This work is part of the H2020 European project ASINA which aims to promote Safe-by-Design (SbD) solutions for new nanomaterials (NMs) across all life cycle stages. The adoption of SbD novel approaches for new NMs is of pivotal importance in the framework of the European Commission’s Green Deal. Over the past decades, the production and use of NMs have seen substantial growth, raising concerns regarding the potential risk associated with their release and population exposure. Silver (Ag) and titania (TiO2) nanoparticles (NPs) are among the most widely used NMs and their applications in various products, due to the antibacterial properties of the first and photocatalytic activity of the latter, are increasing. The aim of this work is to identify the potential hazard posed by newly developed metal based NMs, designed according to a SbD approach, toward human health during their production and use. The initial step involved testing the newly developed Ag and TiO2 NPs coated with different agents to compare their toxicological effects to reference uncoated NPs. The endpoints were selected according to an Adverse Outcome Pathway (AOP) approach that enabled the identification of the main events linked to the toxicity of these NPs. Cell viability, inflammatory response, reactive oxygen species levels, oxidative DNA damage, and cell-NPs interactions were analyzed in the alveolar in vitro model, A549. Furthermore, a New Approach Methodology (NAM) was developed to identify and evaluate the hazard of the safer NPs (based on the results of the first part of this study) considering the potential human exposure during NMs production. The goal was to improve their hazard and risk definition. The selection of appropriate exposure doses for hazard assessment has gained increasing importance in the risk assessment framework. To this end, starting from monitoring campaign data and applying the MPPD model to determine the lung retained dose of NPs, it was possible to estimate the doses representative of a chronic human exposure. The selected in vitro model, consisted in a human cell contact co-culture (A549 and THP-1 cells differentiated in macrophages) representative of the alveolar space, cultured, and exposed at the Air-Liquid-Interface (ALI). Exposure to aerosolized NPs was performed by Vitrocell® and Cultex® commercial systems. This approach allows for more reliable results than by traditional submerged culture systems due to a closer replication of the human physiology. Before exposing the model to the calculated doses, the deposition efficiency (DE) of each NP was determined by using a quartz crystal microbalance (QCM). The results demonstrated that the functionalization of the NPs has a primary role in driving their toxicity. Moreover, the method of NPs production significantly influenced their toxicity. Different NPs also showed different DE depending on their characteristics. The results obtained from the co-culture indicates the absence of significant hazard for chronic inhalation exposure at environmentally relevant doses, confirming the safety of the NMs and the process developed.
Questo lavoro fa parte del progetto Europeo H2020 ASINA, il quale mira a promuovere delle soluzioni Safe-by-design (SbD) per nuovi nanomateriali (NMs) lungo tutto il loro ciclo vitale. L’adozione di innovativi approcci SbD è di grande importanza nel quadro del Green Deal della Commissione Europea. Negli ultimi decenni, la produzione e l’uso di NMs ha subito una sostanziale crescita, sollevando preoccupazioni a proposito del potenziale rischio associato al loro rilascio e all’esposizione della popolazione. Le nanoparticelle (NPs) di argento (Ag) e di biossido di titanio (TiO2) sono tra i NMs più ampiamente utilizzati e le loro applicazioni in vari prodotti, a causa delle proprietà antibatteriche delle prime e dell’attività fotocatalitica delle seconde, stanno aumentando. Lo scopo di questo lavoro è stato di identificare il pericolo posto da NMs metallici di nuova sintesi, disegnati secondo un approccio SbD, verso la salute umana durante la loro produzione e il loro utilizzo. Il primo passo ha previsto il test delle nuove NPs di Ag e TiO2 con diversi rivestimenti per confrontare i loro effetti tossicologici con NPs di riferimento senza alcun rivestimento. Gli endpoint sono stati selezionati secondo un approccio Adverse Outcome Pathway (AOP), il quale ha consentito l’identificazione dei principali eventi connessi alla tossicità di queste NPs. La vitalità cellulare, la risposta infiammatoria, i livelli di specie reattive dell’ossigeno, il danno ossidativo al DNA, e le interazioni tra cellule e NPs sono state analizzate nel modello alveolare in vitro A549. In seguito, un New Approach Methodology (NAM) è stato sviluppato per identificare e valutare il rischio delle NPs identificate come più sicure nella prima parte dello studio considerando la potenziale esposizione umana durante la loro produzione. Lo scopo è stato quello di definire meglio il pericolo di queste NPs. La selezione di dosi di esposizione appropriate per la definizione del pericolo ha un’importanza crescente nel quadro del risk