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Background: The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective. This knowledge gap greatly affects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nano-sized particles (1-100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes., Results: We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR., Conclusion: Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.

Background: Silica nanoparticles (SiNPs) have numerous beneficial properties and are extensively used in cosmetics and food industries as anti-caking, densifying and hydrophobic agents. However, the increasing exposure levels experienced by the general population and the ability of SiNPs to penetrate cells and tissues have raised concerns about possible toxic effects of this material. Although SiNPs are known to affect the function of the airway epithelium, the molecular targets of these particles remain largely unknown. Given that SiNPs interact with the plasma membrane of epithelial cells we hypothesized that they may affect the function of Transient Receptor Potential Vanilloid 4 (TRPV4), a cation-permeable channel that regulates epithelial barrier function. The main aims of this study were to evaluate the effects of SiNPs on the activation of TRPV4 and to determine whether these alter the positive modulatory action of this channel on the ciliary beat frequency in airway epithelial cells., Results: Using fluorometric measurements of intracellular Ca 2+ concentration ([Ca 2+ ] i ) we found that SiNPs inhibit activation of TRPV4 by the synthetic agonist GSK1016790A in cultured human airway epithelial cells 16HBE and in primary cultured mouse tracheobronchial epithelial cells. Inhibition of TRPV4 by SiNPs was confirmed in intracellular Ca 2+ imaging and whole-cell patch-clamp experiments performed in HEK293T cells over-expressing this channel. In addition to these effects, SiNPs were found to induce a significant increase in basal [Ca 2+ ] i , but in a TRPV4-independent manner. SiNPs enhanced the activation of the capsaicin receptor TRPV1, demonstrating that these particles have a specific inhibitory action on TRPV4 activation. Finally, we found that SiNPs abrogate the increase in ciliary beat frequency induced by TRPV4 activation in mouse airway epithelial cells., Conclusions: Our results show that SiNPs inhibit TRPV4 activation, and that this effect may impair the positive modulatory action of the stimulation of this channel on the ciliary function in airway epithelial cells. These findings unveil the cation channel TRPV4 as a primary molecular target of SiNPs.

Large-scale production and use of amorphous silica nanoparticles (SiNPs) have increased the risk of human exposure to SiNPs, while their health effects remain unclear. In this review, scientific papers from 2010 to 2016 were systematically selected and sorted based on in vitro and in vivo studies: to provide an update on SiNPs toxicity and to address the knowledge gaps indicated in the review of Napierska (Part Fibre Toxicol 7:39, 2010). Toxicity of SiNPs in vitro is size, dose, and cell type dependent. SiNPs synthesized by wet route exhibited noticeably different biological effects compared to thermal route-based SiNPs. Amorphous SiNPs (particularly colloidal and stöber) induced toxicity via mechanisms similar to crystalline silica. In vivo, route of administration and physico-chemical properties of SiNPs influences the toxicokinetics. Adverse effects were mainly observed in acutely exposed animals, while no significant signs of toxicity were noted in chronically dosed animals. The correlation between in vitro and in vivo toxicity remains less well established mainly due to improper-unrealistic-dosing both in vitro and in vivo. In conclusion, notwithstanding the multiple studies published in recent years, unambiguous linking of physico-chemical properties of SiNPs types to toxicity, bioavailability, or human health effects is not yet possible.

Conclusion of the Opinion: The SCCS has concluded that the evidence, both provided in the submission and that available in scientific literature, is inadequate and insufficient to allow drawing any firm conclusion either for or against the safety of any of the individual SAS material, or any of the SAS categories that are intended for use in cosmetic products. As the SCCS has not been able to conclude on the safety of the synthetic amorphous silica (SAS) materials included in the current submission, the Applicant is advised to follow the SCCS Guidance on Risk Assessment of Nanomaterials (SCCS/1484/12). A brief summary is provided to enable/facilitate future evaluation of the SAS materials in cosmetic products., (Copyright © 2015. Published by Elsevier Inc.)

