About the Transient Effects of Synthetic Amorphous Silica: An In Vitro Study on Macrophages.

Silica (either crystalline or amorphous) is widely used for different applications and its toxicological assessment depends on its characteristics and intended use. As sustained inflammation induced by crystalline silica is at the root of silicosis, investigating the inflammatory effects induced by amorphous silicas and their persistence is needed. For the development of new grades of synthetic amorphous silicas, it is also desirable to be able to understand better the factors underlying potential adverse effects. Therefore, we used an optimized in vitro macrophage system to investigate the effects of amorphous silicas, and their persistence. By using different amorphous silicas, we demonstrated that the main driver for the adverse effects is a low size of the overall particle/agglomerate; the second driver being a low size of the primary particle. We also demonstrated that the effects were transient. By using silicon dosage in cells, we showed that the transient effects are coupled with a decrease of intracellular silicon levels over time after exposure. To further investigate this phenomenon, a mild enzymatic cell lysis allowed us to show that amorphous silicas are degraded in macrophages over time, explaining the decrease in silicon content and thus the transiency of the effects of amorphous silicas on macrophages.

Oral Toxicokinetics, Tissue Distribution, and 28-Day Oral Toxicity of Two Differently Manufactured Food Additive Silicon Dioxides.

(1) Background: Synthetic amorphous silica (SAS) is widely used as a food additive and contains nano-sized particles. SAS can be produced by fumed and precipitated methods, which may possess different physiochemical properties, toxicokinetics, and oral toxicity. (2) Methods: The toxicokinetics of fumed SAS and precipitated SAS were evaluated following a single-dose oral administration in rats. The tissue distribution and fate of both SAS particles were assessed after repeated oral administration in rats for 28 d, followed by recovery period for 90 d. Their 28-d repeated oral toxicity was also evaluated. (3) Results: Precipitated SAS showed higher oral absorption than fumed SAS, but the oral absorption of both SAS particles was low (<4%), even at 2000 mg/kg. Our tissue-distribution study revealed that both SAS particles, at a high dose (2000 mg/kg), were accumulated in the liver after repeated administration for 28 d, but the increased concentrations returned to normal levels at 29 d, the first day of the recovery period. A higher distribution level of precipitated SAS than fumed SAS and decomposed particle fates of both SAS particles were found in the liver at 28 d. No significant toxicological findings were observed after 28-d oral administration, suggesting their low oral toxicity. (4) Conclusions: Different manufacturing methods of SAS can, therefore, affect its oral toxicokinetics and tissue distribution, but not oral toxicity.

Investigating the effects of differently produced synthetic amorphous silica (E 551) on the integrity and functionality of the human intestinal barrier using an advanced in vitro co-culture model.

E 551, also known as synthetic amorphous silica (SAS), is the second most produced food additive. However, according to the re-evaluation of E 551 by the European Food Safety Authority (EFSA) in 2018, the amount of available data on the oral toxicity of food grade E 551 is still insufficient for reliable risk assessment. To close this gap, this study aimed to investigate six food-grade SAS with distinct physicochemical properties on their interaction with the intestinal barrier using advanced in vitro intestinal co-cultures and to identify potential structure-activity relationships. A mucus-secreting Caco-2/HT-29/Raji co-culture model was treated with up to 50 µg/ml SAS for 48 h, which represents a dose range relevant to dietary exposure. No effects on cell viability, barrier integrity, microvilli function or the release of inflammatory cytokine were detected after acute exposure. Slight biological responses were observed for few SAS materials on iron uptake and gene expression levels of mucin 1 and G-protein coupled receptor 120 (GPR120). There was no clear correlation between SAS properties (single or combined) and the observed biological responses. Overall, this study provides novel insights into the short-term impact of food-relevant SAS with distinct characteristics on the intestinal epithelium including a range of intestine-specific functional endpoints. In addition, it highlights the importance of using advanced intestinal co-cultures embracing relevant cell types as well as a protective mucus barrier to achieve a comprehensive understanding of the biological response of food additives at the intestinal barrier in vitro.

Is aggregated synthetic amorphous silica toxicologically relevant?

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.

MyD88-dependent pro-interleukin-1ß induction in dendritic cells exposed to food-grade synthetic amorphous silica.

