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Inhalation exposure to iron oxide occurs in many workplaces and respirable aerosols occur during thermal processes (e.g. welding, casting) or during abrasion of iron and steel products (e.g. cutting, grinding, machining, polishing, sanding) or during handling of iron oxide pigments. There is limited evidence of adverse effects in humans specifically linked to inhalation of iron oxides. This contrasts to oxides of other metals used to alloy or for coating of steel and iron of which several have been classified as being hazardous by international and national agencies. Such metal oxides are often present in the air at workplaces. In general, iron oxides might therefore be regarded as low-toxicity, low-solubility (LTLS) particles, and are often considered to be nontoxic even if very high and prolonged inhalation exposures might result in diseases. In animal studies, such exposures lead to cancer, fibrosis and other diseases. Our hypothesis was that pulmonary-workplace exposure during manufacture and handling of SPION preparations might be harmful. We therefore conducted a systematic review of the relevant literature to understand how iron oxides deposited in the lung are related to acute and subchronic pulmonary inflammation. We included one human and several in vivo animal studies published up to February 2023. We found 25 relevant studies that were useful for deriving occupational exposure limits (OEL) for iron oxides based on an inflammatory reaction. Our review of the scientific literature indicates that lowering of health-based occupational exposure limits might be considered.

Nanomaterials (NMs) offer plenty of novel functionalities. Moreover, their physicochemical properties can be fine-tuned to meet the needs of specific applications, leading to virtually unlimited numbers of NM variants. Hence, efficient hazard and risk assessment strategies building on New Approach Methodologies (NAMs) become indispensable. Indeed, the design, the development and implementation of NAMs has been a major topic in a substantial number of research projects. One of the promising strategies that can help to deal with the high number of NMs variants is grouping and read-across. Based on demonstrated structural and physicochemical similarity, NMs can be grouped and assessed together. Within an established NM group, read-across may be performed to fill in data gaps for data-poor variants using existing data for NMs within the group. Establishing a group requires a sound justification, usually based on a grouping hypothesis that links specific physicochemical properties to well-defined hazard endpoints. However, for NMs these interrelationships are only beginning to be understood. The aim of this review is to demonstrate the power of bioinformatics with a specific focus on Machine Learning (ML) approaches to unravel the NM Modes-of-Action (MoA) and identify the properties that are relevant to specific hazards, in support of grouping strategies. This review emphasizes the following messages: 1) ML supports identification of the most relevant properties contributing to specific hazards; 2) ML supports analysis of large omics datasets and identification of MoA patterns in support of hypothesis formulation in grouping approaches; 3) omics approaches are useful for shifting away from consideration of single endpoints towards a more mechanistic understanding across multiple endpoints gained from one experiment; and 4) approaches from other fields of Artificial Intelligence (AI) like Natural Language Processing or image analysis may support automated extraction and interlinkage of information related to NM toxicity. Here, existing ML models for predicting NM toxicity and for analyzing omics data in support of NM grouping are reviewed. Various challenges related to building robust models in the field of nanotoxicology exist and are also discussed.

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.

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.

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.

Metal oxide nanoparticles are used in a broad range of industrial processes and workers may be exposed to aerosols of the particles both during production and handling. Despite the widespread use of these particles, relatively few studies have been performed to investigate the toxicological effects in the airways following inhalation. In the present study, the acute (24 h) and persistent (13 weeks) effects in the airways after a single exposure to metal oxide nanoparticles were studied using a murine inhalation model. Mice were exposed 60 min to aerosols of either ZnO, TiO2, Al2O3 or CeO2 and the deposited doses in the upper and lower respiratory tracts were calculated. Endpoints were acute airway irritation, pulmonary inflammation based on analyses of bronchoalveolar lavage (BAL) cell composition, DNA damage assessed by the comet assay and pulmonary toxicity assessed by protein level in BAL fluid and histology. All studied particles reduced the tidal volume in a concentration-dependent manner accompanied with an increase in the respiratory rate. In addition, ZnO and TiO2 induced nasal irritation. BAL cell analyses revealed both neutrophilic and lymphocytic inflammation 24-h post-exposure to all particles except TiO2. The ranking of potency regarding induction of acute lung inflammation was Al2O3 = TiO2 < CeO2 ≪ ZnO. Exposure to CeO2 gave rise to a more persistent inflammation; both neutrophilic and lymphocytic inflammation was seen 13 weeks after exposure. As the only particles, ZnO caused a significant toxic effect in the airways while TiO2 gave rise to DNA-strand break as shown by the comet assay.

