative changes soon after 1 h exposure as revealed by the increase in protein carbonylation and reduced-protein-thiol oxidation. In parallel, cell viability significantly decreased, as shown by MTT assay. Such effects were higher for CuO NPs with more crystalline defects and with higher ROS production than for fully crystalline NPs. At these exposure times, although NPs efficiently interacted with cell surface and were taken up by small endocytic vesicles, no ion dissolution was visible inside the lysosomal compartment and no effects were produced by extracellularly dissolved copper ions. In conclusion, a specific NP surface-dependent oxidative cell injury was demonstrated. More detailed studies are required to understand which targets precociously react with CuO NPs, but these results introduce new paradigms for the toxicity of the metal-based NPs, beyond the Lysosomal-Enhanced Trojan horse-related mechanism, and open-up new opportunities to investigate the interactions and effects at the bio-interface for designing safer as well as more effective CuO-based biocides.

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 (R) 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 (R) 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 (R) 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 (R) 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 (R) 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 (R) 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 (R) Cloud12. This inter-laboratory comparison demonstrates that the QCM integrated in the VITROCELL (R) 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 (R) 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.

Background: It is well known that copper oxide nanoparticles (CuO NPs) are heavily toxic on in vitro systems. In human alveolar epithelial cells, the mechanism of toxicity is mostly related to oxidative insults, coming from intracellularly dissolved copper ions, finally leading to apoptotic or autophagic cell death.Our hypothesis is based on possible early oxidative events coming from specific NP surface reactivity able to undermine the cell integrity and to drive cell to death, independently from lysosomal-enhanced Trojan horse mechanism. Two types of CuO NPs, with different oxidative potential, were selected and tested on A549 cells for 1h and 3h at 10, 25, 50 and 100 mg/mL. Cells were then analyzed for viability and oxidative change of the proteome. Oxidative by-products were localized by immunocytochemistry and cell-NP interactions characterized by confocal and electron microscopy techniques. Results: CuO NPs induced oxidative changes soon after 1 h exposure as revealed by the increase in protein carbonylation and reduced-protein-thiol oxidation. In parallel, cell viability significantly decreased, as shown by MTT assay. Such effects were higher for CuO NPs with more crystalline defects and with higher ROS production than for fully crystalline NPs. At these exposure times, although NPs efficiently interacted with cell surface and were taken up by small endocytic vesicles, no ion dissolution is visible inside endo-lysosomal compartment and no effects were produced by extracellularly dissolved copper ions. Conclusions: A specific NP surface-dependent oxidative cell injury was demonstrated. More detailed studies are required to understand which targets precociously react to CuO NPs, but these results introduce new paradigms for the toxicity of the metal-based NPs, beyond the Trojan horse-related mechanism, and opens-up new opportunities to investigate the interactions and effects at the bio-interface for designing safer as well as more effective CuO-based biocides.

Exposure to aerosol from electronic vapor (e-vapor) products has been suggested to result in less risk of harm to smokers than cigarette smoke (CS) exposure. Although many studies on e-vapor products have tested the effects of liquid formulations on cell cultures, few have evaluated the effects of aerosolized formulations. We examined the effects of acute exposure to the aerosol of an e-vapor device that uses the MESH® technology (IQOS® MESH, Philip Morris International) and to CS from the 3R4F reference cigarette on human organotypic bronchial epithelial culture and alveolar triculture models. In contrast to 3R4F CS exposure, exposure to the IQOS MESH aerosol (Classic Tobacco flavor) did not cause cytotoxicity in bronchial epithelial cultures or alveolar tricultures despite its greater concentrations of deposited nicotine (3- and 4-fold, respectively). CS exposure caused a marked decrease in the frequency and active area of ciliary beating in bronchial cultures, whereas IQOS MESH aerosol exposure did not. Global mRNA expression and secreted protein profiles revealed a significantly lower impact of IQOS MESH aerosol exposure than 3R4F CS exposure. Overall, our whole aerosol exposure study shows a clearly reduced impact of IQOS MESH aerosol relative to CS in bronchial and alveolar cultures, even at greater nicotine doses.

