An in vitro alveolar macrophage assay for predicting the short-term inhalation toxicity of nanomaterials

Additional file 1: Table S1. Comparison of significant in vitro LOAECs (significant as compared to the negative benchmark material corundum) to NOAECs and LOAECs recorded in rat STISs. Table S2. Bioactivity of four types of CeO2 NMs in rat STISs as compared to cellular effects recorded in the in vitro NR8383 AM assay

Biokinetics and effects of barium sulfate nanoparticles

Background: Nanoparticulate barium sulfate has potential novel applications and wide use in the polymer and paint industries. A short-term inhalation study on barium sulfate nanoparticles (BaSO4 NPs) was previously published [Part Fibre Toxicol 11:16, 2014]. We performed comprehensive biokinetic studies of 131BaSO4 NPs administered via different routes and of acute and subchronic pulmonary responses to instilled or inhaled BaSO4 in rats. Methods: We compared the tissue distribution of 131Ba over 28 days after intratracheal (IT) instillation, and over 7 days after gavage and intravenous (IV) injection of 131BaSO4. Rats were exposed to 50 mg/m3 BaSO4 aerosol for 4 or 13 weeks (6 h/day, 5 consecutive days/week), and then gross and histopathologic, blood and bronchoalveolar lavage (BAL) fluid analyses were performed. BAL fluid from instilled rats was also analyzed. Results: Inhaled BaSO4 NPs showed no toxicity after 4-week exposure, but a slight neutrophil increase in BAL after 13-week exposure was observed. Lung burden of inhaled BaSO4 NPs after 4-week exposure (0.84 ± 0.18 mg/lung) decreased by 95% over 34 days. Instilled BaSO4 NPs caused dose-dependent inflammatory responses in the lungs. Instilled BaSO4 NPs (0.28 mg/lung) was cleared with a half-life of ≈ 9.6 days. Translocated 131Ba from the lungs was predominantly found in the bone (29%). Only 0.15% of gavaged dose was detected in all organs at 7 days. IV-injected 131BaSO4 NPs were predominantly localized in the liver, spleen, lungs and bone at 2 hours, but redistributed from the liver to bone over time. Fecal excretion was the dominant elimination pathway for all three routes of exposure. Conclusions: Pulmonary exposure to instilled BaSO4 NPs caused dose-dependent lung injury and inflammation. Four-week and 13-week inhalation exposures to a high concentration (50 mg/m3) of BaSO4 NPs elicited minimal pulmonary response and no systemic effects. Instilled and inhaled BaSO4 NPs were cleared quickly yet resulted in higher tissue retention than when ingested. Particle dissolution is a likely mechanism. Injected BaSO4 NPs localized in the reticuloendothelial organs and redistributed to the bone over time. BaSO4 NP exhibited lower toxicity and biopersistence in the lungs compared to other poorly soluble NPs such as CeO2 and TiO2. Electronic supplementary material The online version of this article (doi:10.1186/s12989-014-0055-3) contains supplementary material, which is available to authorized users

Toxicological inhalation studies in rats to substantiate grouping of zinc oxide nanoforms

Background Significant variations exist in the forms of ZnO, making it impossible to test all forms in in vivo inhalation studies. Hence, grouping and read-across is a common approach under REACH to evaluate the toxicological profile of familiar substances. The objective of this paper is to investigate the potential role of dissolution, size, or coating in grouping ZnO (nano)forms for the purpose of hazard assessment. We performed a 90-day inhalation study (OECD test guideline no. (TG) 413) in rats combined with a reproduction/developmental (neuro)toxicity screening test (TG 421/424/426) with coated and uncoated ZnO nanoforms in comparison with microscale ZnO particles and soluble zinc sulfate. In addition, genotoxicity in the nasal cavity, lungs, liver, and bone marrow was examined via comet assay (TG 489) after 14-day inhalation exposure. Results ZnO nanoparticles caused local toxicity in the respiratory tract. Systemic effects that were not related to the local irritation were not observed. There was no indication of impaired fertility, developmental toxicity, or developmental neurotoxicity. No indication for genotoxicity of any of the test substances was observed. Local effects were similar across the different ZnO test substances and were reversible after the end of the exposure. Conclusion With exception of local toxicity, this study could not confirm the occasional findings in some of the previous studies regarding the above-mentioned toxicological endpoints. The two representative ZnO nanoforms and the microscale particles showed similar local effects. The ZnO nanoforms most likely exhibit their effects by zinc ions as no particles could be detected after the end of the exposure, and exposure to rapidly soluble zinc sulfate had similar effects. Obviously, material differences between the ZnO particles do not substantially alter their toxicokinetics and toxicodynamics. The grouping of ZnO nanoforms into a set of similar nanoforms is justified by these observations