Lung deposition of multi-walled carbon nanotubes (MWCNT) induces pulmonary toxicity. Commercial MWCNT vary greatly in physicochemical properties and consequently in biological effects. To identify determinants of MWCNT-induced toxicity, we analyzed the effects of pulmonary exposure to 10 commercial MWCNT (supplied in three groups of different dimensions, with one pristine and two/three surface modified in each group). We characterized morphology, chemical composition, surface area and functionalization levels. MWCNT were deposited in lungs of female C57BL/6J mice by intratracheal instillation of 0, 6, 18 or 54 μg/mouse. Pulmonary inflammation (neutrophil influx in bronchoalveolar lavage (BAL)) and genotoxicity were determined on day 1, 28 or 92. Histopathology of the lungs was performed on day 28 and 92. All MWCNT induced similar histological changes. Lymphocytic aggregates were detected for all MWCNT on day 28 and 92. Using adjusted, multiple regression analyses, inflammation and genotoxicity were related to dose, time and physicochemical properties. The specific surface area (BET) was identified as a positive predictor of pulmonary inflammation on all post-exposure days. In addition, length significantly predicted pulmonary inflammation, whereas surface oxidation (-OH and -COOH) was predictor of lowered inflammation on day 28. BET surface area, and therefore diameter, significantly predicted genotoxicity in BAL fluid cells and lung tissue such that lower BET surface area or correspondingly larger diameter was associated with increased genotoxicity. This study provides information on possible toxicity-driving physicochemical properties of MWCNT. The results may contribute to safe-by-design manufacturing of MWCNT, thereby minimizing adverse effects.

ENPRA was one of the earlier multidisciplinary European Commission FP7-funded projects aiming to evaluate the risks associated with nanomaterial (NM) exposure on human health across pulmonary, cardiovascular, hepatic, renal, and developmental systems. The outputs from this project have formed the basis of this review. A retrospective interpretation of the findings across a wide range of in vitro and in vivo studies was performed to identify the main highlights from the project. In particular, focus was placed on informing what advances were made in the hazard assessment of NM, as well as offering some suggestions on the future of "nanotoxicology research" based on these observations, shortcomings, and lessons learned from the project. A number of issues related to the hazard assessment of NM are discussed in detail and include use of appropriate NM for nanotoxicology investigations; characterization and dispersion of NM; use of appropriate doses for all related investigations; need for the correct choice of experimental models for risk assessment purposes; and full understanding of the test systems and correct interpretation of data generated from in vitro and in vivo systems. It is hoped that this review may assist in providing information in the implementation of guidelines, model systems, validation of assessment methodology, and integrated testing approaches for risk assessment of NM. It is vital to learn from ongoing and/or completed studies to avoid unnecessary duplication and offer suggestions that might improve different aspects of experimental design.

Engineered nanomaterials (NMs) offer great technological advantages but their risks to human health are still not fully understood. An increasing body of evidence suggests that some NMs are capable of distributing from the site of exposure to a number of secondary organs. The research into the toxicity posed by the NMs in these secondary organs is expanding due to the realisation that some materials may reach and accumulate in these target sites. The translocation to secondary organs includes, but is not limited to, the hepatic, central nervous, cardiovascular and renal systems. Current data indicates that pulmonary exposure is associated with low (inhalation route-0.00001-1% of total applied dose-24 h) translocation of virtually insoluble NMs such as iridium, carbon black, gold and polystyrene, while slightly higher translocation has been observed for NMs with either slow (e.g., silver, cerium dioxide and quantum dots) or fast (e.g., zinc oxide) solubility. The translocation of NMs following intratracheal, intranasal and pharyngeal aspiration is higher (up to 10% of administered dose), however the relevance of these routes for risk assessment is questionable. Uptake of the materials from the gastrointestinal tract seems to follow the same pattern as inhalation translocation, whereas the dermal uptake of NMs is generally reported to be low. The toxicological effects in secondary organs include oxidative stress, inflammation, cytotoxicity and dysfunction of cellular and physiological processes. For toxicological and risk evaluation, further information on the toxicokinetics and persistence of NMs is crucial. The overall aim of this review is to outline the data currently available in the literature on the biokinetics, accumulation, toxicity and eventual fate of NMs in order to assess the potential risks posed by NMs to secondary organs.