Nano-silicon dioxide (SiO2) is used nowadays in several biomedical applications such as drug delivery and cancer therapy, and is produced on an industrial scale as additive to paints and coatings, cosmetics and food. Data regarding the long-term biokinetics of SiO2 engineered nanoparticles (ENPs) is lacking. In this study, the whole-body biodistribution of SiO2 core-shell ENPs containing a paramagnetic core of Fe3O4 was investigated after a single exposure via intravenous injection or intratracheal instillation in mice. The distribution and accumulation in different organs was evaluated for a period of 84 days using several techniques, including magnetic resonance imaging, inductively coupled plasma mass spectrometry, X-ray fluorescence and X-ray absorption near edge structure spectroscopy. We demonstrated that intravenously administered SiO2 ENPs mainly accumulate in the liver, and are retained in this tissue for over 84 days. After intratracheal instillation, an almost complete particle clearance from the lung was seen after 84 days with distribution to spleen and kidney. Furthermore, we have strong evidence that the ENPs retain their original core-shell structure during the whole observation period. This work gives an insight into the whole-body biodistribution of SiO2 ENPs and will provide guidance for further toxicity studies.

The unique physical and chemical properties of nanomaterials have led to their increased use in many industrial applications, including as a paint additive. For example, titanium dioxide (TiO2) engineered nanoparticles (ENPs) have well-established anti-UV, self-cleaning, and air purification effects. Silver (Ag) ENPs are renowned for their anti-microbial capabilities and silicon dioxide (SiO2) ENPs are used as fire retardants and anti-scratch coatings. In this study, the toxic effects and biodistribution of three pristine ENPs (TiO2, Ag, and SiO2), three aged paints containing ENPs (TiO2, Ag, and SiO2) along with control paints without ENPs were compared. BALB/c mice were oropharyngeally aspirated with ENPs or paint particles (20 μg/aspiration) once a week for 5 weeks and sacrificed either 2 or 28 days post final aspiration treatment. A bronchoalveolar lavage was performed and systemic blood toxicity was evaluated to ascertain cell counts, induction of inflammatory cytokines, and key blood parameters. In addition, the lung, liver, kidney, spleen, and heart were harvested and metal concentrations were determined. Exposure to pristine ENPs caused subtle effects in the lungs and negligible alterations in the blood. The most pronounced toxic effects were observed after Ag ENPs exposure; an increased neutrophil count and a twofold increase in pro-inflammatory cytokine secretion (keratinocyte chemoattractant (KC) and interleukin-1ß (IL-1ß)) were identified. The paint containing TiO2 ENPs did not modify macrophage and neutrophil counts, but mildly induced KC and IL-1ß. The paints containing Ag or SiO2 did not show significant toxicity. Biodistribution experiments showed distribution of Ag and Si outside the lung after aspiration to respectively pristine Ag or SiO2 ENPs. In conclusion, we demonstrated that even though direct exposure to ENPs induced some toxic effects, once they were embedded in a complex paint matrix little to no adverse toxicological effects were identified., (© The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

There is evidence that nanoparticles can induce endothelial dysfunction. Here, the effect of monodisperse amorphous silica nanoparticles (SiO(2)-NPs) of different diameters on endothelial cells function is examined. Human endothelial cell line (EA.hy926) or primary human pulmonary artery endothelial cells (hPAEC) are seeded in inserts introduced or not above triple cell co-cultures (pneumocytes, macrophages, and mast cells). Endothelial cells are incubated with SiO(2)-NPs at non-cytotoxic concentrations for 12 h. A significant increase (up to 2-fold) in human monocytes adhesion to endothelial cells is observed for 18 and 54 nm particles. Exposure to SiO(2)-NPs induces protein expression of adhesion molecules (ICAM-1 and VCAM-1) as well as significant up-regulation in mRNA expression of ICAM-1 in both endothelial cell types. Experiments performed with fluorescent-labelled monodisperse amorphous SiO(2)-NPs of similar size evidence nanoparticle uptake into the cytoplasm of endothelial cells. It is concluded that exposure of human endothelial cells to amorphous silica nanoparticles enhances their adhesive properties. This process is modified by the size of the nanoparticle and the presence of other co-cultured cells., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104 nm) at concentrations of 5 μg/cm² cell culture SA or 10 cm² particle SA/cm². A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-α, IL-6, IL-8 and MIP-1α were measured. Significant secretion of all cytokines was observed for the 2 nm particles at both concentrations and in both compartments. Larger particles of 60 nm induced significant cytokine secretion at the dose of 10 cm² particle SA/cm². The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Amorphous silica nanoparticles (SiO₂-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO₂-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO₂-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO₂-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 μg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO₂-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 μg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.