The use of CuO nanoparticles (NPs) has increased greatly and their potential effects on human health need to be investigated. Differentiated Caco‐2 cells were treated from the apical (Ap) and the basolateral (Bl) compartment with different concentrations (0, 10, 50 and 100 μg/mL) of commercial or sonochemically synthesized (sono) CuO NPs. Sono NPs were prepared in ethanol (CuOe) or in water (CuOw), obtaining CuO NPs differing in size and shape. The effects on the Caco‐2 cell barrier were assessed via transepithelial electrical resistance (TEER) evaluation just before and after 1, 2 and 24 hours of exposure and through the analysis of cytokine release and biomarkers of oxidative damage to proteins after 24 hours. Sono CuOe and CuOw NPs induced a TEER decrease with a dose‐dependent pattern after Bl exposure. Conversely, TEER values were not affected by the Ap exposure to commercial CuO NPs and, concerning the Bl exposure, only the lowest concentration tested (10 μg/mL) caused a TEER decrease after 24 hours of exposure. An increased release of interleukin‐8 was induced by sono CuO NPs after the Ap exposure to 100 μg/mL and by sono and commercial CuO after the Bl exposure to all the concentrations. No effects of commercial and sono CuO NPs on interleukin‐6 (with the only exception of 100 μg/mL Bl commercial CuO) and tumor necrosis factor‐α release were observed. Ap treatment with commercial and CuOw NPs was able to induce significant alterations on specific biomarkers of protein oxidative damage (protein sulfhydryl group oxidation and protein carbonylation).

The present study aimed to evaluate the potential differences in the biological effects of two types of spherical silver particles of 20 and 200 nm (Ag20 and Ag200), and of PVP-coated silver nanowires (AgNWs) with a diameter of 50 nm and length up to 50 μm, using a complex 3D model representative for the alveolar barrier cultured at air-liquid interface (ALI). The alveolar model was exposed to 0.05, 0.5 and 5 μg/cm2 of test compounds at ALI using a state-of-the-art exposure system (Vitrocell™Cloud System). Endpoints related to the oxidative stress induction, anti-oxidant defence mechanisms, pro-inflammatory responses and cellular death were selected to evaluate the biocompatibility of silver particles and nanowires (AgNMs) and to further ascribe particular biological effects to the different morphologic properties between the three types of AgNMs evaluated.

The aim of the study was to develop an in vitro model that mimics the alveolar-capillary barrier and that allows assessment of the respiratory sensitizing potential of respiratory sensitizers. The 3D in vitro model cultured at the air liquid interface consists of alveolar type II epithelial cells (A549), endothelial cells (EA.hy926), macrophage-like cells (PMA-differentiated THP-1) and dendritic-like cells (non-differentiated THP-1). This alveolar model was exposed apically to nebulized chemical respiratory sensitizers (Phthalic Anhydride (PA) and TriMellitic Anhydride (TMA)) or irritants (Methyl Salicylate (MeSa) and Acrolein (Acr)) at concentrations inducing at maximum 25% of cytotoxicity. The exposure to respiratory sensitizers induced dendritic cells activation and a specific cytokine release pattern, while the irritants did not. In addition, the cell surface marker OX40L was determined for dendritic like cells activation to identify high molecular weight allergens. With this in vitro model we can postulate a set of promising markers based on the studied compounds that allow the discrimination of chemical respiratory sensitizers from irritants.