BACKGROUND: Dendritic cells (DCs) are specialized first-line sensors of foreign materials invading the organism. These sentinel cells rely on pattern recognition receptors such as Nod-like or Toll-like receptors (TLRs) to launch immune reactions against pathogens, but also to mediate tolerance to self-antigens and, in the intestinal milieu, to nutrients and commensals. Since inappropriate DC activation contributes to inflammatory diseases and immunopathologies, a key question in the evaluation of orally ingested nanomaterials is whether their contact with DCs in the intestinal mucosa disrupts this delicate homeostatic balance between pathogen defense and tolerance. Here, we generated steady-state DCs by incubating hematopoietic progenitors with feline McDonough sarcoma-like tyrosine kinase 3 ligand (Flt3L) and used the resulting immature DCs to test potential biological responses against food-grade synthetic amorphous silica (SAS) representing a common nanomaterial generally thought to be safe. RESULTS: Interaction of immature and unprimed DCs with food-grade SAS particles and their internalization by endocytic uptake fails to elicit cytotoxicity and the release of interleukin (IL)-1α or tumor necrosis factor-α, which were identified as master regulators of acute inflammation in lung-related studies. However, the display of maturation markers on the cell surface shows that SAS particles activate completely immature DCs. Also, the endocytic uptake of SAS particles into these steady-state DCs leads to induction of the pro-IL-1ß precursor, subsequently cleaved by the inflammasome to secrete mature IL-1ß. In contrast, neither pro-IL-1ß induction nor mature IL-1ß secretion occurs upon internalization of TiO2 or FePO4 nanoparticles. The pro-IL-1ß induction is suppressed by pharmacologic inhibitors of endosomal TLR activation or by genetic ablation of MyD88, a downstream adapter of TLR pathways, indicating that endosomal pattern recognition is responsible for the observed cytokine response to food-grade SAS particles. CONCLUSIONS: Our results unexpectedly show that food-grade SAS particles are able to directly initiate the endosomal MyD88-dependent pathogen pattern recognition and signaling pathway in steady-state DCs. The ensuing activation of immature DCs with de novo induction of pro-IL-1ß implies that the currently massive use of SAS particles as food additive should be reconsidered.

Critical review of the safety assessment of nano-structured silica additives in food.

The development of nano-materials is viewed as one of the most important technological advances of the 21st century and new applications of nano-sized particles in the production, processing, packaging or storage of food are expected to emerge soon. This trend of growing commercialization of engineered nano-particles as part of modern diet will substantially increase oral exposure. Contrary to the proven benefits of nano-materials, however, possible adverse health effects have generally received less attention. This problem is very well illustrated by nano-structured synthetic amorphous silica (SAS), which is a common food additive since several decades although the relevant risk assessment has never been satisfactorily completed. A no observed adverse effect level of 2500 mg SAS particles/kg body weight per day was derived from the only available long-term administration study in rodents. However, extrapolation to a safe daily intake for humans is problematic due to limitations of this chronic animal study and knowledge gaps as to possible local intestinal effects of SAS particles, primarily on the gut-associated lymphoid system. This uncertainty is aggravated by digestion experiments indicating that dietary SAS particles preserve their nano-sized structure when reaching the intestinal lumen. An important aspect is whether food-borne particles like SAS alter the function of dendritic cells that, embedded in the intestinal mucosa, act as first-line sentinels of foreign materials. We conclude that nano-particles do not represent a completely new threat and that most potential risks can be assessed following procedures established for conventional chemical hazards. However, specific properties of food-borne nano-particles should be further examined and, for that purpose, in vitro tests with decision-making cells of the immune system are needed to complement existing in vivo studies.

The safety of nanostructured synthetic amorphous silica (SAS) as a food additive (E 551).