Silica nanoparticles (SNPs) are produced on an industrial scale and are an addition to a growing number of commercial products. SNPs also have great potential for a variety of diagnostic and therapeutic applications in medicine. Contrary to the well-studied crystalline micron-sized silica, relatively little information exists on the toxicity of its amorphous and nano-size forms. Because nanoparticles possess novel properties, kinetics and unusual bioactivity, their potential biological effects may differ greatly from those of micron-size bulk materials. In this review, we summarize the physico-chemical properties of the different nano-sized silica materials that can affect their interaction with biological systems, with a specific emphasis on inhalation exposure. We discuss recent in vitro and in vivo investigations into the toxicity of nanosilica, both crystalline and amorphous. Most of the in vitro studies of SNPs report results of cellular uptake, size- and dose-dependent cytotoxicity, increased reactive oxygen species levels and pro-inflammatory stimulation. Evidence from a limited number of in vivo studies demonstrates largely reversible lung inflammation, granuloma formation and focal emphysema, with no progressive lung fibrosis. Clearly, more research with standardized materials is needed to enable comparison of experimental data for the different forms of nanosilicas and to establish which physico-chemical properties are responsible for the observed toxicity of SNPs.

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Identifying the physico-chemical characteristics of nanoparticles (NPs) that drive their toxic activity is the key to conducting hazard assessment and guiding the design of safer nanomaterials. Here we used a set of 17 stable suspensions of monodisperse amorphous silica nanoparticles (SNPs) with selected variations in size (diameter, 2-335 nm), surface area (BET, 16-422 m(2)/g) and microporosity (micropore volume, 0-71 microl/g) to assess with multiple regression analysis the physico-chemical determinants of the cytotoxic activity in four different cell types (J774 macrophages, EAHY926 endothelial cells, 3T3 fibroblasts and human erythrocytes). We found that the response to these SNPs is governed by different physico-chemical parameters which vary with cell type: In J774 macrophages, the cytotoxic activity (WST1 assay) increased with external surface area (alphas method) and decreased with micropore volume (r(2) of the model, 0.797); in EAHY926 and 3T3 cells, the cytotoxic activity of the SNPs (MTT and WST1 assay, respectively) increased with surface roughness and small diameter (r(2), 0.740 and 0.872, respectively); in erythrocytes, the hemolytic activity increased with the diameter of the SNP (r(2), 0.860). We conclude that it is possible to predict with good accuracy the in vitro cytotoxic potential of SNPs on the basis of their physico-chemical characteristics. These determinants are, however, complex and vary with cell type, reflecting the pleiotropic interactions of nanoparticles with biological systems.

For the investigation of the interaction of nanoparticles with biomolecules, cells, organs, and animal models there is a need for well-characterized nanoparticle suspensions. In this paper we report the preparation of monodisperse dense amorphous silica nanoparticles (SNP) suspended in physiological media that are sterile and sufficiently stable against aggregation. SNP sols with various particle sizes (2-335 nm) were prepared via base-catalyzed hydrolysis and polymerization of tetraethyl orthosilicate under sterile conditions using either ammonia (Stober process (1) ) or lysine catalyst (Lys-Sil process (2) ). The series was complemented with commercial silica sols (Ludox). Silica nanoparticle suspensions were purified by dialysis and dispersed without using any dispersing agent into cell culture media (Dulbecco's Modified Eagle's medium) containing antibiotics. Particle sizes were determined by dynamic light scattering. SNP morphology, surface area, and porosity were characterized using electron microscopy and nitrogen adsorption. The SNP sols in cell culture medium were stable for several days. The catalytic activity of the SNP in the conversion of hydrogen peroxide into hydroxyl radicals was investigated using electron paramagnetic resonance. The catalytic activity per square meter of exposed silica surface area was found to be independent of particle size and preparation method. Using this unique series of nanoparticle suspensions, the relationship between cytotoxicity and particle size was investigated using human endothelial and mouse monocyte-macrophage cells. The cytotoxicity of the SNP was strongly dependent on particle size and cell type. This unique methodology and the collection of well-characterized SNP will be useful for further in vitro studies exploring the physicochemical determinants of nanoparticle toxicity.

