The increasing manufacture and use of products based on nanotechnology raises concerns for both workers and consumers. Various studies report induction of pulmonary inflammation after inhalation exposure to nanoparticles, which can vary in aspects such as size, shape, charge, crystallinity, chemical composition, and dissolution rate. Each of these aspects can affect their toxicity, although it is largely unknown to what extent. The aim of the current review is to analyse published data on inhalation of nanoparticles to identify and evaluate the contribution of their physicochemical characteristics to the onset and development of pulmonary inflammation. Many physicochemical characteristics of nanoparticles affect their lung deposition, clearance, and pulmonary response that, in combination, ultimately determine whether pulmonary inflammation will occur and to what extent. Lung deposition is mainly determined by the physical properties of the aerosol (size, density, shape, hygroscopicity) in relation to airflow and the anatomy of the respiratory system, whereas clearance and translocation of nanoparticles are mainly determined by their geometry and surface characteristics. Besides size and chemical composition, other physicochemical characteristics influence the induction of pulmonary inflammation after inhalation. As some nanoparticles dissolve, they can release toxic ions that can damage the lung tissue, making dissolution rate an important characteristic that affects lung inflammation. Fibre-shaped materials are more toxic to the lungs compared to spherical shaped nanoparticles of the same chemical composition. In general, cationic nanoparticles are more cytotoxic than neutral or anionic nanoparticles. Finally, surface reactivity correlates well with observed pulmonary inflammation. With all these characteristics affecting different stages of the events leading to pulmonary inflammation, no unifying dose metric could be identified to describe pulmonary inflammation for all nanomaterials, although surface reactivity might be a useful measure. To determine the extent to which the various characteristics influence the induction of pulmonary inflammation, the effect of these characteristics on lung deposition, clearance, and pulmonary response should be systematically evaluated. The results can then be used to facilitate risk assessment by categorizing nanoparticles according to their characteristics.

Although silver nanoparticles are currently used in more than 400 consumer products, it is not clear to what extent they induce adverse effects after inhalation during production and use. In this study, we determined the lung burden, tissue distribution, and the induction and recovery of adverse effects after short-term inhalation exposure to 15 nm and 410 nm silver nanoparticles. Rats were nose-only exposed to clean air, 15 nm silver nanoparticles (179 μg/m3) or 410 nm silver particles (167 μg/m3) 6 hours per day, for four consecutive days. Tissue distribution and the induction of pulmonary toxicity were determined at 24 hours and 7 days after exposure and compared with the internal alveolar dose. Presence of silver nanoparticles in lung cells was visualized by transmission electron microscopy (TEM). Exposure to 15 nm silver nanoparticles induced moderate pulmonary toxicity compared to the controls, indicated by a 175-fold increased influx of neutrophils in the lungs, a doubling of cellular damage markers in the lungs, a 5-fold increase in pro-inflammatory cytokines, and a 1.5-fold increase in total glutathione at 24 hours after exposure. All the observed effects disappeared at 7 days after exposure. No effects were observed after exposure to 410 nm silver particles. The internal alveolar mass dose of the 15 nm nanoparticles was 3.5 times higher compared to the 410 nm particles, which equals to a 66,000 times higher particle number. TEM analysis revealed 15 nm nanoparticles in vesicles and nuclei of lung cells, which were decreased in size to <5 nm at 24 hours after exposure. This demonstrates substantial dissolution of the silver nanoparticles. The results show a clear size-dependent effect after inhalation of similar mass concentrations of 15 nm and 410 nm silver (nano)particles. This can be partially explained by the difference in the internal alveolar dose between the 15 nm and 410 nm silver (nano)particles as well as by a difference in the release rate of silver ions.

Introduction: Here, we describe the generation of hypotheses for grouping nanoforms (NFs) after inhalation exposure and the tailored Integrated Approaches to Testing and Assessment (IATA) with which each specific hypothesis can be tested. This is part of a state-of-the-art framework to support the hypothesis-driven grouping and read-across of NFs, as developed by the EU-funded Horizon 2020 project GRACIOUS. Development of Grouping Hypotheses and IATA: Respirable NFs, depending on their physicochemical properties, may dissolve either in lung lining fluid or in acidic lysosomal fluid after uptake by cells. Alternatively, NFs may also persist in particulate form. Dissolution in the lung is, therefore, a decisive factor for the toxicokinetics of NFs. This has led to the development of four hypotheses, broadly grouping NFs as instantaneous, quickly, gradually, and very slowly dissolving NFs. For instantaneously dissolving NFs, hazard information can be derived by read-across from the ions. For quickly dissolving particles, as accumulation of particles is not expected, ion toxicity will drive the toxic profile. However, the particle aspect influences the location of the ion release. For gradually dissolving and very slowly dissolving NFs, particle-driven toxicity is of concern. These NFs may be grouped by their reactivity and inflammation potency. The hypotheses are substantiated by a tailored IATA, which describes the minimum information and laboratory assessments of NFs under investigation required to justify grouping. Conclusion: The GRACIOUS hypotheses and tailored IATA for respiratory toxicity of inhaled NFs can be used to support decision making regarding Safe(r)-by-Design product development or adoption of precautionary measures to mitigate potential risks. It can also be used to support read-across of adverse effects such as pulmonary inflammation and subsequent downstream effects such as lung fibrosis and lung tumor formation after long-term exposure.

A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105 µg silver/m³ air) of 18, 34, 60 and 160 nm silver particles for four consecutive days and sacrificed at 24 h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose–response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.

The Safe-by-Design (SbD) concept aims to facilitate the development of safer materials/products, safer production, and safer use and end-of-life by performing timely SbD interventions to reduce hazard, exposure, or both. Early hazard screening is a crucial first step in this process. In this review, for the first time, commonly used in vitro assays are evaluated for their suitability for SbD hazard testing of nanomaterials (NMs). The goal of SbD hazard testing is identifying hazard warnings in the early stages of innovation. For this purpose, assays should be simple, cost-effective, predictive, robust, and compatible. For several toxicological endpoints, there are indications that commonly used in vitro assays are able to predict hazard warnings. In addition to the evaluation of assays, this review provides insights into the effects of the choice of cell type, exposure and dispersion protocol, and the (in)accurate determination of dose delivered to cells on predictivity. Furthermore, compatibility of assays with challenging advanced materials and NMs released from nano-enabled products (NEPs) during the lifecycle is assessed, as these aspects are crucial for SbD hazard testing. To conclude, hazard screening of NMs is complex and joint efforts between innovators, scientists, and regulators are needed to further improve SbD hazard testing.

The Risk Assessment Committee of the European Chemicals Agency issued an opinion on classifying titanium dioxide (TiO₂) as a suspected human carcinogen upon inhalation. Recent animal studies indicate that TiO₂ may be carcinogenic through the oral route. There is considerable uncertainty on the carcinogenicity of TiO₂, which may be decreased if its mechanism of action becomes clearer. Here we consider adverse outcome pathways and present the available information on each of the key events (KEs). Inhalation exposure to TiO₂ can induce lung tumors in rats via a mechanism that is also applicable to other poorly soluble, low-toxicity particles. To reduce uncertainties regarding human relevance, we recommend gathering information on earlier KEs such as oxidative stress in humans. For oral exposure, insufficient information is available to conclude whether TiO₂ can induce intestinal tumors. An oral carcinogenicity study with well-characterized (food-grade) TiO₂ is needed, including an assessment of toxicokinetics and early KEs.