The production and use of nanomaterials (NMs) has increased over the last decades posing relevant questions on their risk after release and exposure of the population or sub-populations. In this context, the safe and sustainable by design (SSbD) approach framework requires to assess the potential hazard connected with intrinsic properties of the material along the whole life cycle of the NM and/or of the nano enabled products. Moreover, in the last years, the use of new advanced methodologies (NAMs) has increasingly gained attention for the use of alternative methods in obtaining relevant information on NMs hazard and risk. Considering the SSbD and the NAMs frameworks, within the ASINA H2020 project, we developed new NAMs devoted at improving the hazard and risk definition of different Ag and TiO2 NPs. The NAMs are developed considering two air liquid interface exposure systems, the Vitrocell Cloud-α and the Cultex Compact module and the relevant steps to obtain reproducible exposures are described. The new NAMs build on the integration of environmental monitoring campaigns at nano-coating production sites, allowing the quantification by the multiple-path particle dosimetry (MPPD) model of the expected lung deposited dose in occupational settings. Starting from this information, laboratory exposures to the aerosolized NPs are performed by using air liquid interface exposure equipment and human alveolar cells (epithelial cells and macrophages), replicating the doses of exposure estimated in workers by MPPD. Preliminary results on cell viability and inflammatory responses are reported. The proposed NAMs may represent possible future reference procedures for assessing the NPs inhalation toxicology, supporting risk assessment at real exposure doses.

Airborne particulate matter (PM) is the primary environmental cause of death worldwide. The understanding of the toxicological mechanism has improved over the last decades, nonetheless now novel exposure systems allow the possibility to mimic the in vivo interaction between particles or particle constituents and lung cells. Here we report the results obtained after exposure to the organic extract of fine PM (PM_2.5-0.3) of the BEAS-2B cells cultured in submerged or at the air liquid interface (ALI) conditions. Deposition efficiency of both PM and its organic extract was followed. Cell viability, inflammatory responses and gene expression were then evaluated. The comparison of submerged vs ALI exposure revealed significant differences in the expression of specific genes such as CYP1A1 and NQO1 6h after exposure and CYP1B1 and NFkB1 24h post exposure. Also, the results demonstrate the importance of evaluating the efficiency of deposition of the treatment to avoid misinterpretation of the results. PM particles determined an extremely low deposition efficiency, making it impossible to use this particle for ALI exposure tests. Although biological results, such as gene expression, may vary between the two exposure approaches (submerged or ALI), the ALI exposure should be preferred as it is more representative of human exposure via the respiratory route.

Silver nanoparticles (Ag NPs) are among the most widely used metal-based nanomaterials (NMs) and their applications in different products, also as antibacterial additives, are increasing. In the present manuscript, according to an adverse outcome pathway (AOP) approach, we tested two safe-by-design (SbD) newly developed Ag NPs coated with hydroxyethyl cellulose (HEC), namely AgHEC powder and AgHEC solution. These novel Ag NPs were compared to two reference Ag NPs (naked and coated with polyvinylpyrrolidone—PVP). Cell viability, inflammatory response, reactive oxygen species, oxidative DNA damage, cell cycle, and cell–particle interactions were analyzed in the alveolar in vitro model, A549 cells. The results show a different toxicity pattern of the novel Ag NPs compared to reference NPs and that between the two novel NPs, the AgHEC solution is the one with the lower toxicity and to be further developed within the SbD framework.