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Background: C60 fullerenes and single-walled carbon nanotubes (SWCNT) are projected to be used in medicine and consumer products with potential human exposure. The hazardous effects of these particles are expected to involve oxidative stress with generation of oxidatively damaged DNA that might be the initiating event in the development of cancer. Objective: In this study we investigated the effect of a single oral administration of C60 fullerenes and SWCNT. Methods: We measured the level of oxidative damage to DNA as the premutagenic 8-oxo-7,8- dihydro-2´-deoxyguanosine (8-oxodG) in the colon mucosa, liver, and lung of rats after intragastric administration of pristine C60 fullerenes or SWCNT (0.064 or 0.64 mg/kg body weight) suspended in saline solution or corn oil. We investigated the regulation of DNA repair systems toward 8-oxodG in liver and lung tissue. Results: Both doses of SWCNT increased the levels of 8-oxodG in liver and lung. Administration of C60 fullerenes increased the hepatic level of 8-oxodG, whereas only the high dose generated 8-oxodG in the lung. We detected no effects on 8-oxodG in colon mucosa. Suspension of particles in saline solution or corn oil yielded a similar extent of genotoxicity, whereas corn oil per se generated more genotoxicity than the particles. Although there was increased mRNA expression of 8-oxoguanine DNA glycosylase in the liver of C60 fullerene-treated rats, we found no significant increase in repair activity. Conclusions: Oral exposure to low doses of C60 fullerenes and SWCNT is associated with elevated levels of 8-oxodG in the liver and lung, which is likely to be caused by a direct genotoxic ability rather than an inhibition of the DNA repair system. [ABSTRACT FROM AUTHOR]

Studies on in vitro-in vivo correlations of inflammatory and genotoxic responses are needed to advance new approach methodologies. Here, we assessed pro-inflammatory and genotoxic responses by 13 nanosized metal oxides (nMeOx) and quartz (DQ12) in alveolar epithelial cells (A549) and macrophages (THP-1a) exposed in submerged conditions, and in A549:THP-1a co-cultures in air-liquid interface (ALI) system. Soluble nMeOx produced the highest IL-8 expression in A549 and THP-1a cells in submerged conditions (≥2-fold, p < 0.05), whereas only CuO caused a strong response in co-cultures exposed in the ALI system (13-fold, p < 0.05). IL-8 expression in A549 cells with concentrations as nMeOx specific surface area (SSA) correlated with neutrophil influx in mice (r = 0.89-0.98, p < 0.05). Similarly, IL-8 expression in THP-1a cell with concentrations as mass and SSA (when excluding soluble nMeOx) correlated with neutrophil influx in mice (r = 0.81-0.84, p < 0.05). DNA strand breaks (SB) was measured by the comet assay. We used a scoring system that categorizes effects in standard deviation units for comparison of genotoxicity in different models. Concordant genotoxicity was observed between SB levels in vitro (A549 and co-culture) and in vivo (broncho-alveolar lavage fluid cells and lung tissue). In conclusion, this study shows in vitro-in vivo correlations of nMeOx-induced inflammatory and genotoxic responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many in vitro and in vivo studies have shown that exposure to carbon nanotubes (CNTs) is associated with inflammation, oxidative stress and genotoxicity, although there is a paucity of studies on these effects in the pleural cavity. In the present study, we investigated adverse outcomes of pleural exposure to multi-walled CNTs (MWCNT-7, NM-401 and NM-403) and single-walled CNTs (NM-411). Female C57BL/6 mice were exposed to 0.2 or 5 µg of CNTs by intra-pleural injection and sacrificed one-year post-exposure. Exposure to long and straight types of MWCNTs (i.e. MWCNT-7 and NM-401) was associated with decreased number of macrophages and increased number of neutrophils and eosinophils in pleural lavage fluid. Increased protein content in the pleural lavage fluid was also observed in mice exposed to MWCNT-7 and NM-401. The concentration of mesothelin was increased in mice exposed to MWCNT-7 and NM-411. Levels of DNA strand breaks and DNA oxidation damage, measured by the comet assay, were unaltered in cells from pleural scrape. Extra-pleural effects were seen in CNT exposed mice, including enlarged and pigmented mediastinal lymph nodes (all four types of CNTs), pericardial plaques (MWCNT-7 and NM-401), macroscopic abnormalities on the liver (MWCNT-7) and ovaries/uterus (NM-411). In conclusion, the results demonstrate that intra-pleural exposure to long and straight MWCNTs is associated with adverse outcomes. Certain observations such as increased content of mesothelin in pleural lavage fluid and ovarian/uterine abnormalities in mice exposed to NM-411 suggests that exposure to SWCNTs may also be associated with some adverse outcomes., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Peter Moller reports financial support was provided by Independent Research Fund Denmark. Ulla Vogel reports financial support was provided by Danish Government., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

This study collects toxicity data from animal inhalation studies of some nanomaterials and their bulk and ionic counterparts. To allow potential grouping and interpretations, we retrieved the primary physicochemical and exposure data to the extent possible for each of the materials. Reviewed materials are compounds (mainly elements, oxides and salts) of carbon (carbon black, carbon nanotubes, and graphene), silver, cerium, cobalt, copper, iron, nickel, silicium (amorphous silica and quartz), titanium (titanium dioxide), and zinc (chemical symbols: Ag, C, Ce, Co, Cu, Fe, Ni, Si, Ti, TiO 2 , and Zn). Collected endpoints are: a) pulmonary inflammation, measured as neutrophils in bronchoalveolar lavage (BAL) fluid at 0-24 hours after last exposure; and b) genotoxicity/carcinogenicity. We present the dose descriptors no-observed-adverse-effect concentrations (NOAECs) and lowest-observed-adverse-effect concentrations (LOAECs) for 88 nanomaterial investigations in data-library and graph formats. We also calculate 'the value where 25% of exposed animals develop tumors' (T25) for carcinogenicity studies. We describe how the data may be used for hazard assessment of the materials using carbon black as an example. The collected data also enable hazard comparison between different materials. An important observation for poorly soluble particles is that the NOAEC for neutrophil numbers in general lies around 1 to 2 mg/m 3 . We further discuss why some materials' dose descriptors deviate from this level, likely reflecting the effects of the ionic form and effects of the fiber-shape. Finally, we discuss that long-term studies, in general, provide the lowest dose descriptors, and dose descriptors are positively correlated with particle size for near-spherical materials.

Tungsten is used in several applications and human exposure may occur. To assess its pulmonary toxicity, we exposed male mice to nose-only inhalation of tungsten particles at 9, 23 or 132 mg/m 3 (Low, Mid and High exposure) (45 min/day, 5 days/week for 2 weeks). Increased genotoxicity (assessed by comet assay) was seen in bronchoalveolar (BAL) fluid cells at Low and High exposure. We measured acellular ROS production, and cannot exclude that ROS contributed to the observed genotoxicity. We saw no effects on body weight gain, pulmonary inflammation, lactate dehydrogenase or protein in BAL fluid, pathology of liver or kidney, or on sperm counts. In conclusion, tungsten showed non-dose dependent genotoxicity in the absence of inflammation and therefore interpreted to be primary genotoxicity. Based on genotoxicity, a Lowest Observed Adverse Effect Concentration (LOAEC) could be set at 9 mg/m 3 . It was not possible to establish a No Adverse Effect Concentration (NOAEC)., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Niels Hadrup reports financial support was provided by The Danish Working Environment Research Fund. Ulla Vogel reports financial support was provided by The Danish Government., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO 2 ) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 μg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints., Competing Interests: Declaration of Competing Interest The authors declare no competing interests and confirm the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We spend most of our time indoors; however, little is known about the effects of exposure to aerosol particles indoors. We aimed to determine differences in relative toxicity and physicochemical properties of PM 2.5 collected simultaneously indoors (PM 2.5 INDOOR ) and outdoors (PM 2.5 OUTDOOR ) in 15 occupied homes in southern Sweden. Collected particles were extracted from filters, pooled (indoor and outdoor separately), and characterized for chemical composition and endotoxins before being tested for toxicity in mice via intratracheal instillation. Various endpoints including lung inflammation, genotoxicity, and acute-phase response in lung and liver were assessed 1, 3, and 28 days post-exposure. Chemical composition of particles used in toxicological assessment was compared to particles analyzed without extraction. Time-resolved particle mass and number concentrations were monitored. PM 2.5 INDOOR showed higher relative concentrations (μg mg -1 ) of metals, PAHs, and endotoxins compared to PM 2.5 OUTDOOR . These differences may be linked to PM 2.5 INDOOR causing significantly higher lung inflammation and lung acute-phase response 1 day post-exposure compared to PM 2.5 OUTDOOR and vehicle controls, respectively. None of the tested materials caused genotoxicity. PM 2.5 INDOOR displayed higher relative toxicity than PM 2.5 OUTDOOR under the studied conditions, that is, wintertime with reduced air exchange rates, high influence of indoor sources, and relatively low outdoor concentrations of PM. Reducing PM 2.5 INDOOR exposure requires reduction of both infiltration from outdoors and indoor-generated particles., (© 2022 The Authors. Indoor Air published by John Wiley & Sons Ltd.)

