Eosinophilic globules (syn. hyaline inclusions) and protein crystalloids in the nasal mucosa of rats treated with synthetic amorphous silica, an unspecific chitinase-like-protein-positive background lesion.

In a 13-week inhalation toxicity study with three recovery periods (3, 6, and 12 months), Crl: WI rats were allocated to nine groups, each containing 25 animals per sex. Eight groups were treated daily by inhalation with the test items at concentrations of 0.5, 1.0, 2.5, or 5.0 mg/m3 (SAS 1 groups 2, 3, 4, or 5, respectively; SAS 2 groups 6, 7, 8, or 9, respectively). Controls (group 1) were treated with air only. In nasal cavities, the major lesions consisted of increased eosinophilic globules and chitinase-3-like-protein-positive crystalloids* in the nasal mucosa, mainly in nasal cavity levels 2-4 up to week 26 of recovery without any further injury in olfactory mucosa, mainly in SAS 1-treated animals. Eosinophilic globules in the rodent nasal cavity are common and increase with age; they represent a particular finding of the rodent nasal mucosa. The relevance of chitinase-3-like protein (Ym1 + Ym2) expression in the rodent nasal mucosa is unknown but is normal in control animals. Both findings developed without any indicator for inflammatory processes. The increase of these unspecific background findings is considered an indicator of minor irritative effects. Due to the clear lack of nasal tissue injury or concurrent changes (degeneration, necrosis, inflammatory infiltrate, dysplasia, and/or neoplasia) following repeated inhalation exposure to SAS, it is deemed that the eosinophilic globules (hyaline inclusions) combined with the formation of eosinophilic protein crystalloids in this study represent an adaptive response.

Enhanced study design for acute inhalation studies with hydrophobic surface-treated particles to determine toxicological effects including suffocation.

High concentrations of low-density particles may cause effects in acute inhalation toxicity studies which can be easily underestimated or misinterpreted following strictly the OECD TG 436, i.e., limited parameters as mortality and gross lesions will be evaluated only. Seven particle types (synthetic amorphous silica (SAS) HMDZ-SAS, silica gel, pyrogenic SAS, and precipitated SAS, calcium carbonate, aluminum oxide pyrogenic alumina, organic red pigment) were chosen at the highest technically feasible concentration of approximately 500 mg/m3 for acute inhalation studies with an expanded endpoint setup. Therefore additional parameters and a thorough histopathological evaluation of an extensive set of organs, including the respiratory tract emphasizing the nasal cavities were added. Six Crl:WI rats per study were exposed for four hours from which three animals were sacrificed after 24 hours and three animals after 14 days. HMDZ-SAS caused early death in all animals due to blockage of the nasal passages caused by its hydrophobicity. For all other Si-containing compounds, histology revealed minor inflammatory and reactive lesions in lungs after 24 hours that were still present after 14 days, except in silica gel-treated animals. After 14 days, for pyrogenic SAS, precipitated SAS, and pyrogenic alumina, granulomas formed in the BALT and lung-associated lymph nodes. In contrast, the calcium carbonate induced almost no findings, and the red pigment (also tested for the additional dose of 1000 mg/m3) stuck partially to the nasal mucosa without causing pathological damage and partly entered the lungs without showing any adverse effects. The results of the present study highlight the advantage of improving the rather simple study design of acute inhalation studies by implementing an extended study design.

Regenerative and progressing lesions in lungs and lung-associated lymph nodes from fourteen 90-day inhalation studies with chemically different particulate materials.

Rat lungs and lung-associated lymph nodes from 14 inhalation studies with chemically different particulate materials were histopathologically re-evaluated, and the bronchoalveolar lavage fluid (BALF) data and lung burden analyses were compared. All investigated substances caused similar lesions. For most substances, 1 mg/m3 of respirable particulate matter was established as the borderline for adverse morphological changes after the 90-day exposure period, confirmed by the increase in polymorphonuclear neutrophils in BALF. Possible reversibility was demonstrated when recovery groups are included in the study especially allowing the differentiation between regeneration or progressing of inflammatory changes during the recovery period. It was concluded, that the major driver of toxicity is not an intrinsic chemical property of the particle but a particle effect. Concerning classification for specific target organ toxicant (STOT) repeated exposure (RE), this paper highlights that merely comparing the lowest concentration, at which adverse effects were observed, with the Classification Labelling and Packaging (CLP) regulation (EC) no. 1272/2008 guidance values is inappropriate and might lead to a STOT classification under CLP for a large part of the substances discussed in this paper, on the basis of typically mild to moderate findings in rat lung and lung-associated lymph nodes on day 1 after exposure. An in-depth evaluation of the pathologic findings is required and an expert judgement has to be included in the decision on classification and labeling, evaluating the type and severity of effects and comparing these with the classification criteria.