assessment. A tale scopo, partendo dai dati ottenuti da una campagna di monitoraggio e applicando il modello MPPD per determinare la dose di NPs ritenuta a livello alveolare, è stato possibile stimare le dosi rappresentative di un’esposizione umana cronica. Il modello in vitro selezionato è composto da una co-coltura (A549 e THP-1 differenziate in macrofagi) rappresentative dello spazio alveolare, coltivate ed esposte all’Air-Liquid-Interface (ALI). L’esposizione all’aerosol di NPs è stata effettuata tramite due sistemi commerciali di Vitrocell® e Cultex®. Questo approccio consente di avere risultati più affidabili rispetto alle tradizionali colture sommerse a causa di una migliore similarità con la fisiologia umana. Prima di esporre il modello alle dosi calcolate, l’efficienza di deposizione (DE) di ogni NP è stata determinata tramite l’uso di una microbilancia. I risultati dimostrano che la funzionalizzazione delle NPs ha un ruolo primario del determinare la loro tossicità. Inoltre, anche il metodo di produzione è in grado di influenzare in maniera significativa la loro tossicità. Diverse NPs hanno dimostrato diversa DE in base alle loro caratteristiche. I risultati ottenuti dalla co-coltura indicano l’assenza di un pericolo significativo in seguito ad esposizione a dosi rappresentative di una reale inalazione cronica, confermando la sicurezza dei NMs e dei processi sviluppati.
(2024). New approach methodologies-oriented toxicology in in vitro systems for implementing the safety-by-design of new nanomaterials. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2024).
New approach methodologies-oriented toxicology in in vitro systems for implementing the safety-by-design of new nanomaterials
MOTTA, GIULIA
2024
Abstract
This work is part of the H2020 European project ASINA which aims to promote Safe-by-Design (SbD) solutions for new nanomaterials (NMs) across all life cycle stages. The adoption of SbD novel approaches for new NMs is of pivotal importance in the framework of the European Commission’s Green Deal. Over the past decades, the production and use of NMs have seen substantial growth, raising concerns regarding the potential risk associated with their release and population exposure. Silver (Ag) and titania (TiO2) nanoparticles (NPs) are among the most widely used NMs and their applications in various products, due to the antibacterial properties of the first and photocatalytic activity of the latter, are increasing. The aim of this work is to identify the potential hazard posed by newly developed metal based NMs, designed according to a SbD approach, toward human health during their production and use. The initial step involved testing the newly developed Ag and TiO2 NPs coated with different agents to compare their toxicological effects to reference uncoated NPs. The endpoints were selected according to an Adverse Outcome Pathway (AOP) approach that enabled the identification of the main events linked to the toxicity of these NPs. Cell viability, inflammatory response, reactive oxygen species levels, oxidative DNA damage, and cell-NPs interactions were analyzed in the alveolar in vitro model, A549. Furthermore, a New Approach Methodology (NAM) was developed to identify and evaluate the hazard of the safer NPs (based on the results of the first part of this study) considering the potential human exposure during NMs production. The goal was to improve their hazard and risk definition. The selection of appropriate exposure doses for hazard assessment has gained increasing importance in the risk assessment framework. To this end, starting from monitoring campaign data and applying the MPPD model to determine the lung retained dose of NPs, it was possible to estimate the doses representative of a chronic human exposure. The selected in vitro model, consisted in a human cell contact co-culture (A549 and THP-1 cells differentiated in macrophages) representative of the alveolar space, cultured, and exposed at the Air-Liquid-Interface (ALI). Exposure to aerosolized NPs was performed by Vitrocell® and Cultex® commercial systems. This approach allows for more reliable results than by traditional submerged culture systems due to a closer replication of the human physiology. Before exposing the model to the calculated doses, the deposition efficiency (DE) of each NP was determined by using a quartz crystal microbalance (QCM). The results demonstrated that the functionalization of the NPs has a primary role in driving their toxicity. Moreover, the method of NPs production significantly influenced their toxicity. Different NPs also showed different DE depending on their characteristics. The results obtained from the co-culture indicates the absence of significant hazard for chronic inhalation exposure at environmentally relevant doses, confirming the safety of the NMs and the process developed.File | Dimensione | Formato | |
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Descrizione: New approach methodologies-oriented toxicology in in vitro systems for implementing the safety-by-design of new nanomaterials
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