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An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Following inhalation and deposition in the alveolar region at sufficient dose, biopersistent (nano)materials generally provoke pulmonary inflammation. Alveolar macrophages (AMs) are mediators of pulmonary immune responses and were broadly categorized in pro-inflammatory M1 and anti-inflammatory M2 macrophages. This study aimed at identifying AM phenotype as M1 or M2 upon short-term inhalation exposure to different (nano)materials followed by a postexposure period. Phenotyping of AM was retrospectively performed using immunohistochemistry. M1 (CD68+iNOS+) and M2 (CD68+CD206+and CD68+ArgI+) AMs were characterized in formalin-fixed, paraffin-embedded lung tissue of rats exposed for 6 hours/day for 5 days to air, 100 mg/m3nano-TiO2, 25 mg/m3nano-CeO2, 32 mg/m3multiwalled carbon nanotubes, or 100 mg/m3micron-sized quartz. During acute inflammation, relative numbers of M1 AMs were markedly increased, whereas relative numbers of M2 were generally decreased compared to control. Following an exposure-free period, changes in iNOS or CD206 expression correlated with persistence, regression, or progression of inflammation, suggesting a role of M1/M2 AMs in the pathogenesis of pulmonary inflammation. However, no clear correlation of AM subpopulations with qualitatively distinct histopathological findings caused by different (nano)materials was found. A more detailed understanding of the processes underlaying these morphological changes is needed to identify biomarkers for different histopathological outcomes.

Numerous recent reviews have highlighted the urgent need for methods to determine the biodissolution of nanomaterials in relevant lung fluids, and to validate the results against the bioprocessing in vivo. Moreover, it is largely unknown to what extent (nano)forms of a substance that differ in size, shape, or coating also differ in biodissolution. Here we apply a previously optimized abiotic flow-through method to 24 (nano)forms of 6 substances and compare the results with alveolar macrophage-assisted biodissolution of a subset of these nanomaterials in vitro and short-term inhalation results in vivo. As a main result we found that the results obtained with the flow-through method for the lung were consistent to the results of in vivo studies and were not improved by measuring alveolar macrophage-assisted biodissolution for up to 48 h. Based on selected benchmark materials we propose four groups of materials according to quantitative biodissolution rates (1 ng/cm2/h to 100 ng/cm2/h cutoffs) and qualitative transformation parameters, as detected by TEM analysis. These groups were also reflected by different lung clearance rates, as previously determined in short term inhalation studies. Biodissolution was similar within substance (nano)forms of Fe2O3, SiO2, CeO2, ZnO, though slightly varied upon surface area/coating. But the difference of biodissolution between the substances was in some cases >1000-fold. Among the Cu-containing materials, the behavior of the two CuPhthalocyanin nanoforms was similar with each other, but completely different than the dissolution and transformation of Cu salts. Different production routes and/or surface coatings significantly modulated biodissolution, whereas effects of shape or size were limited. In summary, we refined a protocol for the abiotic determination of biodissolution along with an integrated assessment of nanomaterial transformation. The protocol is suggested as tier 2 methodology for grouping and read-across purposes.