Periodic Reporting for period 3 - PATROLS (Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment)
Période du rapport: 2021-01-01 au 2021-09-30
Classical hazard testing strategies to define the human & environmental health impact of engineered nanomaterials (ENM) commonly apply unrealistic acute, high-dose exposures to models that do not reflect the in vivo environment. Furthermore, existing in vitro & in silico hazard detection methods are not highly predictive. Hence there is an urgent need to address these limitations by developing improved in vitro & in silico tools tailored to realistic ENM exposure scenarios. To overcome these challenges, PATROLS has established & standardised a battery of next generation physiologically anchored, hazard assessment tools that more accurately predict adverse human health & environmental effects caused by long-term, low dose ENM exposure to support regulatory risk decision-making. The project has developed: 1) more realistic in vitro 3D lung, gastrointestinal tract (GIT) & liver models for mechanism-based hazard assessment, that have been anchored against chronic in vivo exposure outcomes & report on mechanistic endpoints linked to adverse outcome pathways (AOPs); 2) novel methods to evaluate long-term exposure hazard endpoints in ecologically relevant test systems & organisms, selected according to their position in the food chain; these endpoints have been based on key events associated with AOPs; 3) robust in silico methods for exposure & dosimetry modelling, as well as in vitro-to-in vivo extrapolation (IVIVE) and hazard prediction; 4) improved understanding of ENM physico-chemical identity & dosimetry in physiologically relevant exposure scenarios dictated by the advanced human & environmental models developed. Iterative training, testing, stakeholder feedback & pre-validation phases have supported the provision of robust, fit-for-purpose test systems. PATROLS has therefore provided an innovative & effective set of models, bioassays & computational tools to more reliably predict potential hazards resulting from realistic ENM exposures. These tools will minimise the necessity for animal testing to promote the 3Rs, support the European Chemicals Strategy & will also support future nanosafety risk assessment requirements.
PATROLS has developed a versatile toolbox of in vitro, in silico & environmental models, plus physico-chemical characterisation methods, to comprehensively assess the effects of sub-chronically exposed ENM in advanced human tissue models & organisms across a food-chain, using a multi-faceted approach.
Key scientific & technical advances include:
• Defined the primary physico-chemical characteristics of ENM used in PATROLS & completed extensive characterisation of secondary ENM features under experimental conditions relevant to both human cell culture & environmental models (e.g. dissolution, redox potential, size distribution etc). Methods for testing ENM solubility & dissolution in biologically relevant media have been standardised.
• Completed a comprehensive database of all existing long-term in vivo oral & inhalation toxicity studies, to benchmark the advanced in vitro models under development. Effects after exposure, NOAEL/LOAELs, biodistribution & data gaps have been highlighted. The gaps have been filled through 1) new biodistribution analysis on tissues available from previously completed in vivo studies; 2) targeted in vivo studies to facilitate development of PBPK & IVIVE in silico models.
• Defined AOPs to link in vitro with in vivo data have been completed, focusing on lung inflammation, fibrosis, cancer & mesothelioma; and liver inflammation, fibrosis & cancer. This has provided important information on new hazard biomarkers that were utilised to increase the in vitro lung & liver models' predictive potential.
• Designed a suite of more physiologically relevant 3D & multi-cellular co-culture lung, liver & GIT tract models, tested with low-dose, repeated ENM exposures. The lung model supports aerosol ENM exposures & can be incorporated into a bioreactor introducing both fluid flow and tissue flexing to mimic more realistic movement of human alveoli. Two healthy liver model systems incorporating multiple cell-types have been developed; a primary cell system supporting repeated ENM exposures for up to 3-weeks & a cell line-based 3D model that enables genotoxicity testing. A mucus producing, intestinal triple GIT culture has been established using enterocytes, goblet cells & macrophages and has been exposed repeatedly to low-dose ENM.
• In addition to the healthy lung, liver & GIT in vitro models, novel disease models have been developed for each of the tissue types. Upon long-term ENM testing, the disease models demonstrate greater sensitivity for adverse effects than their equivalent healthy in vitro tissue models.
• Long-term (multi-generation) exposure studies have been conducted in environmental organisms, where more significant toxic responses were observed when compared to acute exposures. A transgenic fish line for early signalling of ENM exposure has been developed & a better understanding of ecological microbiome–host interactions has been provided.
• A novel transcriptomic based & AOP-anchored NanoQSAR model has been developed for predicting pulmonary pathology induced by MWCNTs.