Issues in the inhalation toxicity testing and hazard assessment for low density particulate materials such as synthetic amorphous silica (SAS).

Inhalation toxicity testing of particulate materials is mandated for classification. According to CLP, particulate materials should be tested as marketed and many particulate materials are marketed as non-respirable particles. However, OECD TG 413 requires exposure to particle sizes that are respirable and reach the alveoli. The requirement for exposure of rats to respirable particles is thus in contrast to CLP and requires the application of high shear forces. The exposure to artificially small particles causes a number of issues that hamper the interpretation of the results of the testing. These issues are aerosol altering in the exposure system, assessment of the adversity of the inflammatory lung responses, inclusion of recovery groups, and extrapolation of the results to humans exposed under occupational condition. In addition, effects of many particulate materials after testing according to OECD 413 are not intrinsic properties, but a general reaction of the lung to the deposited material, show very similar NOAECs for chemical diverse materials, and often are completely reversible.

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.

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.

Serum Lowers Bioactivity and Uptake of Synthetic Amorphous Silica by Alveolar Macrophages in a Particle Specific Manner.

Various cell types are compromised by synthetic amorphous silica (SAS) if they are exposed to SAS under protein-free conditions in vitro. Addition of serum protein can mitigate most SAS effects, but it is not clear whether this is solely caused by protein corona formation and/or altered particle uptake. Because sensitive and reliable mass spectrometric measurements of SiO2 NP are cumbersome, quantitative uptake studies of SAS at the cellular level are largely missing. In this study, we combined the comparison of SAS effects on alveolar macrophages in the presence and absence of foetal calf serum with mass spectrometric measurement of 28Si in alkaline cell lysates. Effects on the release of lactate dehydrogenase, glucuronidase, TNFα and H2O2 of precipitated (SIPERNAT® 50, SIPERNAT® 160) and fumed SAS (AEROSIL® OX50, AEROSIL® 380 F) were lowered close to control level by foetal calf serum (FCS) added to the medium. Using a quantitative high resolution ICP-MS measurement combined with electron microscopy, we found that FCS reduced the uptake of particle mass by 9.9% (SIPERNAT® 50) up to 83.8% (AEROSIL® OX50). Additionally, larger particle agglomerates were less frequent in cells in the presence of FCS. Plotting values for lactate dehydrogenase (LDH), glucuronidase (GLU) or tumour necrosis factor alpha (TNFα) against the mean cellular dose showed the reduction of bioactivity with a particle sedimentation bias. As a whole, the mitigating effects of FCS on precipitated and fumed SAS on alveolar macrophages are caused by a reduction of bioactivity and by a lowered internalization, and both effects occur in a particle specific manner. The method to quantify nanosized SiO2 in cells is a valuable tool for future in vitro studies.

Effects of Ultrasonic Dispersion Energy on the Preparation of Amorphous SiO2 Nanomaterials for In Vitro Toxicity Testing.