KEY MESSAGES: Particle sizes of E 551 products are in the micrometre range. The typical external diameters of the constituent particles (aggregates) are greater than 100 nm. E 551 does not break down under acidic conditions such as in the stomach, but may release dissolved silica in environments with higher pH such as the intestinal tract. E 551 is one of the toxicologically most intensively studied substances and has not shown any relevant systemic or local toxicity after oral exposure. Synthetic amorphous silica (SAS) meeting the specifications for use as a food additive (E 551) is and has always been produced by the same two production methods: the thermal and the wet processes, resulting in E 551 products consisting of particles typically in the micrometre size range. The constituent particles (aggregates) are typically larger than 100 nm and do not contain discernible primary particles. Particle sizes above 100 nm are necessary for E 551 to fulfil its technical function as spacer between food particles, thus avoiding the caking of food particles. Based on an in-depth review of the available toxicological information and intake data, it is concluded that the SAS products specified for use as food additive E 551 do not cause adverse effects in oral repeated-dose studies including doses that exceed current OECD guideline recommendations. In particular, there is no evidence for liver toxicity after oral intake. No adverse effects have been found in oral fertility and developmental toxicity studies, nor are there any indications from in vivo studies for an immunotoxic or neurotoxic effect. SAS is neither mutagenic nor genotoxic in vivo. In intact cells, a direct interaction of unlabelled and unmodified SAS with DNA was never found. Differences in the magnitude of biological responses between pyrogenic and precipitated silica described in some in vitro studies with murine macrophages at exaggerated exposure levels seem to be related to interactions with cell culture proteins and cell membranes. The in vivo studies do not indicate that there is a toxicologically relevant difference between SAS products after oral exposure. It is noted that any silicon dioxide product not meeting established specifications, and/or produced to provide new functionality in food, requires its own specific safety and risk assessment.

Cytotoxic and genotoxic evaluation of different synthetic amorphous silica nanomaterials in the V79 cell line.

The nature of occupational risks and hazards in industries that produce or use synthetic amorphous silica (SAS) nanoparticles is still under discussion. Manufactured SAS occur in amorphous form and can be divided into two main types according to the production process, namely, pyrogenic silica (powder) and precipitated silica (powder, gel or colloid). The physical and chemical properties of SAS may vary in terms of particle size, surface area, agglomeration state or purity, and differences in their toxicity potential might therefore be expected. The aim of this study was to compare the cytotoxicity and genotoxicity of representative manufactured SAS samples in Chinese hamster lung fibroblasts (V79 cells). Five samples from industrial SAS producers were evaluated, that is, two pyrogenic SAS powders (with primary particle sizes of 20 nm and 25/70 nm), one precipitated SAS powder (20 nm) and two precipitated SAS colloids (15 and 40/80 nm). V79 cell cultures were treated with different concentrations of SAS pre-dispersed in bovine serum albumin -water medium. Pyr (pyrogenic) 20, Pre (precipitated) 20 and Col (colloid) 15 significantly decreased the cell viability after 24 h of exposure, whilst Pyr 25/70 and Col 40/80 had negligible effects. The cytotoxicity of Pyr 20, Pre 20 and Col 15 was revealed by the induction of apoptosis, and Pyr 20 and Col 15 also produced DNA damage. However, none of the SAS samples generated intracellular reactive oxidative species, micronuclei or genomic mutations in V79 cells after 24 h of exposure. Overall, the results of this study show that pyrogenic, precipitated and colloidal manufactured SAS of around 20 nm primary particle size can produce significant cytotoxic and genotoxic effects in V79 cells. In contrast, the coarser-grained pyrogenic and colloid SAS (approximately 50 nm) yielded negligible toxicity, despite having been manufactured by same processes as their finer-grained equivalents. To explain these differences, the influence of particle agglomeration and oxidative species formation is discussed.

Reproduction toxicity study with the synthetic amorphous silica SYLOID® AL-1 FP, HDK® N20, LUDOX® P T-40 F and SYLOID® MX 107 in the earthworm species Eisenia fetida.

Ecotoxicology studies were performed in the earthworm Eisenia fetida with four different synthetic amorphous silica (SAS) (SYLOID® AL-1 FP, SYLOID® MX 107, LUDOX® P T-40 F, and HDK® N20) mixed into artificial soil to determine a NOEC/LOEC for effects on reproduction (56 days after application), mortality and biomass development (28 days after application) using a standardized artificial soil with 10% peat. The LC50 for test-item effects on adult mortality, and an EC10 and EC50 for reproduction were also determined. Furthermore, earthworms underwent histopathology evaluation, and the amount of silica in different organs from these organisms was evaluated using EDX (Energy Dispersive X-ray Spectroscopy). Histopathology revealed no findings in any organ of the earthworms, except for desiccated dissepiments in evaluated decedents at extremely high SAS doses. To measure SAS uptake into the organs, a fully quantitative method for silica was established and validated using standards containing known concentrations of silica to ensure the accuracy of the analyses undertaken. Results from EDX analysis demonstrated the negligible presence of silicon within the brain ganglia and gonads of adult earthworms comparable to controls. Therefore, any deposition of the test items within these two organs was excluded. In contrast, traces of silicon higher than in controls were found in the intestinal lumina of the earthworms due to ingestion of SAS with soil and feed, but not in other organs.