ZnO nanoparticles (NPs) are widely used nowadays, thus the gastrointestinal exposure to ZnO NPs is likely to be relevant and the effects on the intestinal barrier should be investigated. Polarized Caco‐2 cells were exposed from the apical (Ap) and basolateral (Bl) compartments to increasing concentrations (0, 10, 50 and 100 μg/mL) of sonochemical (sono) and commercial ZnO NPs. The transepithelial electrical resistance (TEER), cell viability, proinflammatory cytokine release and presence of protein oxidative damage were evaluated after exposure. TEER was not significantly affected by Ap exposure to either sono or commercial ZnO NPs at any tested concentrations. After Bl exposure to sono ZnO NPs (all the concentrations) and to 100 μg/mL of commercial ZnO NPs TEER was decreased ( P < 0.05). Ap and Bl exposure to 100 μg/mL sono ZnO NPs and Ap exposure to 50 μg/mL commercial ZnO NPs induced a significant ( P < 0.05) release of interleukin‐6. A significant ( P < 0.05) release of interleukin‐8 was observed after Ap exposure to ZnO NPs at 100 μg/mL and after Bl exposure to sono ZnO NPs at 100 μg/mL. Ap or Bl exposure to sono or commercial ZnO NPs did not affect tumour necrosis factor‐alpha secretion or protein sulphydryl oxidation. In conclusion, the ZnO NP exposure from the Ap compartment appeared almost safe, while the exposure through the basal compartment appeared to be more hazardous and the different NP size and crystallinity seem to affect the mode of action, but further studies are necessary to elucidate better these toxicity mechanisms.

To more accurately model inhalation toxicity in vitro, we developed a tetra-culture system that combines lung alveolar epithelial cells, endothelial cells, macrophages, and mast cells in a three-dimensional orientation. We characterized the influence of the added complexity using network perturbation analysis and gene expression data. This will allow us to gain insight into the steady-state profile of the assembled, complete three-dimensional model using all four cell types and of simpler models of one, two, or three cell types. Gene expression data were analyzed using cause-and-effect biological network models, together with a quantitative network-scoring algorithm, to determine the biological impact of co-culturing the various cell types. In the assembled tetra-culture, macrophages appeared to be the largest contributors to overall network perturbations, promoting high basal levels of oxidative stress and inflammation. This finding led to further optimization of the model using rested macrophages; the addition of rested macrophages decreased the basal inflammatory and cell stress status of the co-culture. Finally, we compared transcriptional profiles from publicly available datasets of conventional in vitro models representative of the airways and of healthy human lung tissues to assess similarities between our model and other in vitro models and the human lung. On the transcriptional level, we found an increasing correlation between airway models and normal human lung tissue, particularly as cell types became more physiologically relevant and the complexity of the system increased. This indicates that the combination of multiple lung-relevant cell types in vitro does indeed increase similarity to the physiological counterpart.

Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis.

The aim of the current study was to evaluate the responses of a 3D tetra-culture alveolar model cultivated at the air-liquid-interface (ALI) after apical exposure to diesel exhaust particulate matter (DEPM) based on the three tiered oxidative stress concept. The alveolar model exposed to increasing doses of DEPM (1.75-5 mu g/cm(2)) responded with increasing activity of the anti-oxidant defense mechanisms (Nrf2 translocation, increased gene expression for anti-oxidant proteins and increased HMOX-1 synthesis) (tier 1). Higher exposure generated a proinflammatory response (NF-kB translocation, increased gene expression of pro-inflammatory cytokines and adhesion molecules, and increased IL-6 and IL-8 synthesis) (tier 2) and, finally, the highest doses applied resulted in a decrease of cell viability due to necrosis (extra-cellular release of LDH) or apoptosis (increased expression of the pro-apoptotic genes CASP7 and FAS) (tier 3). Overall, the results of our study demonstrate that the 3D tetra culture model when directly exposed to DEPM potently generates a realistic response according to the three tiered oxidative stress concept. Further evaluation and benchmarking against currently used in vivo rodent models is needed to show its suitability, and to serve in the future as an alternative for in vivo studies in the hazard evaluation of inhalable irritants.

Due to the increasing concerns regarding the transferability of data from animal studies to potential human health effects, the development of complex in vitro cellular models which could potentially replace the ethically-debatable in vivo studies and fill the existing gap between in vivo and in vitro data has received considerable attention. The current review focuses on pulmonary in vitro cellular models available for studying the biological effects elicited by inhaled chemicals. The advantages and disadvantages of the 2D monoculture system and the more complex 3D models such as co-cultures and organ-on-a-chip platforms are discussed. Moreover, recent advancements in the field of respiratory toxicology such as the development of airliquid interface systems that better mimic the in vivo respiratory exposure are reviewed. We conclude with future perspectives of the in vitro cellular models in respiratory toxicology.