Animal experiments are highly relevant models for the assessment of toxicological effects of engineered nanomaterials (ENMs), due to lack of biomonitoring and epidemiological studies. However, the expanding number of ENMs with different physico-chemical properties strains this approach, as there are ethical concerns and economical challenges with the use of animals in toxicology. There is an urgent need for cell culture models that predict the level of toxicological responses in vivo, consequently reducing or replacing the use of animals in nanotoxicology. However, there is still a limited number of studies on in vitro-in vivo correlation of toxicological responses following ENMs exposure. In this review, we collected studies that have compared in vitro and in vivo toxic effects caused by ENMs. We discuss the influence of cell culture models and exposure systems on the predictability of in vitro models to equivalent toxic effects in animal lungs after pulmonary exposure to ENMs. In addition, we discuss approaches to qualitatively or quantitatively compare the effects in vitro and in vivo. The magnitude of toxicological responses in cells that are exposed in submerged condition is not systematically different from the response in cells exposed in air-liquid interface systems, and there appears to be similar ENMs hazard ranking between the two exposure systems. Overall, we show that simple in vitro models with cells exposed to ENMs in submerged condition can be used to predict toxic effects in vivo, and identify future strategies to improve the associations between in vitro and in vivo ENMs-induced pulmonary toxicity. This article is categorized under: Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials., (© 2022 The Authors. WIREs Nanomedicine and Nanobiotechnology published by Wiley Periodicals LLC.)

Some implantable medical devices contain silver. We aimed to assess at what amount implanted silver becomes toxic. Silver was elevated in bodily fluids and tissues surrounding silver-containing implants. Silver released from implants also distributes to blood and other tissues; there is evidence to suggest silver can pass the blood-brain-barrier. Silver can be deposited as nano-sized particles in various tissues. Such particles, in addition to silver, often contain other elements too, e.g., selenium and sulfur. Silver released from implants seems to stay in the body for long periods (years). Reported excretion pathways following implantation are urinary and fecal ones. Reported toxicological effects were virtually all local reactions surrounding the implants. Argyria is a blue-gray discoloration of the skin due to deposited silver granules. Localized argyria has been described after the implantation of acupuncture needles and silver-coated prostheses, although the presence of silver was tested only for and shown in the former. Other toxicological effects include local tissue reactivity and examples of neurotoxic and vascular effects. We did not include genotoxicity studies in the present publication as we recently evaluated silver to be genotoxic. Carcinogenicity studies were absent. We conclude that local toxicity of implanted silver can be foreseen in some situations.

The inclusion of nanoparticles can increase the quality of certain products. One application is the inclusion of Zinc oxide (ZnO) nanoparticles in a glass coating matrix to produce a UV-absorbing coating for glass sheets. Yet, the question is whether the inclusion of ZnO in the matrix induces toxicity at low exposure levels. To test this, mice were given single intratracheal instillation of 1) ZnO powder (ZnO), 2) ZnO in a glass matrix coating in its liquid phase (ZnO-Matrix), and 3) the matrix with no ZnO (Matrix). Doses of ZnO were 0.23, 0.67, and 2 µg ZnO/mouse. ZnO Matrix doses had equal amounts of ZnO, while Matrix was adjusted to have an equal volume of matrix as ZnO Matrix. Post-exposure periods were 1, 3, or 28 d. Endpoints were pulmonary inflammation as bronchoalveolar lavage (BAL) fluid cellularity, genotoxicity in lung and liver, measured by comet assay, histopathology of lung and liver, and global gene expression in lung using microarrays. Neutrophil numbers were increased to a similar extent with ZnO and ZnO-Matrix at 1 and 3 d. Only weak genotoxicity without dose-response effects was observed in the lung. Lung histology showed an earlier onset of inflammation in material-exposed groups as compared to controls. Microarray analysis showed a stronger response in terms of the number of differentially regulated genes in ZnO-Matrix exposed mice as compared to Matrix only. Activated canonical pathways included inflammatory and cardiovascular ones. In conclusion, the pulmonary toxicity of ZnO was not changed by formulation in a liquid matrix for glass coating.

Background: The EU-project GRACIOUS developed an Integrated Approach to Testing and Assessment (IATA) to support grouping high aspect ratio nanomaterials (HARNs) presenting a similar inhalation hazard. Application of grouping reduces the need to assess toxicity on a case-by-case basis and supports read-across of hazard data from substances that have the data required for risk assessment (source) to those that lack such data (target). The HARN IATA, based on the fibre paradigm for pathogenic fibres, facilitates structured data gathering to propose groups of similar HARN and to support read-across by prompting users to address relevant questions regarding HARN morphology, biopersistence and inflammatory potential. The IATA is structured in tiers, allowing grouping decisions to be made using simple in vitro or in silico methods in Tier1 progressing to in vivo approaches at the highest Tier3. Here we present a case-study testing the applicability of GRACIOUS IATA to form an evidence-based group of multiwalled carbon nanotubes (MWCNT) posing a similar predicted fibre-hazard, to support read-across and reduce the burden of toxicity testing., Results: The case-study uses data on 15 different MWCNT, obtained from the published literature. By following the IATA, a group of 2 MWCNT was identified (NRCWE006 and NM-401) based on a high degree of similarity. A pairwise similarity assessment was subsequently conducted between the grouped MWCNT to evaluate the potential to conduct read-across and fill data gaps required for regulatory hazard assessment. The similarity assessment, based on expert judgement of Tier 1 assay results, predicts both MWCNT are likely to cause a similar acute in vivo hazard. This result supports the possibility for read-across of sub-chronic and chronic hazard endpoint data for lung fibrosis and carcinogenicity between the 2 grouped MWCNT. The implications of accepting the similarity assessment based on expert judgement of the MWCNT group are considered to stimulate future discussion on the level of similarity between group members considered sufficient to allow regulatory acceptance of a read-across argument., Conclusion: This proof-of-concept case-study demonstrates how a grouping hypothesis and IATA may be used to support a nuanced and evidence-based grouping of 'similar' MWCNT and the subsequent interpolation of data between group members to streamline the hazard assessment process., (© 2022. The Author(s).)