An estimated 1% or less of nanoparticles (NPs) deposited in the lungs translocate to systemic circulation and enter other organs; however, this estimation may not be accurate given the low sensitivity of existing in vivo NP detection methods. Moreover, the biological effects of such low levels of translocation are unclear. We employed a nano-scale hyperspectral microscope to spatially observe and spectrally profile NPs in tissues and blood following pulmonary deposition in mice. In addition, we characterized effects occurring in blood, liver and heart at the mRNA and protein level following translocation from the lungs. Adult female C57BL/6 mice were exposed via intratracheal instillation to 18 or 162 µg of industrially relevant titanium dioxide nanoparticles (nano-TiO2) alongside vehicle controls. Using the nano-scale hyperspectral microscope, translocation to heart and liver was confirmed at both doses, and to blood at the highest dose, in mice analyzed 24 h post-exposure. Global gene expression profiling and ELISA analysis revealed activation of complement cascade and inflammatory processes in heart and specific activation of complement factor 3 in blood, suggesting activation of an early innate immune response essential for particle opsonisation and clearance. The liver showed a subtle response with changes in the expression of genes associated with acute phase response. This study characterizes the subtle systemic effects that occur in liver and heart tissues following pulmonary exposure and low levels of translocation of nano-TiO2 from lungs.

Effects on the liver C3A cell line treated with a panel of engineered nanomaterials (NMs) consisting of two zinc oxide particles (ZnO; coated 100 nm and uncoated 130 nm), two multi-walled carbon nanotubes (MWCNTs), one silver (Ag < 20 nm), one 7 nm anatase, two rutile TiO2 nanoparticles (10 and 94 nm) and two derivatives with positive and negative covalent functionalisation of the 10 nm rutile were evaluated. The silver particles elicited the greatest level of cytotoxicity (24 h LC50 - 2 µg/cm(2)). The silver was followed by the uncoated ZnO (24 h LC50 - 7.5 µg/cm(2)) and coated ZnO (24 h LC50 - 15 µg/cm(2)) particles with respect to cytotoxicity. The ZnO NMs were found to be about 50-60% soluble which could account for their toxicity. By contrast, the Ag was <1% soluble. The LC50 was not attained in the presence of any of the other engineered NMs (up to 80 µg/cm(2)). All NMs significantly increased IL-8 production. Meanwhile, no significant change in TNF-α, IL-6 or CRP was detected. Urea and albumin production were measured as indicators of hepatic function. These markers were only altered by the coated and uncoated ZnO, which significantly decreased albumin production.

Nanoparticles in primary form and nanoproducts might elicit different toxicological responses. We compared paint-related nanoparticles with respect to effects on endothelial oxidative stress, cytotoxicity and cell adhesion molecule expression. Primary human umbilical vein endothelial cells were exposed to primary nanoparticles (fine, photocatalytic or nanosized TiO(2), aluminium silicate, carbon black, nano-silicasol or axilate) and dust from sanding reference- or nanoparticle-containing paints. Most of the samples increased cell surface expressions of vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1), but paint sanding dust samples generally generated less response than primary particles of TiO(2) and carbon black. We found no relationship between the expression of adhesion molecules, cytotoxicity and production of reactive oxygen species. In conclusion, sanding dust from nanoparticle-containing paint did not generate more oxidative stress or expression of cell adhesion molecules than sanding dust from paint without nanoparticles, whereas the primary particles had the largest effect on mass basis.