Nine human epidemiological studies on synthetic amorphous silica and respiratory health.

The respiratory health effects of Synthetic Amorphous Silica (SAS) have been studied in human epidemiological research. This article presents a historical overview and review of nine occupational worker studies that have been conducted so far on this topic. The combined study population of all of these studies included 1172 employees, and exposure concentrations ranged from < 1 mg/m3 to 100 mg/m3. In two studies with a total of 293 workers, the incidence of silicosis was investigated after long-term exposure to precipitated SAS, and no cases of silicosis were found (Plunkett and Dewitt, 1962; Volk, 1960). In another study, the spirometry results of 40 workers were normal (Vitums et al., 1977). In a study of 28 workers, 4 cases of silicosis were identified, but it is possible that contamination with cristobalite occurred and detailed information about the amorphous silica origin was not provided (Mohrmann and Kahn, 1985). Ferch et al. (1987) found that lung impairment was associated with confounding factors (smoking) but not with exposure to precipitated SAS in a study of 143 workers. Choudat et al. (1990) reported a reduction in forced expiratory flow in a group exposed to precipitated SAS compared to a control group. Still, they found no correlation between the extent of exposure and pulmonary function was found in a study of 131 workers. Wilson et al. (1979) also failed to show a significant association between the degree of exposure to precipitated SAS and annual changes in lung function in a study of 165 workers. In the most recent and most extensive study (Taeger et al., 2016; Yong et al., 2022) in Germany, involving 462 factory workers, no association between inhalable or respirable SAS dust exposure and respiratory health was reported. Based on the available data, there is no evidence-base to support a relationship between SAS and respiratory health in humans.

Investigation on the skin penetration of synthetic amorphous silica (SAS) used in cosmetic products.

Synthetic amorphous silica (SAS) is used as additive in a variety of industrial applications for many decades and has been approved to be used in food, food contact materials, pharmaceuticals, and cosmetics. Due its internal structure, SAS is considered as a nanomaterial, thus it is affected by a general safety discussion. Based on the production process, SAS for cosmetic application is a nanomaterial by the EU Recommendation, although it was not considered as such, because the solely size-dependent definitions of the term "nanomaterial" emerged in recent times first in Recommendation 2011/696/EU. Therefore, former physicochemical and toxicological evaluations of SAS were already performed on nanomaterials, however, without being addressed as such. Safety concerns can only emerge if two criteria, (toxicological) hazard and exposure towards the substance is fulfilled at the same time. In case of SAS, the Scientific Committee on Consumer Safety (SCCS) challenged provided data to be insufficient to draw a conclusion regarding the safety of SAS and thus, requested further investigations, in particular by exploring skin penetration of particulate SAS.Investigation of specific particulate substances in skin penetration tests is an analytical challenge. The number of available analytical techniques that are capable to detect nanomaterials in complex matrices, like receptor fluids from skin penetration testing, are limited and still emerging. In the new studies, a comprehensive set of analytical techniques were used to investigate the skin penetration potential of SAS. Particle-sensitive, element and particle-specific combinations of techniques and different sample preparation procedures, that respected the particulate nature of SAS, were used to detect SAS in receptor fluids directly. In addition, electron microscopic techniques were used to examine different layers of skin to detect adsorbed SAS.The combination of Asymmetric Flow Field-Flow Fractionation (AF4) in combination with Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for examination of receptor fluids and Scanning Electron Microscopy coupled with Energy Dispersive X-ray spectroscopy (SEM/EDX) for examination of skin itself, were identified as suitable techniques for the detection of SAS in skin penetration tests. Data from literature was used to compare the results of the studies with the outcome of other test systems (other particles, other techniques). Both, the test results, and literature evaluation led to the conclusion, that SAS does not penetrate skin. Based on this outcome and local and systemic dermal toxicity review of SAS, it can be concluded that dermal application of SAS in cosmetic formulations is negligible.

Pyrogenic synthetic amorphous silica (NM-203): Genotoxicity in rats following sub-chronic oral exposure.