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are formed as a result of natural cellular processes, intracellular signaling, or as adverse responses associated with diseases or exposure to oxidizing chemical and non-chemical stressors. The action of ROS and RNS, collectively referred to as reactive oxygen and nitrogen species (RONS), has recently become highly relevant in a number of adverse outcome pathways (AOPs) that capture, organize, evaluate and portray causal relationships pertinent to adversity or disease progression. RONS can potentially act as a key event (KE) in the cascade of responses leading to an adverse outcome (AO) within such AOPs, but are also known to modulate responses of events along the AOP continuum without being an AOP event itself. A substantial discussion has therefore been undertaken in a series of workshops named "Mystery or ROS" to elucidate the role of RONS in disease and adverse effects associated with exposure to stressors such as nanoparticles, chemical, and ionizing and non-ionizing radiation. This review introduces the background for RONS production, reflects on the direct and indirect effects of RONS, addresses the diversity of terminology used in different fields of research, and provides guidance for developing a harmonized approach for defining a common event terminology within the AOP developer community., Competing Interests: KL and HY were employed by the company Philip Morris International (PMI). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tanabe, O’Brien, Tollefsen, Kim, Chauhan, Yauk, Huliganga, Rudel, Kay, Helm, Beaton, Filipovska, Sovadinova, Garcia-Reyero, Mally, Poulsen, Delrue, Fritsche, Luettich, La Rocca, Yepiskoposyan, Klose, Danielsen, Esterhuizen, Jacobsen, Vogel, Gant, Choi and FitzGerald.)

Carbon black exposure causes oxidative stress, inflammation and genotoxicity. The objective of this systematic review was to assess the contributions of primary (i.e. direct formation of DNA damage) and secondary genotoxicity (i.e., DNA lesions produced indirectly by inflammation) to the overall level of DNA damage by carbon black. The database is dominated by studies that have measured DNA damage by the comet assay. Cell culture studies indicate a genotoxic action of carbon black, which might be mediated by oxidative stress. Many in vivo studies originate from one laboratory that has investigated the genotoxic effects of Printex 90 in mice by intra-tracheal instillation. Meta-analysis and pooled analysis of these results demonstrate that Printex 90 exposure is associated with a slightly increased level of DNA strand breaks in bronchoalveolar lavage cells and lung tissue. Other types of genotoxic damage have not been investigated as thoroughly as DNA strand breaks, although there is evidence to suggest that carbon black exposure might increase the mutation frequency and cytogenetic endpoints. Stratification of studies according to concurrent inflammation and DNA damage does not indicate that carbon black exposure gives rise to secondary genotoxicity. Even substantial pulmonary inflammation is at best only associated with a weak genotoxic response in lung tissue. In conclusion, the review indicates that nanosized carbon black is a weak genotoxic agent and this effect is more likely to originate from a primary genotoxic mechanism of action, mediated by e.g., oxidative stress, than inflammation-driven (secondary) genotoxicity., Competing Interests: Conflict of interest The authors report no conflicts of interests., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Improved strategies are required for testing nanomaterials (NMs) to make hazard and risk assessment more efficient and sustainable. Including reduced reliance on animal models, without decreasing the level of human health protection. Acellular detection of reactive oxygen species (ROS) may be useful as a screening assay to prioritize NMs of high concern. To improve reliability and reproducibility, and minimize uncertainty, a standard operating procedure (SOP) has been developed for the detection of ROS using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH 2 -DA) assay. The SOP has undergone an inter- and intra-laboratory comparison, to evaluate robustness, reliability, and reproducibility, using representative materials (ZnO, CuO, Mn 2 O 3 , and BaSO 4 NMs), and a number of calibration tools to normalize data. The SOP includes an NM positive control (nanoparticle carbon black (NPCB)), a chemical positive control (SIN-1), and a standard curve of fluorescein fluorescence. The interlaboratory comparison demonstrated that arbitrary fluorescence units show high levels of partner variability; however, data normalization improved variability. With statistical analysis, it was shown that the SIN-1 positive control provided an extremely high level of reliability and reproducibility as a positive control and as a normalization tool. The NPCB positive control can be used with a relatively high level of reproducibility, and in terms of the representative materials, the reproducibility CuO induced-effects was better than for Mn 2 O 3 . Using this DCFH 2 -DA acellular assay SOP resulted in a robust intra-laboratory reproduction of ROS measurements from all NMs tested, while effective reproduction across different laboratories was also demonstrated; the effectiveness of attaining reproducibility within the interlaboratory assessment was particle-type-specific.

Carbon black nanoparticles (CBNPs) have a large surface area/volume ratio and are known to generate oxidative stress and inflammation that may result in genotoxicity and cancer. Here, we evaluated the primary and inflammatory response-driven (i.e. secondary) genotoxicity of two CBNPs, Flammruss101 (FL101) and PrintexXE2B (XE2B) that differ in size and specific surface area (SSA), and cause different amounts of reactive oxygen species. Three doses (low, medium and high) of FL101 and XE2B were assessed in vitro in the lung epithelial (A549) and activated THP-1 (THP-1a) monocytic cells exposed in submerged conditions for 6 and 24 h, and in C57BL/6 mice at day 1, 28 and 90 following intratracheal instillation. In vitro , we assessed pro-inflammatory response as IL-8 and IL-1β gene expression, and in vivo , inflammation was determined as inflammatory cell infiltrates in bronchial lavage (BAL) fluid and as histological changes in lung tissue. DNA damage was quantified in vitro and in vivo as DNA strand breaks levels by the alkaline comet assay. Inflammatory responses in vitro and in vivo correlated with dosed CBNPs SSA. Both materials induced DNA damage in THP-1a (correlated with dosed mass), and only XE2B in A549 cells. Non-statistically significant increase in DNA damage in vivo was observed in BAL cells. In conclusion, this study shows dosed SSA predicted inflammation both in vivo and in vitro , whereas dosed mass predicted genotoxicity in vitro in THP-1a cells. The observed lack of correlation between CBNP surface area and genotoxicity provides little evidence of inflammation-driven genotoxicity in vivo and in vitro .

Expectations for safer and sustainable chemicals and products are growing to comply with the United Nations and European strategies for sustainability. The application of Safe(r) by Design (SbD) in nanotechnology implies an iterative process where functionality, human health and safety, environmental and economic impact and cost are assessed and balanced as early as possible in the innovation process and updated at each step. The EU H2020 NanoReg2 project was the first European project to implement SbD in six companies handling and/or manufacturing nanomaterials (NMs) and nano-enabled products (NEP). The results from this experience have been used to develop these guidelines on the practical application of SbD. The SbD approach foresees the identification, estimation, and reduction of human and environmental risks as early as possible in the development of a NM or NEP, and it is based on three pillars: (i) safer NMs and NEP; (ii) safer use and end of life and (iii) safer industrial production. The presented guidelines include a set of information and tools that will help deciding at each step of the innovation process whether to continue, apply SbD measures or carry out further tests to reduce uncertainty. It does not intend to be a prescriptive protocol where all suggested steps have to be followed to achieve a SbD NM/NEP or process. Rather, the guidelines are designed to identify risks at an early state and information to be considered to identify those risks. Each company adapts the approach to its specific needs and circumstances as company decisions influence the way forward., (Copyright © 2022 Elsevier B.V. All rights reserved.)

With ever-increasing production and use of nanoparticles (NPs), there is a necessity to evaluate the probability of consequential adverse effects in individuals exposed to these particles. It is now understood that a proportion of NPs can translocate from primary sites of exposure to a range of secondary organs, with the liver, kidneys and spleen being some of the most important. In this study, we carried out a comprehensive toxicological profiling (inflammation, changes in serum biochemistry, oxidative stress, acute phase response and histopathology) of Ag NP induced adverse effects in the three organs of interest following acute exposure of the materials at identical doses via intravenous (IV), intratracheal (IT) instillation and oral administration. The data clearly demonstrated that bioaccumulation and toxicity of the particles were most significant following the IV route of exposure, followed by IT. However, oral exposure to the NPs did not result in any changes that could be interpreted as toxicity in any of the organs of interest within the confines of this investigation. The finding of this study clearly indicates the importance of the route of exposure in secondary organ hazard assessment for NPs. Finally, we identify Connexin 32 (Cx32) as a novel biomarker of NP-mediated hepatic damage which is quantifiable both ( in vitro) and in vivo following exposure of physiologically relevant doses.