Synthetic amorphous silica (SAS) constitute a large group of industrial nanomaterials (NM). Based on their different production processes, SAS can be distinguished as precipitated, fumed, gel and colloidal. The biological activity of SAS, e.g., cytotoxicity or inflammatory potential in the lungs is low but has been shown to depend on the particle size, at least for colloidal silica. Therefore, the preparation of suspensions from highly aggregated or agglomerated SAS powder materials is critical. Here we analyzed the influence of ultrasonic dispersion energy on the biologic activity of SAS using NR8383 alveolar macrophage (AM) assay. Fully characterized SAS (7 precipitated, 3 fumed, 3 gel, and 1 colloidal) were dispersed in H2O by stirring and filtering through a 5 µm filter. Aqueous suspensions were sonicated with low or high ultrasonic dispersion (USD) energy of 18 or 270 kJ/mL, respectively. A dose range of 11.25⁻90 µg/mL was administered to the AM under protein-free conditions to detect particle-cell interactions without the attenuating effect of proteins that typically occur in vivo. The release of lactate dehydrogenase (LDH), glucuronidase (GLU), and tumor necrosis factor α (TNF) were measured after 16 h. Hydrogen peroxide (H2O2) production was assayed after 90 min. The overall pattern of the in vitro response to SAS (12/14) was clearly dose-dependent, except for two SAS which showed very low bioactivity. High USD energy gradually decreased the particle size of precipitated, fumed, and gel SAS whereas the low adverse effect concentrations (LOECs) remained unchanged. Nevertheless, the comparison of dose-response curves revealed slight, but uniform shifts in EC50 values (LDH, and partially GLU) for precipitated SAS (6/7), gel SAS (2/3), and fumed SAS (3/3). Release of TNF changed inconsistently with higher ultrasonic dispersion (USD) energy whereas the induction of H2O2 was diminished in all cases. Electron microscopy and energy dispersive X-ray analysis showed an uptake of SAS into endosomes, lysosomes, endoplasmic reticulum, and different types of phagosomes. The possible effects of different uptake routes are discussed. The study shows that the effect of increased USD energy on the in vitro bioactivity of SAS is surprisingly small. As the in vitro response of AM to different SAS is highly uniform, the production process per se is of minor relevance for toxicity.

The Facial Adipose System: Its Role in Facial Aging and Approaches to Volume Restoration.

BACKGROUND: Volume loss in facial adipose tissue plays a critical role in the aesthetics of facial aging. Furthermore, the facial adipose system is a complex network of distinct compartments, and a detailed understanding of these compartments is essential for optimal facial volume restoration. OBJECTIVE: To review the facial adipose system, age-related changes, and the role of volume restoration products for facial rejuvenation. METHODS: Publications including deceased donors' dissection studies and more recent studies using computed tomography were reviewed to provide an up-to-date understanding of the facial adipose system anatomy and age-related changes. Current volume restoration treatment options including hyaluronic acid, calcium hydroxylapatite, and poly-L-lactic acid are discussed. RESULTS: Facial aging is associated with volume loss in superficial and deep adipose compartments, including those of the forehead, cheek, lip, chin, and jowl areas. Volume restoration products can be used to address the age-related changes of the facial adipose compartments. CONCLUSION: Understanding the complex network of facial adipose compartments and their age-related changes allows for the optimal use of injectable volume restoration products for facial rejuvenation that can be customized to the anatomical needs of each patient.

Cellulite: an evidence-based review.

BACKGROUND: Cellulite is a multifactorial condition that is present in 80-90 % of post-pubertal women. Despite its high prevalence, it remains a major cosmetic concern for women. A wide range of products and treatments for cellulite reduction is available; however, no systematic review has been performed so far to evaluate the efficacy of the available treatment options for cellulite. OBJECTIVE: The objective of this review is to provide a systematic evaluation of the scientific evidence of the efficacy of treatments for cellulite reduction. METHODS: This systematic review followed the PRISMA guidelines for reporting systematic reviews and meta-analyses. Only original articles in English or German reporting data on the efficacy of cellulite treatments from in vivo human studies were considered. In total, 67 articles were analyzed for the following information: therapy, presence of a control group, randomization, blinding, sample size, description of statistical methods, results, and level of evidence. RESULTS: Most of the evaluated studies, including laser- and light-based modalities, radiofrequency, and others had important methodological flaws; some did not use cellulite severity as an endpoint or did not provide sufficient statistical analyses. Of the 67 studies analyzed in this review, only 19 were placebo-controlled studies with randomization. Some evidence for potential benefit was only seen for acoustic wave therapy (AWT) and the 1440 nm Nd:YAG minimally invasive laser. CONCLUSION: This article provides a systematic evaluation of the scientific evidence of the efficacy of treatment for cellulite reduction. No clear evidence of good efficacy could be identified in any of the evaluated cellulite treatments.

Patient-reported outcomes after incobotulinumtoxinA treatment for upper facial wrinkles.