Pathway-based predictive approaches for non-animal assessment of acute inhalation toxicity

New approaches are needed to assess the effects of inhaled substances on human health. These approaches will be based on mechanisms of toxicity, an understanding of dosimetry, and the use of in silico modeling and in vitro test methods. In order to accelerate wider implementation of such approaches, development of adverse outcome pathways (AOPs) can help identify and address gaps in our understanding of relevant parameters for model input and mechanisms, and optimize non-animal approaches that can be used to investigate key events of toxicity. This paper describes the AOPs and the toolbox of in vitro and in silico models that can be used to assess the key events leading to toxicity following inhalation exposure. Because the optimal testing strategy will vary depending on the substance of interest, here we present a decision tree approach to identify an appropriate non-animal integrated testing strategy that incorporates consideration of a substance's physicochemical properties, relevant mechanisms of toxicity, and available in silico models and in vitro test methods. This decision tree can facilitate standardization of the testing approaches. Case study examples are presented to provide a basis for proof-of-concept testing to illustrate the utility of non-animal approaches to inform hazard identification and risk assessment of humans exposed to inhaled substances

Deposition behavior of inhaled nanostructured TiO2 in rats: fractions of particle diameter below 100 nm (nanoscale) and the slicing bias of transmission electron microscopy

Context: In experimental studies with nanomaterials where translocation to secondary organs was observed, the particle sizes were smaller than 20 nm and were mostly produced by spark generators. Engineered nanostructured materials form microsize aggregates/agglomerates. Thus, it is unclear whether primary nanoparticles or their small aggregates/agglomerates occur in non-negligible concentrations after exposure to real-world materials in the lung. Objective: We dedicated an inhalation study with nanostructured TiO2 to the following research question: Does the particle size distribution in the lung contain a relevant subdistribution of nanoparticles? Methods: Six rats were exposed to 88 mg/m(3) TiO2 over 5 days with 20% (count fraction) and <0.5% (mass fraction) of nanoscaled objects. Three animals were sacrificed after cessation of exposure (5 days), others after a recovery period of 14 days. Particle sizes were determined morphometrically by transmission electron microscopy (TEM) of ultra-thin lung slices. Since the particles visible are two-dimensional surrogates of three-dimensional structures we developed a model to estimate expected numbers of particle diameters below 100 nm due to the TEM slicing bias. Observed and expected numbers were contrasted in 2 x 2 tables by odds ratios. Results: Comparisons of observed and expected numbers did not present evidence in favor of the presence of nanoparticles in the rat lungs. In simultaneously exposed satellite animals agglomerates of nanostructured TiO2 were observed in the mediastinal lymph nodes but not in secondary organs. Conclusions: For nanostructured TiO2, the deposition of nanoscaled particles in the lung seem to play a negligible role

Hazard Identification of Inhaled Nanomaterials: Making use of Short-term Inhalation Studies

A major health concern for nanomaterials is their potential toxic effect after inhalation of dusts. Correspondingly, the core element of tier 1 in the currently proposed Integrated Testing Strategy (ITS) is a short-term rat inhalation study (STIS) for this route of exposure. STIS comprises a comprehensive scheme of biological effects and marker determination in order to generate appropriate information on early key elements of pathogenesis, such as inflammatory reactions in the lung and indications of effects in other organs. Within the STIS information on the persistence, progression and/or regression of effects is obtained. The STIS also addresses organ burden in the lung and potential translocation to other tissues. Up to now STIS was performed in research projects and routine testing of nanomaterials. Meanwhile rat STIS results for more than 20 nanomaterials are available including the representative nanomaterials listed by the Organization for Economic Cooperation and Development (OECD) Working Party on Manufactured Nanomaterials (WPMN), which has endorsed a list of representative Manufactured Nanomaterials (MN) as well as a set of relevant endpoints to be addressed. Here, results of STIS carried out with different nanomaterials are discussed as case studies. The ranking of different nanomaterials potential to induce adverse effects and the ranking of the respective NOAEC is the same among the STIS and the corresponding sub-chronic and chronic studies. In another case study, a translocation of a coated silica nanomaterial was judged critical for its safety assessment. Thus, STIS enables application of the proposed ITS, as long as reliable and relevant in vitro methods for the tier 1 testing are still missing. Compared to traditional subacute and subchronic inhalation testing (according to OECD test guidelines (TG) 412 and 413), STIS uses less animals and resources and offers additional information on organ burden and pro-/regression of potential effects.JRC.D-Institute for Reference Materials and Measurements (Geel

An Integrated Approach to Testing and Assessment to Support Grouping and Read-Across of Nanomaterials after Inhalation Exposure

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