We investigated effects of maternal pulmonary exposure to titanium dioxide (UV-Titan) on prenatally exposed offspring. Time-mated mice (C57BL/6BomTac) were inhalation exposed (1 h/day to 42 mg UV-Titan/m(3) aerosolised powder or filtered air) during gestation days (GDs) 8-18. We evaluated DNA strand breaks using the comet assay in bronchoalveolar lavage (BAL) cells and livers of the time-mated mice (5 and 26-27 days after inhalation exposure), and in livers of the offspring (post-natal days (PND) 2 and 22). We also analysed hepatic gene expression in newborns using DNA microarrays. UV-Titan exposure did not induce DNA strand breaks in time-mated mice or their offspring. Transcriptional profiling of newborn livers revealed changes in the gene expression related to the retinoic acid signalling pathway in the females, while gene expression in male offspring was unaffected. Changes may be a secondary response to maternal inflammation although no direct link was evident through gene expression analysis.

PARTICLE&#95;RISK was one of the first multidisciplinary projects funded by the European Commission's Framework Programme that was responsible for evaluating the implications of nanomaterial (NM) exposure on human health. This project was the basis for this review which identifies the challenges that exist within the assessment of NM risk. We have retrospectively reflected on the findings of completed nanotoxicology studies to consider what progress and advances have been made within the risk assessment of NMs, as well as discussing the direction that nanotoxicology research is taking and identifying the limitations and failings of existing research. We have reflected on what commonly encountered challenges exist and explored how these issues may be resolved. In particular, the following is discussed (i) NM selection (ii) NM physico-chemical characterisation; (iii) NM dispersion; (iv) selection of relevant doses and concentrations; (v) identification of relevant models, target sites and endpoints; (vi) development of alternatives to animal testing; and (vii) NM risk assessment. These knowledge gaps are relatively well recognised by the scientific community and recommendations as to how they may be overcome in the future are provided. It is hoped that this will help develop better defined hypothesis driven research in the future that will enable comprehensive risk assessments to be conducted for NMs. Importantly, the nanotoxicology community has responded and adapted to advances in knowledge over recent years to improve the approaches used to assess NM hazard, exposure and risk. It is vital to learn from existing information provided by ongoing or completed studies to avoid unnecessary duplication of effort, and to offer guidance on aspects of the experimental design that should be carefully considered prior to the start of a new study.

Nanoparticles are increasingly used in paints and lacquers. Little is known of the toxicity of nanoparticles incorporated in complex matrices and released during different phases of the life cycle. DNA damaging activity and inflammogenicity of sanding dust sampled during standardised sanding of boards painted with paints with and without nanoparticles were determined 24 h after intratracheal instillation of a single dose of 54 μg in mice. Dusts from nanoparticle-containing paints and lacquers did not generate pulmonary inflammation or oxidative stress. Sanding dust from both the nanoparticle-containing and the conventional lacquer and the outdoor acrylic-based reference paint increased the level of DNA strand breaks in bronchoalveolar fluid cells. In conclusion, addition of nanoparticles to paint or lacquers did not increase the potential of sanding dust for causing inflammation, oxidative stress or DNA damage, suggesting that the paint/lacquer matrix is more important as determinant of DNA damage than the nanomaterial.

Manufactured nanomaterials are projected to be used on a large scale in paints and lacquers. We selected seven commercially interesting materials: Three titanium dioxide-based (two coated rutile; one uncoated anatase), one carbon black (Flamrüss 101), one kaolinite clay, and two silica products, whereas carbon black, Printex 90, was used as reference material. DNA damaging activity and inflammogenicity (pulmonary cell composition and mRNAs) were determined 24 h after intratracheal instillation of a single dose of 54 μg in mice. Greatest inflammation was induced by Printex 90 and uncoated titanium dioxide. The inflammatory potency correlated with instilled surface area (R(2) = 0.94) but not with material volume (R(2) = 0.17). The coated titanium dioxides induced DNA damage in lung lining fluid cells. The uncoated titanium dioxide was not DNA damaging by the comet assay 24 h after exposure despite being highly inflammogenic. This suggests that inflammation is not a prerequisite to DNA damage in titanium dioxide-based products.