The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EAHY926 cell line) is investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (as indicated by lactate dehydrogenase (LDH) release) and a decrease in cell survival (as determined by the tetrazolium reduction, MTT, assay) in the EAHY926 cell line in a dose-related manner. Concentrations leading to a 50% reduction in cell viability (TC(50)) for the smallest particles tested (14-, 15-, and 16-nm diameter) ranging from 33 to 47 microg cm(-2) of cell culture differ significantly from values assessed for the bigger nanoparticles: 89 and 254 microg cm(-2) (diameter of 19 and 60 nm, respectively). Two fine silica particles with diameters of 104 and 335 nm show very low cytotoxic response compared to nanometer-sized particles with TC(50) values of 1095 and 1087 microg cm(-2), respectively. The smaller particles also appear to affect the exposed cells faster with cell death (by necrosis) being observed within just a few hours. The surface area of the tested particles is an important parameter in determining the toxicity of monodisperse amorphous silica nanoparticles.

Our previous in vitro studies indicated that emulsion polyvinyl chloride (PVC) particles (PVC-E3), with a mean diameter of 2 microm, exhibited a moderate toxicity in different pulmonary cell cultures. The in vitro cytotoxicity and pro-inflammatory potential of PVC-E3 particles were reduced when the additives had been "washed off" (PVC-W3), indicating that PVC-particle associated toxicity is probably related to the residual additives. In the present study, male Wistar rats (230 +/- 18 g) received a single intratracheal instillation of vehicle, crystalline silica particles [Min-U-Sil, 10 mg/kg body weight (BW)], PVC-E3 (10 or 50 mg/kg BW), or PVC-W3 (10 or 50 mg/kg BW). After 2, 7, 28, or 90 days, the rats were sacrificed (n = 6) and pulmonary injury and inflammation were determined by measuring lung weight, lactate dehydrogenase (LDH) activity and protein concentrations in bronchoalveolar lavage fluid (BALF), differential BALF cell count, and histopathology. Silica exposure resulted in pulmonary inflammation and damage at all time points with a progressive deterioration. Exposure to high concentrations of PVC particles caused pulmonary inflammation and damage, which were similar to the silica-exposed group at 2 days, but at 90 days, most parameters had returned to the control level, except for minor histopathological lesions. PVC-E3 did not induce more damage than PVC-W3. Two days after exposure, PVC-W3 caused less neutrophil but more eosinophil influx than PVC-E3. Although the pulmonary toxicity of both PVC-E3 and PVC-W3 appeared limited, this in vivo study has not confirmed the conclusion from the in vitro toxicity tests that removal of residual additives reduces the toxicity of PVC-E3 particles.

Occupational exposure to polyvinyl chloride (PVC) dust has been occasionally associated with lung disease. Our aim was to evaluate the in vitro toxicity of various types of PVC particles in relevant cell culture systems. Six samples from the normal industrial suspension process (PVC-S) and eight samples from the emulsion process (PVC-E) were studied. Cytotoxicity was assessed, using the MTT assay, after 20 h of incubation in A549 cells and in primary cultures of alveolar macrophages (AM) and type II pneumocytes (TII) obtained from rats (r-AM, r-TII) or from human surgical specimens (h-AM, h-TII). Hemolysis was assessed after 2 h of incubation with human erythrocytes (h-RBC). A positive control (crystalline SiO(2), Min-U-Sil) and relevant additives of these PVC particles were tested concurrently. No toxicity of PVC-S particles could be established up to 5 mg/ml in the hemolysis test and 2.5 mg/ml in the MTT assay. In contrast, 4 out of 8 PVC-E particles induced significant toxicity, with EC50 values ranging from 0.7 to 3.7 mg/ml in the hemolysis test and from 0.2 to 1.2 mg/ml in primary cells. The most toxic particles were those that contained the additives with the highest in vitro cytotoxicity. There was a good correlation between EC50 values obtained in relevant bioassays. The sensitivity order of the cell systems was A549 < h-RBC approximately h-TII < or = h-AM < r-TII < or = r-AM for the particles. In conclusion, PVC-E particles produced a moderate in vitro toxicity for primary rat and human pulmonary cells, probably because of the residual presence of additives.