• An in vitro partico-kinetics model has been developed to define ENM diffusion, sedimentation & dissolution in cell culture medium as well as cellular uptake. A DosiGUI multi-model graphical user graphical user interface for the in vitro model of ENM deposition has been designed to facilitate ease of use. In silico QSAR, PBPK & IVIVE models developed early in the project have been refined, with supporting web tools.
• The PATROLS website and social media accounts have been highly active sources for rapid dissemination of project outputs. Additionally, strong connections to key stakeholders have been built through industrial events, workshops (targeting industry & policy makers), webinars & integration into OECD activities.
Key scientific & technical advances include:
• Defined the primary physico-chemical characteristics of ENM used in PATROLS & completed extensive characterisation of secondary ENM features under experimental conditions relevant to both human cell culture & environmental models (e.g. dissolution, redox potential, size distribution etc). Methods for testing ENM solubility & dissolution in biologically relevant media have been standardised.
• Completed a comprehensive database of all existing long-term in vivo oral & inhalation toxicity studies, to benchmark the advanced in vitro models under development. Effects after exposure, NOAEL/LOAELs, biodistribution & data gaps have been highlighted. The gaps have been filled through 1) new biodistribution analysis on tissues available from previously completed in vivo studies; 2) targeted in vivo studies to facilitate development of PBPK & IVIVE in silico models.
• Defined AOPs to link in vitro with in vivo data have been completed, focusing on lung inflammation, fibrosis, cancer & mesothelioma; and liver inflammation, fibrosis & cancer. This has provided important information on new hazard biomarkers that were utilised to increase the in vitro lung & liver models' predictive potential.
• Designed a suite of more physiologically relevant 3D & multi-cellular co-culture lung, liver & GIT tract models, tested with low-dose, repeated ENM exposures. The lung model supports aerosol ENM exposures & can be incorporated into a bioreactor introducing both fluid flow and tissue flexing to mimic more realistic movement of human alveoli. Two healthy liver model systems incorporating multiple cell-types have been developed; a primary cell system supporting repeated ENM exposures for up to 3-weeks & a cell line-based 3D model that enables genotoxicity testing. A mucus producing, intestinal triple GIT culture has been established using enterocytes, goblet cells & macrophages and has been exposed repeatedly to low-dose ENM.
• In addition to the healthy lung, liver & GIT in vitro models, novel disease models have been developed for each of the tissue types. Upon long-term ENM testing, the disease models demonstrate greater sensitivity for adverse effects than their equivalent healthy in vitro tissue models.
• Long-term (multi-generation) exposure studies have been conducted in environmental organisms, where more significant toxic responses were observed when compared to acute exposures. A transgenic fish line for early signalling of ENM exposure has been developed & a better understanding of ecological microbiome–host interactions has been provided.
• A novel transcriptomic based & AOP-anchored NanoQSAR model has been developed for predicting pulmonary pathology induced by MWCNTs.
• An in vitro partico-kinetics model has been developed to define ENM diffusion, sedimentation & dissolution in cell culture medium as well as cellular uptake. A DosiGUI multi-model graphical user graphical user interface for the in vitro model of ENM deposition has been designed to facilitate ease of use. In silico QSAR, PBPK & IVIVE models developed early in the project have been refined, with supporting web tools.
• The PATROLS website and social media accounts have been highly active sources for rapid dissemination of project outputs. Additionally, strong connections to key stakeholders have been built through industrial events, workshops (targeting industry & policy makers), webinars & integration into OECD activities.
PATROLS is aimed at advancing the state-of-the-art in ENM human and environmental hazard assessment through the provision of improved and innovative tools that reduce the necessity for animal testing. The project has achieved significant innovative developments that has involved adapting & further advancing cell based ENM hazard testing systems by 1) enhancing their physiological relevance through increased biological complexity; 2) extending their culture time whilst maintaining normal cell function and viability, to better support more realistic longer-term, repeated dosing scenarios; 3) development of a suite of diseased-state models. Ecological assays have also been improved to better support long-term ENM exposures, through generation of novel systems to both support chronic environmental exposure and detect new hazard endpoints with greater sensitivity. Thus, PATROLS has created substantial advances in state-of-the-art for ENM hazard testing. The PATROLS partners have been pro-active in developing relationships and engaging with a range of stakeholders to raise awareness of the project and facilitate its impact. This has involved active interactions with stakeholders through dedicated workshops, webinars, distribution of e-newsletters, public engagement activities and transfer of knowledge generated within the project to on-going OECD Working Party on Manufactured Nanomaterials initiatives & standardisation activities.