Effects of maternal pulmonary exposure to carbon black (Printex 90) on gestation, lactation and DNA strand breaks were evaluated. Time-mated C57BL/6BomTac mice were exposed by inhalation to 42 mg/m(3) Printex 90 for 1 h/day on gestation days (GD) 8-18, or by four intratracheal instillations on GD 7, 10, 15 and 18, with total doses of 11, 54 and 268 μg/animal. Dams were monitored until weaning and some offspring until adolescence. Inflammation was assessed in maternal bronchoalveolar lavage (BAL) 3-5 days after exposure, and at weaning. Levels of DNA strand breaks were assessed in maternal BAL cells and liver, and in offspring liver. Persistent lung inflammation was observed in exposed mothers. Inhalation exposure induced more DNA strand breaks in the liver of mothers and their offspring, whereas intratracheal instillation did not. Neither inhalation nor instillation affected gestation and lactation. Maternal inhalation exposure to Printex 90-induced liver DNA damage in the mothers and the in utero exposed offspring.

Particulates are small particles of solid or liquid suspended in liquid or air. In vitro studies show that particles generate reactive oxygen species, deplete endogenous antioxidants, alter mitochondrial function and produce oxidative damage to lipids and DNA. Surface area, reactivity and chemical composition play important roles in the oxidative potential of particulates. Studies in animal models indicate that particles from combustion processes (generated by combustion of wood or diesel oil), silicate, titanium dioxide and nanoparticles (C60 fullerenes and carbon nanotubes) produce elevated levels of lipid peroxidation products and oxidatively damaged DNA. Biomonitoring studies in humans have shown associations between exposure to air pollution and wood smoke particulates and oxidative damage to DNA, deoxynucleotides and lipids measured in leukocytes, plasma, urine and/or exhaled breath. The results indicate that oxidative stress and elevated levels of oxidatively altered biomolecules are important intermediate endpoints that may be useful markers in hazard characterization of particulates.

DNA repair may prevent increased levels of oxidatively damaged DNA from prolonged oxidative stress induced by, e.g. exposure to diesel exhaust particles (DEP). We studied oxidative damage to DNA in broncho-alveolar lavage cells, lungs, and liver after 4 x 1.5 h inhalations of DEP (20 mg/m3) in Ogg1-/- and wild type (WT) mice with similar extent of inflammation. DEP exposure increased lung levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in Ogg1-/- mice, whereas no effect on 8-oxodG or oxidized purines in terms of formamidopyrimidine DNA glycosylase (FPG) sites was observed in WT mice. In both unexposed and exposed Ogg1-/- mice the level of FPG sites in the lungs was 3-fold higher than in WT mice. The high basal level of FPG sites in Ogg1-/- mice probably saturated the assay and prevented detection of DEP-generated damage. In conclusion, Ogg1-/- mice have elevated pulmonary levels of FPG sites and accumulate genomic 8-oxodG after repeated inhalations of DEP.

The effect of high dietary intake of animal fat and an increased fat energy intake on colon and liver genotoxicity and on markers of oxidative damage and antioxidative defence in colon, liver and plasma was investigated in Big Blue rats. The rats were fed ad libitum with semi-synthetic feed supplemented with 0, 3, 10 or 30% w/w lard. After 3 weeks, the mutation frequency, DNA repair gene expression, DNA damage and oxidative markers were determined in liver, colon and plasma. The mutation frequency of the lambda gene cII did not increase with increased fat or energy intake in colon or liver. The DNA-adduct level measured by 32P-postlabelling decreased in both liver and colon with increased fat intake. In liver, this was accompanied by a 2-fold increase of the mRNA level of nucleotide excision repair (NER) gene ERCC1. In colon, a non-statistically significant increase in the ERCC1 mRNA levels was observed. Intake of lard fat resulted in increased ascorbate synthesis and affected markers of oxidative damage to proteins in liver cytosol, but not in plasma. The effect was observed at all lard doses and was not dose-dependent. However, no evidence of increased oxidative DNA damage was found in liver, colon, or urine. Thus, lard intake at the expense of other nutrients and a large increase in the fat energy consumption affects the redox state locally in the liver cytosol, but does not induce DNA-damage, systemic oxidative stress or a dose-dependent increase in mutation frequency in rat colon or liver.