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Smart Tools for Gauging Nano Hazards

Periodic Reporting for period 3 - SmartNanoTox (Smart Tools for Gauging Nano Hazards)

Reporting period: 2018-09-01 to 2020-09-30

The capacity to assess the safety of novel engineered nanomaterials (NM), matching the rate of their development and introduction to the market, is the defining factor for the progress of nanotechnology. It can be significantly increased using modern methods in biology, materials and data science to identify the mechanisms of action of the NM. SmartNanoTox project used in vivo, in vitro and in silico methods to infer the main respiratory adverse outcome pathways (AOP) induced by exposure to NMs, to relate them to interactions at the bionano interface and to NM's properties. Knowledge of the properties of concern enables the safer-by-design approaches in industry as it allow one to reduce or avoid the undesirable characteristics at the stage of nano-enabled product development. The detailed information about AOPs can be used to perform toxicity screening without animal experimentation.
SmartNanoTox objectives:
• To identify main pulmonary AOs induced by NMs and associated molecular initiating events (MIE), biological key events (KE) and toxicity pathways (TP) leading to the AOs
• To establish relationships between physicochemical properties of NMs and the KEs, and suggest descriptors for grouping of NMs according to their toxicological mode of action
• To create a database of bionano interactions that will enable development of read-across and QSAR tools for the toxicity assessment of new NMs
• To develop a smart screening approach, where predictions of toxicity of a NM can be made on the basis of purely computational or limited in vitro screening tests focused on crucial bionano interactions
SmartNanoTox team has successfully completed the planned research programme and met its main goals. The team demonstrated the possibility to make predictions of chronic AOs based on in vitro and in silico tests only, described several AOPs, developed methods of advanced NM characterisation, created a database of bionano interactions, and proposed suits of smart tests based on the mechanistic picture of the toxicity.
SmartNanoTox research programme addressed respiratory AOs, as they present the largest concern for workplace exposure and urban environment. The consortium contributed to the development of 5 AOPs relevant for pulmonary exposure to NMs:
• AOP 173: Substance interaction with the lung resident cell membrane components leading to lung fibrosis
• AOP (no ID): Interactions with lung tissues leading to lung fibrosis via a pathway involving Trefoil Factor 2
• AOP 237: Pulmonary acute phase response leading to CHD
• AOP 302: Disruption of lung surfactant function – acute inhalation toxicity
• AOP 303: Frustrated phagocytosis-induced lung cancer

SmartNanoTox consortium has selected 61 NM for the toxicity studies, which include the most common industrial NMs:
• Quartz and Silica
• Metal oxides
• Carbon black
• Carbon nanotubes
• Graphene
• Asbestos

Main activities and outcomes:
Research highlights:
• Demonstrated the possibility to predict chronic AOs (chronic lung inflammation) with only in vitro and in silico tests
• Developed a statistical framework for inferring transcription regulatory programs for the identification of crucial transcription factors
• Proposed a suite of in vitro/in silico/in vivo assays matching the AOP-specific MIEs/KEs
• Developed quantitative multiscale models of bionano interface, which can be used for prediction of NM functionalities
• Created a public database of bionano interactions

In vivo:
• Performed animal exposure experiments (inhalation and instillation) with multiple NMs
• Performed toxicology and histology analyses of in vivo samples
• Demonstrated correlation between the total deposited NM surface area and level of inflammation in the lung
• Demonstrated correlation of lung inflammation between intratracheal instillation and inhalation for different NMs

In vitro:
• Performed in vitro NM tracking and characterisation in lung epithelial cells and macrophages
• Developed an advanced in vitro lung barrier model using a realistic surfactant layer
• Evaluated the effect of NM on the lung membrane using Constrained Drop Surfactometer
• Optimized advanced super-resolved imaging techniques for tracking and identification of NM under in vitro and in vivo condition
• Identified new candidates for MIE and KE such as lipid wrapping around NMs
• Exposed cell lines to NMs using submerged or murine dose-controlled ALI setups
• Performed proteomics and lipidomics studies on protein/lipid corona/wraps of NM in the lung

In silico:
• Developed multiscale methods for characterisation of NMs and bionano interactions
• Computed descriptors of NMs responsible for bionano interactions
• Developed a computational model of lung lining fluid and predicted the corona content for selected NMs
• Developed QSARs for prediction of lung inflammation from NM properties
• Developed in silico prediction methods for NM-lipid membrane interaction and lysosomal damage

• Project website containing description of project activities and links to main outcomes, project brochure and outreach video
• Over 70 papers in peer-reviewed journals
• Over 120 presentations at conferences
• OECD expert meeting on the use of AOP framework for nanosafety regulation (Paris, 2019)
• 6 international conferences
• 5 webinars, including stakeholder meeting
• 11 PR presentations (TV/Press)
• 13 Exhibition booths at international conferences
• 3 policy papers
• 16 spec sheets for exploitable outcomes
The project legacy is truly multidimensional as it includes novel concepts in nanosafety assessment, novel methods, models and tools.
• Mechanism-based toxicity assessment paradigm based on the AOP ideology

Methods and SOPs
• NM labelling, tracking inside tissues, post-uptake characterisation
• Air-liquid interface exposure imitation, aerosolisation, exposure quantification
• Bionano interaction modelling
• Pathway analysis, gene regulation network analysis, omics data processing

• QSARs for MIE/KE
• Multiscale models for evaluation of NM properties, protein adsorption and protein corona

We envision that the following project results to make the largest impact:
Research impacts
• Gene expression profiles for in vivo respiratory exposure
• Novel analysis protocols for inference of gene regulation networks
• Novel NM labelling techniques
• Novel protocols for corona analysis
• Novel algorithms for image analysis / colocalization
• Protein corona-based NM fingerprints
• NM tracking techniques, post-uptake characterisation data
• Atomistic, coarse-grained force fields for common NMs
• Multiscale simulation tools for bionano interface
• Novel advanced NM and protein descriptors
• Data in an accessible and searchable database

Industry/Regulation impacts:
• Development and validation a novel testing strategy for risk assessment of new NMs
• Novel ALI systems imitating realistic exposure conditions
• Demonstration of mapping inhalation-instillation
• Demonstration of equivalence between rat/mouse/human models
• Novel toxicity endpoints bound to in vivo AOPs
• Novel in vitro assays targeting MIE/KE based on reporter gene
• Elucidation of toxicity mechanisms for oxides, carbonaceous materials
• Creation of basis for read-across and safety by design through identification of NM properties of concern
• Mechanism-aware QSARs relating NM properties to biological activity

Human impact:
• 7 PhD theses
• 25 postdocs
• 2 training schools
Research concept and outline of the project activities