Nanoclays and graphene oxide nanomaterials represent a class of materials sharing similar shapes constituted of high aspect ratio platelets. The increased production of these materials for various industrial applications increases the risk of occupational exposure, consequently with elevated risk of adverse reactions and development of pulmonary diseases, including lung cancer. In this study, pro-inflammatory responses and genotoxicity were assessed in alveolar epithelial cells (A549) and activated THP-1 macrophages (THP-1a) after exposure to three nanoclays; a pristine (Bentonite) and two surface modified (benzalkonium chloride-coated Nanofil9, and dialkyldimethyl-ammonium-coated NanofilSE3000); graphene oxide (GO) and reduced graphene oxide (r-GO) nanomaterials. The pro-inflammatory response in terms of IL-8 expression was strongest in cells exposed to Bentonite, whereas surface modification resulted in decreased toxicity in both cell lines when exposed to Nanofil9 and NanofilSE3000. GO and r-GO induced a pro-inflammatory response in A549 cells, while no effect was detected with the two nanomaterials on THP-1a cells. The pro-inflammatory response was strongly correlated with in vivo inflammation in mice after intra-tracheal instillation when doses were normalized against surface area. Genotoxicity was assessed as DNA strand breaks, using the alkaline comet assay. In A549 cells, an increase in DNA strand breaks was detected only in cells exposed to Bentonite, whereas Bentonite, NanofilSE3000 and GO caused an increased level of genotoxicity in THP-1a cells. Genotoxicity in THP-1a cells was concordant with the DNA damage in bronchoalveolar lavage fluid cells following 1 and 3 days after intra-tracheal instillation in mice. In conclusion, this study shows that surface modification of pristine nanoclays reduces the inflammatory and genotoxic response in A549 and THP-1a cells, and these in vitro models show comparable toxicity to what seen in previous mouse studies with the same materials., (Copyright © 2021. Published by Elsevier B.V.)

Carbon nanotubes (CNTs) are speculated to cause mesothelioma by persistent inflammation, oxidative stress, tissue injury, and genotoxicity. To investigate the pleural response to CNTs, we exposed C57BL/6 mice by intrapleural injection of 0.2 or 5 µg multiwalled CNTs (MWCNT-7, NM-401, and NM-403) or single-walled CNTs (NM-411). Inflammatory response, cellular reactive oxygen species (ROS) production of pleural lavage cells, and genotoxicity in cells from the mesothelial surface were assessed at days 1 and 90 after the exposure. Long and rigid types of MWCNTs (MWCNT-7 and NM-401) caused acute inflammation, characterized by influx of macrophages, neutrophils, and eosinophils into the pleural cavity. The inflammation was still evident at 90 days after the exposure, although it had reduced dramatically. The cellular ROS production was increased at day 90 after the exposure to MWCNT-7 and NM-401. The short and tangled type of MWCNT (ie NM-403) did not cause pleural inflammation or ROS production in pleural fluid cells. The exposure to NM-411 did not cause consistent inflammation responses or cellular ROS production. Levels of DNA strand breaks and DNA oxidation damage were unaltered, except for NM-411-exposed mice that had increased levels of DNA strand breaks at 90 days after the exposure. In conclusion, the long and rigid CNTs caused prolonged inflammatory response and increased ROS production in pleural lavage cells, yet it was not reflected in higher levels of DNA damage in pleural tissue., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Multi-walled carbon nanotubes (MWCNT) have received attention due to extraordinary properties, resulting in concerns for occupational health and safety. Costs and ethical concerns of animal testing drive a need for in vitro models with predictive power in respiratory toxicity. The aim of this study was to assess pro-inflammatory response (Interleukin-8 expression, IL-8) and genotoxicity (DNA strand breaks) caused by MWCNT with different physicochemical properties in different pulmonary cell models and correlate these to previously published in vivo data. Seven MWCNT were selected; two long/thick (NRCWE-006/Mitsui-7 and NM-401), two short/thin (NM-400 and NM-403), a pristine (NRCWE-040) and two surface modified; hydroxylated (NRCWE-041) and carboxylated (NRCWE-042). Carbon black Printex90 (CB) was included as benchmark material. Human alveolar epithelial cells (A549) and monocyte-derived macrophages (THP-1a) were exposed to nanomaterials (NM) in submerged conditions, and two materials (NM-400 and NM-401) in co-cultures of A549/THP-1a and lung fibroblasts (WI-38) in an air-liquid interface (ALI) system. Effective doses were quantified by thermo-gravimetric-mass spectrometry analysis (TGA-MS). To compare genotoxicity in vitro and in vivo, we developed a scoring system based on a categorization of effects into standard deviation (SD) units (< 1, 1, 2, 3 or 4 standard deviation increases) for the increasing genotoxicity., Results: Effective doses were shown to be 25 to 53%, and 21 to 57% of the doses administered to A549 and THP-1a, respectively. In submerged conditions (A549 and THP-1a cells), all NM induced dose-dependent IL-8 expression. NM-401 and NRCWE-006 caused the strongest pro-inflammatory response. In the ALI-exposed co-culture, only NM-401 caused increased IL-8 expression, and no DNA strand breaks were observed. Strong correlations were found between in vitro and in vivo inflammation when doses were normalized by surface area (also proxy for diameter and length). Significantly increased DNA damage was found for all MWCNT in THP-1a cells, and for short MWCNT in A549 cells. A concordance in genotoxicity of 83% was obtained between THP-1a cells and broncho-alveolar lavaged (BAL) cells., Conclusion: This study shows correlations of pro-inflammatory potential in A549 and THP-1a cells with neutrophil influx in mice, and concordance in genotoxic response between THP-1a cells and BAL cells, for seven MWCNT., (© 2021. The Author(s).)

Moving towards safe and sustainable innovations is an international policy ambition. In the on-hand manuscript, a concept combining safe by design and sustainability was implemented through the integration of human and environmental risk assessment, life cycle assessment as well as an assessment of the economic viability. The result is a nested and iterative process in form of a decision tree that integrates these three elements in order to achieve sustainable, safe and competitive materials, products or services. This approach, embedded into the stage-gate-model for safe by design, allows to reduce the uncertainty related to the assessment of risks and impacts by improving the quality of the data collected along each stage. In the second part of the manuscript, the application is shown for a case study dealing with the application of nanoparticles for Li-Ion batteries. One of the general conclusions out of this case study is that data gaps are a key aspect in view of the reliability of the results., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Carbon nanotubes (CNTs) were the first nanomaterials to be evaluated by the International Agency for Research on Cancer (IARC). The categorization as possibly carcinogenic agent to humans was only applicable to multi-walled carbon nanotubes called MWCNT-7. Other types of CNTs were not classifiable because of missing data and it was not possible to pinpoint unique CNT characteristics that cause cancer. Importantly, the European Commission's Joint Research Centre (JRC) has established a repository of industrially manufactured nanomaterials that encompasses at least four well-characterized MWCNTs called NM-400 to NM-403 (original JRC code). This review summarizes the genotoxic effects of these JRC materials and MWCNT-7. The review consists of 36 publications with results on cell culture experiments (22 publications), animal models (9 publications) or both (5 publications). As compared to the publications in the IARC monograph on CNTs, the current database represents a significant increase as there is only an overlap of 8 publications. However, the results come mainly from cell cultures and/or measurements of DNA strand breaks by the comet assay and the micronucleus assay (82 out of 97 outcomes). A meta-analysis of cell culture studies on DNA strand breaks showed a genotoxic response by MWCNT-7, less consistent effect by NM-400 and NM-402, and least consistent effect by NM-401 and NM-403. Results from other in vitro tests indicate strongest evidence of genotoxicity for MWCNT-7. There are too few observations from animal models and humans to make general conclusions about genotoxicity., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Carbon nanotubes (CNTs) are fiber-like nanomaterials, which are used in various applications with possible exposure to humans. The genotoxicity and carcinogenic potential of CNTs remain to be fully understood. This study assessed the genotoxicity of three different multi-walled carbon nanotubes (MWCNTs) (MWCNT-7, NM-401 and NM-403) and one single-walled carbon nanotube (SWCNT) (NM-411) in FE1-Muta™Mouse lung epithelial (MML) cells using the alkaline comet assay. With the 2',7'-dichlorodihydrofluorescein diacetate fluorescent probe, we assessed the effect of CNT-exposure on the intracellular production of reactive oxygen species (ROS). We measured the effect of a 10-week CNT exposure on telomere length using quantitative PCR. Two of the included MWCNTs (NM-401 and MWCNT-7) and the SWCNT (NM-411) caused a significant increase in the level of DNA damage at concentrations up to 40 µg/ml (all concentrations pooled, p  < 0.05), but no concentration-response relationships were found. All of the CNTs caused an increase in intracellular ROS production compared to unexposed cells ( p trend < 0.05). Results from the long-term exposure showed longer telomere length in cells exposed to MWCNTs compared to unexposed cells ( p  < 0.01). In conclusion, our results indicated that the included CNTs cause ROS production and DNA strand breaks in FE1-MML cells. Moreover, the MWCNTs, but not the SWCNT, had an impact on telomere length in a long-term exposure scenario.