The genotoxicity of nano-structured synthetic amorphous silica (SAS), a common food additive, was investigated in vivo in rats. A 90-day oral toxicity study was performed according to OECD test guideline 408 and the genotoxicity of pyrogenic SAS nanomaterial NM-203 was assessed in several organs, using complementary tests. Adult Sprague-Dawley rats of both sexes were treated orally for 90 days with 0, 2, 5, 10, 20, or 50 mg SAS/kg bw per day. Dose levels were selected to approximate expected human dietary exposures to SAS. DNA strand breaks were evaluated by the comet assay in blood, bone marrow, liver, and spleen according to OECD test guideline 489; mutations induced in bone marrow precursors of erythrocytes were assessed by the Pig-a assay and chromosome/ genome damage by the micronucleus assay in blood (OECD test guideline 474) and colon. No treatment-related increases of gene (Pig-a) or chromosome/genome (micronucleus) mutations were detected in the blood. The percentage of micronucleated cells was not increased in the colon of treated rats. Among the organs analyzed by the comet assay, the spleen was the only target showing a weak but biologically relevant genotoxic effect.

Repeated Exposure of Macrophages to Synthetic Amorphous Silica Induces Adaptive Proteome Changes and a Moderate Cell Activation.

Synthetic amorphous silica (SAS) is a nanomaterial used in a wide variety of applications, including the use as a food additive. Two types of SAS are commonly employed as a powder additive, precipitated silica and fumed silica. Numerous studies have investigated the effects of synthetic amorphous silica on mammalian cells. However, most of them have used an exposure scheme based on a single dose of SAS. In this study, we have used instead a repeated 10-day exposure scheme in an effort to better simulate the occupational exposure encountered in daily life by consumers and workers. As a biological model, we have used the murine macrophage cell line J774A.1, as macrophages are very important innate immune cells in the response to particulate materials. In order to obtain a better appraisal of the macrophage responses to this repeated exposure to SAS, we have used proteomics as a wide-scale approach. Furthermore, some of the biological pathways detected as modulated by the exposure to SAS by the proteomic experiments have been validated through targeted experiments. Overall, proteomics showed that precipitated SAS induced a more important macrophage response than fumed SAS at equal dose. Nevertheless, validation experiments showed that most of the responses detected by proteomics are indeed adaptive, as the cellular homeostasis appeared to be maintained at the end of the exposure. For example, the intracellular glutathione levels or the mitochondrial transmembrane potential at the end of the 10 days exposure were similar for SAS-exposed cells and for unexposed cells. Similarly, no gross lysosomal damage was observed after repeated exposure to SAS. Nevertheless, important functions of macrophages such as phagocytosis, TNFα, and interleukin-6 secretion were up-modulated after exposure, as was the expression of important membrane proteins such as the scavenger receptors, MHC-II, or the MAC-1 receptor. These results suggest that repeated exposure to low doses of SAS slightly modulates the immune functions of macrophages, which may alter the homeostasis of the immune system.

Extended Investigation of Exposure to Respirable Synthetic Amorphous Silica Dust and Its Potential Impact on Non-malignant Respiratory Morbidity.

Objectives: The present analysis aims to study the health impact of an occupational exposure to respirable synthetic amorphous silica (SAS) dusts, based on the available data from the German study. Methods: The effect of cumulative exposure to respirable SAS dust on respiratory morbidity were investigated in 462 exposed male workers. Multiple exposure assessments was performed anchored by a most recent measurement series. Internal regression models in addition to Monte Carlo-Multi Model were fitted. Results: An averaged cumulative respirable SAS dust concentration of 6.44 mg/m3-years was estimated. Internal regression models suggested a reduction of 8.11 ml (95% confidence interval: 0.49-15.73) in forced vital capacity (FVC) per 1 mg/m3-year increase of exposure. But no effect on forced expiratory volume in 1 s (FEV1) and the ratio of the parameters FEV1/FVC was observed in association with exposure to a respirable fraction of SAS. No adverse effects on the occurrence of respiratory diseases were indicated. Conclusion: This study provides no clear evidence of adverse health effects from occupational exposure to respirable SAS. Sponsor: Evonik Operations GmbH/Smart Materials, Cabot Corporation, Wacker Chemie AG.

Surface Treatment With Hydrophobic Coating Reagents (Organosilanes) Strongly Reduces the Bioactivity of Synthetic Amorphous Silica in vitro.