BACKGROUND: Botulinum toxins are used for facial rejuvenation. OBJECTIVE: To investigate the efficacy, patient satisfaction, and skin physiology after incobotulinumtoxinA treatment of the upper face. MATERIALS AND METHODS: Thirty women aged 35 to 55 were enrolled in this single-center prospective 16-week study. Patients and an investigator blinded to visit number assessed wrinkle severity after incobotulinumtoxinA treatment of glabellar, periorbital, and forehead lines using Validated Assessment Scales at Days 0 (baseline), 2, 7, 14, 28, and 112. Responder rates (for each region) and mean wrinkle scores (whole upper face) were calculated. Treatment satisfaction was self-assessed by questionnaire. Transepidermal water loss (TEWL), stratum corneum hydration, and skin pH were measured to assess skin barrier function. RESULTS: Wrinkle severity decreased rapidly after treatment. Responder rates generally peaked at Days 7 to 14. At 112 days, mean scores remained significantly lower than baseline (p < .05). Patients were significantly more satisfied with their appearance after treatment; most felt "younger" and "more attractive" (p < .05). Transepidermal water loss decreased significantly throughout (p < .05), and stratum corneum hydration increased. No treatment-related adverse events occurred. CONCLUSION: IncobotulinumtoxinA rapidly improved wrinkle severity, and improvements were maintained for up to 112 days. Increased stratum corneum hydration and reduced TEWL may improve skin quality. IncobotulinumtoxinA was well tolerated and improved patients' self-perception.

Cryolipolysis for noninvasive body contouring: clinical efficacy and patient satisfaction.

In recent years, a number of modalities have become available for the noninvasive reduction of adipose tissue, including cryolipolysis, radiofrequency, low-level laser, and high-intensity focused ultrasound. Each technology employs a different mechanism of action to cause apoptosis or necrosis of the targeted adipocytes. Among these technologies, cryolipolysis has not only been commercially available for the longest time, but has also been best researched including in vitro and animal models and randomized controlled clinical trials in humans. The principle behind cryolipolysis exploits the premise that adipocytes are more susceptible to cooling than other skin cells. The precise application of cold temperatures triggers apoptosis of the adipocytes, which invokes an inflammatory response and leads to slow digestion by surrounding macrophages. In clinical studies, cryolipolysis was shown to reduce subcutaneous fat at the treatment site by up to 25% after one treatment. Improvements were seen in 86% of treated subjects. At 73%, the patient satisfaction rate is higher than with other technologies used for noninvasive lipolysis. Cryolipolysis has been proven to be a very safe method for body contouring, and is accomplished with only minimal discomfort. Expected side effects are temporary erythema, bruising, and transient numbness that usually resolve within 14 days after treatment. With a prevalence of 0.1%, the most common complaint is late-onset pain, occurring 2 weeks post-procedure, which resolves without intervention. Although no procedure has been accepted as the gold standard for noninvasive body contouring as yet, cryolipolysis is considered to be both safe and efficient with a high patient satisfaction rate.

Quantification of age-related facial wrinkles in men and women using a three-dimensional fringe projection method and validated assessment scales.

BACKGROUND: Whereas the molecular mechanisms of skin aging are well understood, little information is available concerning the clinical onset and lifetime development of facial wrinkles. OBJECTIVES: To perform the first systematic evaluation of the lifetime development of facial wrinkles and sex-specific differences using three-dimensional (3D) imaging and clinical rating. METHODS: 200 men and women aged 20 to 70 were selected. Wrinkle severity of periorbital, glabellar, and forehead lines was evaluated using 3D imaging and validated assessment scales. RESULTS: Wrinkle severity was greater at all assessed locations with older age. In men, wrinkles manifested earlier and were more severe than in women. In women, periorbital lines were the first visible wrinkles, in contrast to the forehead lines in men. In both sexes, glabellar lines did not clinically manifest before the age of 40. CONCLUSION: The results of the present study confirm a progressive increase of crow's feet and forehead and glabellar lines in men and women. Although the development of facial wrinkles happens earlier and is more severe in men, perimenopause seems to particularly affect development in women. Clinical ratings and 3D measurements are suitable methods to assess facial wrinkle severity in men and women.

Aesthetic uses of the botulinum toxin.

The cosmetic use of botulinum toxin (BoNT) is the most common cosmetic procedure performed in the world today. Common adverse events seen in the aesthetic use of the BoNT include swelling, localized bruising, headaches, injection site discomfort, excessive muscle weakness, and unintended paresis of adjacent muscles. BoNT has a wide array of cosmetic uses, including treatment of glabellar lines, chemical browlift, forehead wrinkles, periorbital, and perioral lines. The future formulations and applications of BoNT type A will be plentiful, and are exciting to consider.