Nanotechnology is a key enabling technology with billions of euros in global investment from public funding, which include large collaborative projects that have investigated environmental and health safety aspects of nanomaterials, but the reuse of accumulated data is clearly lagging behind. Here we summarize challenges and provide recommendations for the efficient reuse of nanosafety data, in line with the recently established FAIR (findable, accessible, interoperable and reusable) guiding principles. We describe the FAIR-aligned Nanosafety Data Interface, with an aggregated findability, accessibility and interoperability across physicochemical, bio-nano interaction, human toxicity, omics, ecotoxicological and exposure data. Overall, we illustrate a much-needed path towards standards for the optimized use of existing data, which avoids duplication of efforts, and provides a multitude of options to promote safe and sustainable nanotechnology.

Here we describe the development of an Integrated Approach to Testing and Assessment (IATA) to support the grouping of different types (nanoforms; NFs) of High Aspect Ratio Nanomaterials (HARNs), based on their potential to cause mesothelioma. Hazards posed by the inhalation of HARNs are of particular concern as they exhibit physical characteristics similar to pathogenic asbestos fibres. The approach for grouping HARNs presented here is part of a framework to provide guidance and tools to group similar NFs and aims to reduce the need to assess toxicity on a case-by-case basis. The approach to grouping is hypothesis-driven, in which the hypothesis is based on scientific evidence linking critical physicochemical descriptors for NFs to defined fate/toxicokinetic and hazard outcomes. The HARN IATA prompts users to address relevant questions (at decision nodes; DNs) regarding the morphology, biopersistence and inflammatory potential of the HARNs under investigation to provide the necessary evidence to accept or reject the grouping hypothesis. Each DN in the IATA is addressed in a tiered manner, using data from simple in vitro or in silico methods in the lowest tier or from in vivo approaches in the highest tier. For these proposed methods we provide justification for the critical descriptors and thresholds that allow grouping decisions to be made. Application of the IATA allows the user to selectively identify HARNs which may pose a mesothelioma hazard, as demonstrated through a literature-based case study. By promoting the use of alternative, non-rodent approaches such as in silico modelling, in vitro and cell-free tests in the initial tiers, the IATA testing strategy streamlines information gathering at all stages of innovation through to regulatory risk assessment while reducing the ethical, time and economic burden of testing., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Airway exposure to eg particulate matter is associated with cardiovascular disease including atherosclerosis. Acute phase genes, especially Serum Amyloid A3 (Saa3), are highly expressed in the lung following pulmonary exposure to particles. We aimed to investigate whether the human acute phase protein SAA (a homolog to mouse SAA3) accelerated atherosclerotic plaque progression in Apolipoprotein E knockout (ApoE -/- ) mice. Mice were intratracheally (i.t.) instilled with vehicle (phosphate buffered saline) or 2 µg human SAA once a week for 10 weeks. Plaque progression was assessed in the aorta using noninvasive ultrasound imaging of the aorta arch as well as by en face analysis. Additionally, lipid peroxidation, SAA3, and cholesterol were measured in plasma, inflammation was determined in lung, and mRNA levels of the acute phase genes Saa1 and Saa3 were measured in the liver and lung, respectively. Repeated i.t. instillation with SAA caused a significant progression in the atherosclerotic plaques in the aorta (1.5-fold). Concomitantly, SAA caused a statistically significant increase in neutrophils in bronchoalveolar lavage fluid (625-fold), in pulmonary Saa3 (196-fold), in systemic SAA3 (1.8-fold) and malondialdehyde levels (1.14-fold), indicating acute phase response (APR), inflammation and oxidative stress. Finally, pulmonary exposure to SAA significantly decreased the plasma levels of very low-density lipoproteins - low-density lipoproteins and total cholesterol, possibly due to lipids being sequestered in macrophages or foam cells in the arterial wall. Combined these results indicate the importance of the pulmonary APR and SAA3 for plaque progression., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Materials can be modified for improved functionality. Our aim was to test whether pulmonary toxicity of silica nanomaterials is increased by the introduction of: a) porosity; and b) surface doping with CuO; and whether c) these modifications act synergistically. Mice were exposed by intratracheal instillation and for some doses also oropharyngeal aspiration to: 1) solid silica 100 nm; 2) porous silica 100 nm; 3) porous silica 100 nm with CuO doping; 4) solid silica 300 nm; 5) porous silica 300 nm; 6) solid silica 300 nm with CuO doping; 7) porous silica 300 nm with CuO doping; 8) CuO nanoparticles 9.8 nm; or 9) carbon black Printex 90 as benchmark. Based on a pilot study, dose levels were between 0.5 and 162 µg/mouse (0.2 and 8.1 mg/kg bw). Endpoints included pulmonary inflammation (neutrophil numbers in bronchoalveolar fluid), acute phase response, histopathology, and genotoxicity assessed by the comet assay, micronucleus test, and the gamma-H2AX assay. The porous silica materials induced greater pulmonary inflammation than their solid counterparts. A similar pattern was seen for acute phase response induction and histologic changes. This could be explained by a higher specific surface area per mass unit for the most toxic particles. CuO doping further increased the acute phase response normalized according to the deposited surface area. We identified no consistent evidence of synergism between surface area and CuO doping. In conclusion, porosity and CuO doping each increased the toxicity of silica nanomaterials and there was no indication of synergy when the modifications co-occurred.