Synthetic amorphous silica (SAS) is industrially relevant material whose bioactivity in vitro is strongly diminished, for example, by protein binding to the particle surface. Here, we investigated the in vitro bioactivity of fourteen SAS (pyrogenic, precipitated, or colloidal), nine of which were surface-treated with organosilanes, using alveolar macrophages as a highly sensitive test system. Dispersion of the hydrophobic SAS required pre-wetting with ethanol and extensive ultrasonic treatment in the presence of 0.05% BSA (Protocol 1). Hydrophilic SAS was suspended by moderate ultrasonic treatment (Protocol 2) and also by Protocol 1. The suspensions were administered to NR8383 alveolar macrophages under serum-free conditions for 16 h, and the release of LDH, GLU, H2O2, and TNFα was measured in cell culture supernatants. While seven surface-treated hydrophobic SAS exhibited virtually no bioactivity, two materials (AEROSIL® R 504 and AEROSIL® R 816) had minimal effects on NR8383 cells. In contrast, non-treated SAS elicited considerable increases in LDH, GLU, and TNFα, while the release of H2O2 was low except for CAB-O-SIL® S17D Fumed Silica. Dispersing hydrophilic SAS with Protocol 1 gradually reduced the bioactivity but did not abolish it. The results show that hydrophobic coating reagents, which bind covalently to the SAS surface, abrogate the bioactivity of SAS even under serum-free in vitro conditions. The results may have implications for the hazard assessment of hydrophobic surface-treated SAS in the lung.

Physical Obstruction of Nasal Cavities With Subsequent Asphyxia, Causes Lethality of Rats in an Acute Inhalation Study With Hydrophobic HMDZ Surface-Treated Synthetic Amorphous Silica (SAS).

The aim of the present study was to understand the mechanism of lethality associated with high dose inhalation of a low-density hydrophobic surface-treated SAS observed in some acute inhalation studies. It was demonstrated that physical obstruction of the upper respiratory tract (nasal cavities) caused the effects observed. Hydrophobic surface-treated SAS was inhaled (flow-past, nose-only) by six Wistar rats (three males and three females) in an acute toxicity study at a concentration of ~500 mg/m3 for an intended 4-hr exposure. Under the conditions of the test set-up, the concentration applied was found to be the highest that can be delivered to the test animal port without significant alteration of the aerosol size distribution over time. None of the test- material-exposed animals survived the planned observation time of 4 h; three animals died between 2 34 h after starting exposure and cessation of exposure at 3 14 h, two died after transfer to their cages and the remaining animal was sacrificed due to its poor condition and welfare considerations. Histology accomplished by energy dispersive X-ray (EDX) analysis demonstrated that test material particles agglomerated and formed a gel-like substrate that ultimately blocked the upper respiratory airways, which proved fatal for the rat as an obligatory nose breather. This observation is in line with the findings reported by Hofmann et al. showing a correlation between lethality and hydrophobicity determined by contact angle measurement. The aerosol characterizations associated with this study are provided in detail by Wessely et al.

The TLR4/NFκB-Dependent Inflammatory Response Activated by LPS Is Inhibited in Human Macrophages Pre-Exposed to Amorphous Silica Nanoparticles.

Amorphous silica nanoparticles (ASNP) are present in a variety of products and their biological effects are actively investigated. Although several studies have documented pro-inflammatory effects of ASNP, the possibility that they also modify the response of innate immunity cells to natural activators has not been thoroughly investigated. Here, we study the effects of pyrogenic ASNP on the LPS-dependent activation of human macrophages differentiated from peripheral blood monocytes. In macrophages, 24 h of pre-exposure to non-cytotoxic doses of ASNP markedly inhibited the LPS-dependent induction of pro-inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10). The inhibitory effect was associated with the suppression of NFκB activation and the increased intracellular sequestration of the TLR4 receptor. The late induction of glutamine synthetase (GS) by LPS was also prevented by pre-exposure to ASNP, while GS silencing did not interfere with cytokine secretion. It is concluded that (i) macrophages exposed to ASNP are less sensitive to LPS-dependent activation and (ii) GS induction by LPS is likely secondary to the stimulation of cytokine secretion. The observed interference with LPS effects may point to a dampening of the acute inflammatory response after exposure to ASNP in humans.