Hazard evaluation of graphene-based materials (GBM) is still in its early stage and it is slowed by their large diversity in the physicochemical properties. This study explores transcriptomic differences in the lung and liver after pulmonary exposure to two GBM with similar physical properties, but different surface chemistry. Female C57BL/6 mice were exposed by a single intratracheal instillation of 0, 18, 54 or 162 μg/mouse of graphene oxide (GO) or reduced graphene oxide (rGO). Pulmonary and hepatic changes in the transcriptome were profiled to identify commonly and uniquely perturbed functions and pathways by GO and rGO. These changes were then related to previously analyzed toxicity endpoints. GO exposure induced more differentially expressed genes, affected more functions, and perturbed more pathways compared to rGO, both in lung and liver tissues. The largest differences were observed for the pulmonary innate immune response and acute phase response, and for hepatic lipid homeostasis, which were strongly induced after GO exposure. These changes collective indicate a potential for atherosclerotic changes after GO, but not rGO exposure. As GO and rGO are physically similar, the higher level of hydroxyl groups on the surface of GO is likely the main reason for the observed differences. GO exposure also uniquely induced changes in the transcriptome related to fibrosis, whereas both GBM induced similar changes related to Reactive Oxygen Species production and genotoxicity. The differences in transcriptomic responses between the two GBM types can be used to understand how physicochemical properties influence biological responses and enable hazard evaluation of GBM and hazard ranking of GO and rGO, both in relation to each other and to other nanomaterials., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Surface modification by different quaternary ammonium compounds (QAC) makes nanoclays more compatible with various polymeric matrices, thereby expanding their potential applications. The growing industrial use of nanoclays could potentially pose a health risk for workers. Here, we assessed how surface modification of nanoclays modulates their pulmonary toxicity. An in vitro screening of the unmodified nanoclay Bentonite (montmorillonite) and four organomodified nanoclays (ONC); coated with various QAC, including benzalkonium chloride (BAC), guided the selection of the materials for the in vivo study. Mice were exposed via a single intratracheal instillation to 18, 54, and 162 µg of unmodified Bentonite or dialkyldimethyl-ammonium-coated ONC (NanofilSE3000), or to 6, 18, and 54 µg of a BAC-coated ONC (Nanofil9), and followed for one, 3, or 28 days. All materials induced dose- and time-dependent responses in the exposed mice. However, all doses of Bentonite induced larger, but reversible, inflammation (BAL neutrophils) and acute phase response ( Saa3 gene expression in lung) than the two ONC. Similarly, highest levels of DNA strand breaks were found in BAL cells of mice exposed to Bentonite 1 day post-exposure. A significant increase of DNA strand breaks was detected also for NanofilSE3000, 3 days post-exposure. Only mice exposed to Bentonite showed increased Tgf-β gene expression in lung, biomarker of pro-fibrotic processes and hepatic extravasation, 3 days post-exposure. This study indicates that Bentonite treatment with some QAC changes main physical-chemical properties, including shape and surface area, and may decrease their pulmonary toxicity in exposed mice.

Background: Diesel exhaust is carcinogenic and exposure to diesel particles cause health effects. We investigated the toxicity of diesel exhaust particles designed to have varying physicochemical properties in order to attribute health effects to specific particle characteristics. Particles from three fuel types were compared at 13% engine intake O 2 concentration: MK1 ultra low sulfur diesel (DEP13) and the two renewable diesel fuels hydrotreated vegetable oil (HVO13) and rapeseed methyl ester (RME13). Additionally, diesel particles from MK1 ultra low sulfur diesel were generated at 9.7% (DEP9.7) and 17% (DEP17) intake O 2 concentration. We evaluated physicochemical properties and histopathological, inflammatory and genotoxic responses on day 1, 28, and 90 after single intratracheal instillation in mice compared to reference diesel particles and carbon black., Results: Moderate variations were seen in physical properties for the five particles: primary particle diameter: 15-22 nm, specific surface area: 152-222 m 2 /g, and count median mobility diameter: 55-103 nm. Larger differences were found in chemical composition: organic carbon/total carbon ratio (0.12-0.60), polycyclic aromatic hydrocarbon content (1-27 μg/mg) and acid-extractable metal content (0.9-16 μg/mg). Intratracheal exposure to all five particles induced similar toxicological responses, with different potency. Lung particle retention was observed in DEP13 and HVO13 exposed mice on day 28 post-exposure, with less retention for the other fuel types. RME exposure induced limited response whereas the remaining particles induced dose-dependent inflammation and acute phase response on day 1. DEP13 induced acute phase response on day 28 and inflammation on day 90. DNA strand break levels were not increased as compared to vehicle, but were increased in lung and liver compared to blank filter extraction control. Neutrophil influx on day 1 correlated best with estimated deposited surface area, but also with elemental carbon, organic carbon and PAHs. DNA strand break levels in lung on day 28 and in liver on day 90 correlated with acellular particle-induced ROS., Conclusions: We studied diesel exhaust particles designed to differ in physicochemical properties. Our study highlights specific surface area, elemental carbon content, PAHs and ROS-generating potential as physicochemical predictors of diesel particle toxicity.

Silver is used in a wide range of products, and during their production and use, humans may be exposed through inhalation. Therefore, it is critical to know the concentration levels at which adverse effects may occur. In rodents, inhalation of silver nanoparticles has resulted in increased silver in the lungs, lymph nodes, liver, kidney, spleen, ovaries, and testes. Reported excretion pathways of pulmonary silver are urinary and faecal excretion. Acute effects in humans of the inhalation of silver include lung failure that involved increased heart rate and decreased arterial blood oxygen pressure. Argyria-a blue-grey discoloration of skin due to deposited silver-was observed after pulmonary exposure in 3 individuals; however, the presence of silver in the discolorations was not tested. Argyria after inhalation seems to be less likely than after oral or dermal exposure. Repeated inhalation findings in rodents have shown effects on lung function, pulmonary inflammation, bile duct hyperplasia, and genotoxicity. In our evaluation, the range of NOAEC values was 0.11-0.75 mg/m 3 . Silver in the ionic form is likely more toxic than in the nanoparticle form but that difference could reflect their different biokinetics. However, silver nanoparticles and ions have a similar pattern of toxicity, probably reflecting that the effect of silver nanoparticles is primarily mediated by released ions. Concerning genotoxicity studies, we evaluated silver to be positive based on studies in mammalian cells in vitro and in vivo when considering various exposure routes. Carcinogenicity data are absent; therefore, no conclusion can be provided on this endpoint., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Inhaled nanoparticles constitute a potential health hazard due to their size-dependent lung deposition and large surface to mass ratio. Exposure to high levels contributes to the risk of developing respiratory and cardiovascular diseases, as well as of lung cancer. Particle-induced acute phase response may be an important mechanism of action of particle-induced cardiovascular disease. Here, the authors review new important scientific evidence showing causal relationships between inhalation of particle and nanomaterials, induction of acute phase response, and risk of cardiovascular disease. Particle-induced acute phase response provides a means for risk assessment of particle-induced cardiovascular disease and underscores cardiovascular disease as an occupational disease., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Exposure to metal oxide nanomaterials potentially occurs at the workplace. We investigated the toxicity of two Fe-oxides: Fe 2 O 3 nanoparticles and nanorods; and three MFe 2 O 4 spinels: NiZnFe 4 O 8 , ZnFe 2 O 4 , and NiFe 2 O 4 nanoparticles. Mice were dosed 14, 43 or 128 μg by intratracheal instillation. Recovery periods were 1, 3, or 28 days. Inflammation - neutrophil influx into bronchoalveolar lavage (BAL) fluid - occurred for Fe 2 O 3 rods (1 day), ZnFe 2 O 4 (1, 3 days), NiFe 2 O 4 (1, 3, 28 days), Fe 2 O 3 (28 days) and NiZnFe 4 O 8 (28 days). Conversion of mass-dose into specific surface-area-dose showed that inflammation correlated with deposited surface area and consequently, all these nanomaterials belong to the so-called low-solubility, low-toxicity class. Increased levels of DNA strand breaks were observed for both Fe 2 O 3 particles and rods, in BAL cells three days post-exposure. To our knowledge, this is, besides magnetite (Fe 3 O 4 ), the first study of the pulmonary toxicity of MFe 2 O 4 spinel nanomaterials., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nanomaterial (NM) characteristics may affect the pulmonary toxicity and inflammatory response, including specific surface area, size, shape, crystal phase or other surface characteristics. Grouping of TiO 2 in hazard assessment might be challenging because of variation in physicochemical properties. We exposed C57BL/6 J mice to a single dose of four anatase TiO 2 NMs with various sizes and shapes by intratracheal instillation and assessed the pulmonary toxicity 1, 3, 28, 90 or 180 days post-exposure. The quartz DQ12 was included as benchmark particle. Pulmonary responses were evaluated by histopathology, electron microscopy, bronchoalveolar lavage (BAL) fluid cell composition and acute phase response. Genotoxicity was evaluated by DNA strand break levels in BAL cells, lung and liver in the comet assay. Multiple regression analyses were applied to identify specific TiO 2 NMs properties important for the pulmonary inflammation and acute phase response. The TiO 2 NMs induced similar inflammatory responses when surface area was used as dose metrics, although inflammatory and acute phase response was greatest and more persistent for the TiO 2 tube. Similar histopathological changes were observed for the TiO 2 tube and DQ12 including pulmonary alveolar proteinosis indicating profound effects related to the tube shape. Comparison with previously published data on rutile TiO 2 NMs indicated that rutile TiO 2 NMs were more inflammogenic in terms of neutrophil influx than anatase TiO 2 NMs when normalized to total deposited surface area. Overall, the results suggest that specific surface area, crystal phase and shape of TiO 2 NMs are important predictors for the observed pulmonary effects of TiO 2 NMs., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Inhalation of nanosized zinc oxide (ZnO) induces metal fume fever and systemic acute phase response in humans. Acute phase response activation is a cardiovascular risk factor; we investigated whether pulmonary exposure of mice can be used to assess ZnO-induced acute phase response as well as inflammation and genotoxicity. Uncoated (NM-110) and triethoxycaprylylsilane-coated (NM-111) ZnO nanoparticles were intratracheally instilled once at 0.2, 0.7 or 2 µg/mouse (11, 33 and 100 µg/kg body weight). Serum amyloid A3 mRNA level in lung tissue, bronchoalveolar lavage (BAL) fluid cellularity, and levels of DNA strand breaks in BAL fluid cells, lung and liver tissue were assessed 1, 3 and 28 days post-exposure. Global transcription patterns were assessed in lung tissue using microarrays. The acute-phase response serum amyloid A3 mRNA levels were increased on day 1; for uncoated ZnO nanoparticles at the highest dose and for coated ZnO nanoparticles at medium and highest dose. Neutrophils were increased in BAL fluid only after exposure to coated ZnO nanoparticles. Genotoxicity was observed only in single dose groups, with no dose-response relationship. Most changes in global transcriptional response were observed after exposure to uncoated ZnO nanoparticles and involved cell cycle G2 to M phase DNA damage checkpoint regulation. Although, uncoated and coated ZnO nanoparticles qualitatively exerted similar effects, observed differences are likely explained by differences in solubility kinetics. The finding of serum amyloid A3 induction at low exposure suggests that mouse models can be used to assess the nanoparticle-mediated induction of acute phase responses in humans.

Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the "long and thick" NM-401 and the "short and thin" NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1-3 days and 28-30 days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multi-walled carbon nanotubes (MWCNT) are widely used nanomaterials that cause pulmonary toxicity upon inhalation. The physicochemical properties of MWCNT vary greatly, which makes general safety evaluation challenging to conduct. Identification of the toxicity-inducing physicochemical properties of MWCNT is therefore of great importance. We have evaluated histological changes in lung tissue 1 year after a single intratracheal instillation of 11 well-characterized MWCNT in female C57BL/6N BomTac mice. Genotoxicity in liver and spleen was evaluated by the comet assay. The dose of 54 μg MWCNT corresponds to three times the estimated dose accumulated during a work life at a NIOSH recommended exposure limit (0.001 mg/m 3 ). Short and thin MWCNT were observed as agglomerates in lung tissue 1 year after exposure, whereas thicker and longer MWCNT were detected as single fibres, suggesting biopersistence of both types of MWCNT. The thin and entangled MWCNT induced varying degree of pulmonary inflammation, in terms of lymphocytic aggregates, granulomas and macrophage infiltration, whereas two thick and straight MWCNT did not. By multiple regression analysis, larger diameter and higher content of iron predicted less histopathological changes, whereas higher cobalt content significantly predicted more histopathological changes. No MWCNT-related fibrosis or tumours in the lungs or pleura was found. One thin and entangled MWCNT induced increased levels of DNA strand breaks in liver; however, no physicochemical properties could be related to genotoxicity. This study reveals physicochemical-dependent difference in MWCNT-induced long-term, pulmonary histopathological changes. Identification of diameter size and cobalt content as important for MWCNT toxicity provides clues for designing MWCNT, which cause reduced human health effects following pulmonary exposure., (© 2018 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)

This paper presents a comprehensive review of European Union (EU) legislation addressing the safety of chemical substances, and possibilities within each piece of legislation for applying grouping and read-across approaches for the assessment of nanomaterials (NMs). Hence, this review considers both the overarching regulation of chemical substances under REACH (Regulation (EC) No 1907/2006 on registration, evaluation, authorization, and restriction of chemicals) and CLP (Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures) and the sector-specific pieces of legislation for cosmetic, plant protection and biocidal products, and legislation addressing food, novel food, and food contact materials. The relevant supporting documents (e.g. guidance documents) regarding each piece of legislation were identified and reviewed, considering the relevant technical and scientific literature. Prospective regulatory needs for implementing grouping in the assessment of NMs were identified, and the question whether each particular piece of legislation permits the use of grouping and read-across to address information gaps was answered.

Little is known on the toxicity of nanomaterials in the user phase. Inclusion of nanomaterials in paints is a common nanotechnology application. This study focuses on the toxicity of dusts from sanding of paints containing nanomaterials. We compared the toxicity of titanium dioxide nanomaterials (TiO 2 NMs) and dusts generated by sanding boards coated with paints with different amounts of two different types of uncoated TiO 2 NMs (diameters:10.5 nm and 38 nm). Mice were intratracheally instilled with a single dose of 18, 54 and 162 µg of TiO 2 NMs or 54, 162 and 486 µg of sanding dusts. At 1, 3 and 28 days post-instillation, we evaluated pulmonary inflammation, liver histology and DNA damage in lung and liver. Pulmonary exposure to both pristine TiO 2 NMs and sanding dusts with different types of TiO 2 NMs resulted in dose-dependently increased influx of neutrophils into the lung lumen. There was no difference between the sanding dusts from the two paints. For all exposures but not in vehicle controls, mild histological lesions were observed in the liver. Pulmonary exposure to pristine TiO 2 NMs and paint dusts with TiO 2 NMs caused similar type of histological lesions in the liver.

Background: Little is known about the mechanism underlying the genotoxicity observed in the liver following pulmonary exposure to carbon black (CB) nanoparticles (NPs). The genotoxicity could be caused by the presence of translocated particles or by circulating inflammatory mediators released during pulmonary inflammation and acute-phase response. To address this, we evaluated induction of pulmonary inflammation, pulmonary and hepatic acute-phase response and genotoxicity following exposure to titanium dioxide (TiO 2 ), cerium oxide (CeO 2 ) or CB NPs. Female C57BL/6 mice were exposed by intratracheal instillation, intravenous injection or oral gavage to a single dose of 162 μg NPs/mouse and terminated 1, 28 or 180 days post-exposure alongside vehicle control., Results: Liver DNA damage assessed by the Comet Assay was observed after intravenous injection and intratracheal instillation of CB NPs but not after exposure to TiO 2 or CeO 2 . Intratracheal exposure to NPs resulted in pulmonary inflammation in terms of increased neutrophils influx for all NPs 1 and 28 days post-exposure. Persistent pulmonary acute phase response was detected for all NPs at all three time points while only a transient induction of hepatic acute phase response was observed. All 3 materials were detected in the liver by enhanced darkfield microscopy up to 180 days post-exposure. In contrast to TiO 2 and CeO 2 NPs, CB NPs generated ROS in an acellular assay., Conclusions: Our results suggest that the observed hepatic DNA damage following intravenous and intratracheal dosing with CB NPs was caused by the presence of translocated, ROS-generating, particles detected in the liver rather than by the secondary effects of pulmonary inflammation or hepatic acute phase response.

Intratracheal instillation serves as a model for inhalation exposure. However, for this, materials are dispersed in appropriate media that may influence toxicity. We tested whether different intratracheal instillation dispersion media influence the pulmonary toxicity of different nanomaterials. Rodents were intratracheally instilled with 162 µg/mouse/1620 µg/rat carbon black (CB), 67 µg/mouse titanium dioxide nanoparticles (TiO2) or 54 µg/mouse carbon nanotubes (CNT). The dispersion media were as follows: water (CB, TiO2); 2% serum in water (CB, CNT, TiO2); 0.05% serum albumin in water (CB, CNT, TiO2); 10% bronchoalveolar lavage fluid in 0.9% NaCl (CB), 10% bronchoalveolar lavage (BAL) fluid in water (CB) or 0.1% Tween-80 in water (CB). Inflammation was measured as pulmonary influx of neutrophils into bronchoalveolar fluid, and DNA damage as DNA strand breaks in BAL cells by comet assay. Inflammation was observed for all nanomaterials (except 38-nm TiO2) in all dispersion media. For CB, inflammation was dispersion medium dependent. Increased levels of DNA strand breaks for CB were observed only in water, 2% serum and 10% BAL fluid in 0.9% NaCl. No dispersion medium-dependent effects on genotoxicity were observed for TiO2, whereas CNT in 2% serum induced higher DNA strand break levels than in 0.05% serum albumin. In conclusion, the dispersion medium was a determinant of CB-induced inflammation and genotoxicity. Water seemed to be the best dispersion medium to mimic CB inhalation, exhibiting DNA strand breaks with only limited inflammation. The influence of dispersion media on nanomaterial toxicity should be considered in the planning of intratracheal investigations., (© The Author(s) 2017. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society.)

The utilisation of nanoparticles as the means of targeted delivery of therapeutics and/or imaging agents could greatly enhance the specific transport of biologically active payloads to target tissues while avoiding or reducing undesired side-effects. To allow for this to become a reality, the question of potential toxicological effects needs to be addressed. In the present investigation, a cationic liposome with prospective for medical applications was constructed and thoroughly assessed for any material-induced hepatic adverse effects in vivo - in healthy and alcoholic hepatic disease models and in vitro - (HepG2 cells). The data demonstrated that intravenous injection of liposomes did not cause any significant in vivo hepatic toxicity (inflammation, alterations in blood parameters, anti-oxidant depletion, acute phase response and histopathology) at doses of 200 μg per mouse in either healthy or chronically alcohol fed mice. Additionally, the in vitro material-induced adverse effects (cytotoxicity, inflammation or albumin secretion) were all also minimal. The data from this study demonstrated that the intravenous injection of cationic liposomes does not cause hepatic toxicity. This investigation is important as it investigates the toxicity of a nano-sized material in a model of alcoholic hepatic disease in vitro and in vivo . This is an area of research in the field of nanotoxicology that is currently almost entirely overlooked.

Carbon nanotube (CNT) is a nanomaterial that has received interest because of its high-tensile strength and low weight. Although CNTs differ substantially in physico-chemical properties, they share high aspect ratio which resembles that of asbestos and other fibers causing lung cancer and mesothelioma. One type of multi-walled CNTs (i.e. MWCNT-7) has been classified as possibly carcinogenic to humans by IARC (Group 2B) based on experimental animal data, whereas other types of MWCNTs and single-walled CNTs (SWCNT) could not be classified due to lack of data from epidemiologic studies and insufficient mechanistic evidence. Damage to DNA is considered to be a key mechanistic step in the development of fiber-induced cancer. Thus, the genotoxic potential can be a cornerstone in the evaluation of hazards of CNTs. The present study used a weight of evidence (WoE) analysis to evaluate the genotoxicity of different types of CNTs. Genotoxicity endpoints close to cancer (mutations and chromosome aberrations) and animal models had highest weight in the WoE analysis. Eight CNT materials out of 130, which had been assessed in several studies, were evaluated in the WoE analysis. The results demonstrated that MWCNT-7 has strongest WoE for a genotoxic hazard among the MWCNTs. Two types of SWCNTs have a similar WoE for genotoxicity as MWCNT-7. Several reference materials from the Joint Research Centre have less WoE for genotoxicity. The WoE analysis demonstrates a difference in genotoxicity for CNTs, but further research is required to unravel the physico-chemical characteristics that govern the differences in genotoxic hazard.

The International Agency for Research on Cancer has classified one type of multi-walled carbon nanotubes (MWCNTs) as possibly carcinogenic to humans. However, the underlying mechanisms of MWCNT- induced carcinogenicity are not known. In this study, the genotoxic, mutagenic, inflammatory, and fibrotic potential of MWCNTs were investigated. Muta™Mouse adult females were exposed to 36±6 or 109±18μg/mouse of Mitsui-7, or 26±2 or 78±5μg/mouse of NM-401, once a week for four consecutive weeks via intratracheal instillations, alongside vehicle-treated controls. Samples were collected 90days following the first exposure for measurement of DNA strand breaks, lacZ mutant frequency, p53 expression, cell proliferation, lung inflammation, histopathology, and changes in global gene expression. Both MWCNT types persisted in lung tissues 90days post-exposure, and induced lung inflammation and fibrosis to similar extents. However, there was no evidence of DNA damage as measured by the comet assay following Mitsui-7 exposure, or increases in lacZ mutant frequency, for either MWCNTs. Increased p53 expression was observed in the fibrotic foci induced by both MWCNTs. Gene expression analysis revealed perturbations of a number of biological processes associated with cancer including cell death, cell proliferation, free radical scavenging, and others in both groups, with the largest response in NM-401-treated mice. The results suggest that if the two MWCNT types were capable of inducing DNA damage, strong adaptive responses mounted against the damage, resulting in efficient and timely elimination of damaged cells through cell death, may have prevented accumulation of DNA damage and mutations at the post-exposure time point investigated in the study. Thus, MWCNT-induced carcinogenesis may involve ongoing low levels of DNA damage in an environment of persisting fibres, chronic inflammation and tissue irritation, and parallel increases or decreases in the expression of genes involved in several pro-carcinogenic pathways., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The many interesting physical and chemical properties of carbon nanotubes (CNT) make it one of the most commercially attractive materials in the era of nanotechnology. Here, we review the recent publications on in vivo biodistribution of pristine and functionalized forms of single-walled and multi-walled CNT. Pristine CNT remain in the lung for months or even years after pulmonary deposition. If cleared, the majority of CNT move to the gastrointestinal (GI) tract via the mucociliary escalator. However, there appears to be no uptake of CNT from the GI tract, with a possible exception of the smallest functionalized SWCNT. Importantly, a significant fraction of CNT translocate from the alveolar space to the near pulmonary region including lymph nodes, subpleura and pleura (<7% of the pulmonary deposited dose) and to distal organs including liver, spleen and bone marrow (~1%). These results clearly demonstrate the main sites of long-term CNT accumulation, which also includes pleura, a major site for fibre-induced pulmonary diseases. Studies on intravenous injection show that CNT in blood circulation are cleared relatively fast with a half-life of minutes or hours. The major target organs were the same as identified after pulmonary exposure with the exception of urine excretion of especially functionalized SWCNT and accumulation in lung tissue. Overall, there is evidence that CNT will primarily be distributed to the liver where they appear to be present at least one year after exposure., (© 2016 The Authors. Basic & Clinical Pharmacology & Toxicology published by John Wiley & Sons Ltd on behalf of Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).)