A Systematic Review on the Hazard Assessment of Amorphous Silica Based on the Literature From 2013 to 2018.

Background: Nanomaterials are suspected of causing health problems, as published studies on nanotoxicology indicate. On the other hand, some of these materials, such as nanostructured pyrogenic and precipitated synthetic amorphous silica (SAS) and silica gel, have been used for decades without safety concerns in industrial, commercial, and consumer applications. However, in addition to many in vivo and in vitro studies that have failed to demonstrate the intrinsic toxicity of SAS, articles periodically emerge, in which biological effects of concern have been described. Even though most of these studies do not meet high-quality standards and do not always use equivalent test materials or standardized test systems, the results often trigger substance re-evaluation. To put the results into perspective, an extensive literature study was carried out and an example of amorphous silica will be used to try to unravel the reliability from the unreliable results. Methods: A systematic search of studies on nanotoxicological effects has been performed covering the years 2013 to 2018. The identified studies have been evaluated for their quality regarding material and method details, and the data have been curated and put into a data collection. This review deals only with investigations on amorphous silica. Results: Of 18,162 publications 1,217 have been selected with direct reference to experiments with synthetically produced amorphous silica materials. The assessment of these studies based on defined criteria leads to a further reduction to 316 studies, which have been included in this systematic review. Screening for quality with well-defined quantitative criteria following the GUIDE nano concept reveals only 27.3% has acceptable quality. Overall, the in vitro and in vivo data showed low or no toxicity of amorphous silica. The data shown do not support the hypothesis of dependency of biological effects on the primary particle size of the tested materials. Conclusion: This review demonstrates the relatively low quality of most studies published on nanotoxicological issues in the case of amorphous silica. Moreover, mechanistic studies are often passed off or considered toxicological studies. In general, standardized methods or the Organization for Economic Cooperation and Development (OECD) guidelines are rarely used for toxicological experiments. As a result, the significance of the published data is usually weak and must be reevaluated carefully before using them for regulatory purposes.

Comparison of Biogenic Amorphous Silicas Found in Common Horsetail and Oat Husk With Synthetic Amorphous Silicas.

The present study summarizes the current literature on the presence and the structure of biogenic amorphous silica (BAS) in nature. Based on this review, it is shown that BAS is ubiquitous in nature and exhibits a structure that cannot be differentiated from the structure of synthetic amorphous silica (SAS). The structural similarity of BAS and SAS is further supported by our investigations-in particular, specific surface area (BET) and electron microscope techniques-on oat husk and common horsetail. Many food products containing BAS are considered to be beneficial to health. In the context of the use of SAS in specific applications (e.g., food, feed, and cosmetics), this is of particular interest for discussions of the safety of these uses.

Genome-Wide DNA Methylation Alterations and Potential Risk Induced by Subacute and Subchronic Exposure to Food-Grade Nanosilica in Mice.

The intensive application of nanomaterials in the food industry has raised concerns about their potential risks to human health. However, limited data are available on the biological safety of nanomaterials in food, especially at the epigenetic level. This study examined the implications of two types of synthetic amorphous silica (SAS), food-grade precipitated silica (S200) and fumed silica Aerosil 200F (A200F), which are nanorange food additives. After 28-day continuous and intermittent subacute exposure to these SAS via diet, whole-genome methylation levels in mouse peripheral leukocytes and liver were significantly altered in a dose- and SAS type-dependent manner, with minimal toxicity detected by conventional toxicological assessments, especially at a human-relevant dose (HRD). The 84-day continuous subchronic exposure to all doses of S200 and A200F induced liver steatosis where S200 accumulated in the liver even at HRD. Genome-wide DNA methylation sequencing revealed that the differentially methylated regions induced by both SAS were mainly located in the intron, intergenic, and promoter regions after 84-day high-dose continuous exposure. Bioinformatics analysis of differentially methylated genes indicated that exposure to S200 or A200F may lead to lipid metabolism disorders and cancer development. Pathway validation experiments indicated both SAS types as potentially carcinogenic. While S200 inhibited the p53-mediated apoptotic pathway in mouse liver, A200F activated the HRAS-mediated MAPK signaling pathway, which is a key driver of hepatocarcinogenesis. Thus, caution must be paid to the risk of long-term exposure to food-grade SAS, and epigenetic parameters should be included as end points during the risk assessment of food-grade nanomaterials.