Opinion of the Scientific Committee on Consumer Safety (SCCS) - Revision of the opinion on the safety of the use of Silica, Hydrated Silica, and Silica Surface Modified with Alkyl Silylates (nano form) in cosmetic products.

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.

Nano-TiO2 modulates the dermal sensitization potency of dinitrochlorobenzene after topical exposure.

BACKGROUND: Little is known about the impact of engineered nanoparticles (ENPs) on skin sensitization caused by chemicals. OBJECTIVES: We determined the ability of different ENPs (TiO2 , Ag and SiO2 ) and aged paint particles containing ENPs to modulate dermal sensitization by a known potent dermal sensitizer. METHODS: The fur of BALB/c mice in the area around the ears was cut with scissors 1 day prior to topical exposure to ENPs (0·4, 4 or 40 mg mL(-1) ), paint particles containing ENPs (4 mg mL(-1) ) or vehicle (day 0). On days 1, 2 and 3, the mice received dermal applications on the back of both ears of 2,4-dinitrochlorobenzene (DNCB) or vehicle. The stimulation index (SI) was calculated on day 6. RESULTS: Topical exposure to TiO2 , Ag or SiO2 ENPs, or aged paint particles followed by vehicle treatment as a control, did not influence the SI. When 4 mg mL(-1) TiO2 ENPs were applied prior to DNCB sensitization, we found an increased SI compared with vehicle-exposed mice prior to DNCB sensitization. Furthermore, an increased titanium concentration was found in the draining lymph node cells of this group. Topical exposure to Ag or SiO2 ENPs or aged paint particles prior to DNCB sensitization did not influence the SI. CONCLUSIONS: We have demonstrated that topical exposure to TiO2 ENPs increases chemical-induced dermal sensitization.

Lung exposure to nanoparticles modulates an asthmatic response in a mouse model.

The aim of this study was to investigate the modulation of an asthmatic response by titanium dioxide (TiO2) or gold (Au) nanoparticles (NPs) in a murine model of diisocyanate-induced asthma. On days 1 and 8, BALB/c mice received 0.3% toluene diisocyanate (TDI) or the vehicle acetone-olive oil (AOO) on the dorsum of both ears (20 µL). On day 14, the mice were oropharyngeally dosed with 40 µL of a NP suspension (0.4 mg·mL⁻¹ (∼0.8 mg·kg⁻¹) TiO2 or Au). 1 day later (day 15), the mice received an oropharyngeal challenge with 0.01% TDI (20 µL). On day 16, airway hyperreactivity (AHR), bronchoalveolar lavage (BAL) cell and cytokine analysis, lung histology, and total serum immunoglobulin E were assessed. NP exposure in sensitised mice led to a two- (TiO2) or three-fold (Au) increase in AHR, and a three- (TiO2) or five-fold (Au) increase in BAL total cell counts, mainly comprising neutrophils and macrophages. The NPs taken up by BAL macrophages were identified by energy dispersive X-ray spectroscopy. Histological analysis revealed increased oedema, epithelial damage and inflammation. In conclusion, these results show that a low, intrapulmonary doses of TiO2 or Au NPs can aggravate pulmonary inflammation and AHR in a mouse model of diisocyanate-induced asthma.

Immunological determinants in a mouse model of chemical-induced asthma after multiple exposures.

In a mouse model of chemical-induced asthma, we investigated the effects of multiple challenges, using toluene diisocyanate (TDI), a known cause of occupational asthma. On days 1 and 7, BALB/c mice received TDI or vehicle (acetone/olive oil). On days 10, 13 and 16 the mice received an intranasal instillation of TDI. Ventilatory function (Penh) was monitored by whole body plethysmography for 40 min after each challenge. Reactivity to methacholine was measured 22 h later. Pulmonary inflammation, TNF-alpha and MIP-2 levels were assessed 24 h after the last challenge by broncho-alveolar lavage (BAL). Other immunological parameters included total IgE, lymphocyte sub-populations in auricular and cervical lymph nodes, and IL-4, IFN-gamma and IL-13 levels in supernatants of lymph node cells, cultured with or without concanavalin A. Early ventilatory function and airway reactivity increased in all groups that received a dermal application and one or multiple intranasal challenges of TDI. After multiple challenges, lung inflammation was characterized by neutrophils (approximately 15%), and eosinophils (approximately 4%), along with an increase in BAL MIP-2 and TNF-alpha levels. The auricular and cervical lymph node cells of all sensitized mice showed an increase in B cells, Th cells and an increased concentration of in vitro release of IL-4, IFN-gamma and IL-13 after stimulation with concanavalin A. Total serum IgE was elevated in dermally TDI-sensitized mice. This protocol including multiple challenges results in a model that resembles human asthma, indicating that responses found in the model using a single challenge could be a good first indication for the development of asthma.

Co-cultures of multiple cell types mimic pulmonary cell communication in response to urban PM10.

The current authors evaluated whether a system of co-cultures of relevant cells (pneumocytes (A549), macrophages (THP-1), mast cells (HMC-1) and endothelial cells (EAHY926)) would mimic the responses to particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)) previously reported in vivo. The role of mast cells was considered of special interest. Single cultures, bicultures (A549 + HMC-1 in a 10:1 ratio; THP-1 + HMC-1 in a 2:1 ratio) and tricultures (A549 + THP-1 + HMC-1 in a 10:2:1 ratio) were exposed to urban PM(10) (24 h at 0, 10, 30 or 100 microg x cm(-2)). Additionally, EAHY926 cells were introduced in inserts above the tricultures. The released cytokines were evaluated with a fluorescence-activated cell sorter array system. THP-1 + HMC-1 bicultures and the tricultures released more granulocyte colony-stimulating factor (G-CSF), macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-1beta, IL-8, IL-6, tumour necrosis factor-alpha and MIP-1alpha in response to PM(10) than the sum of the single cultures. Tricultures with EAHY926 released more G-CSF, MIP-1alpha, IL-8 and MIP-1beta than the EAHY926 single culture. The bicultures, tricultures and tricultures with EAHY926 provide results that are consistent with the local and systemic effects previously described for particulate matter effects, i.e. inflammation, endothelial dysfunction and bone marrow cell mobilisation. Mast cells seem to play a significant role in the co-culture responses.

Optimisation of culture conditions to develop an in vitro pulmonary permeability model.

An in vitro model to study pulmonary translocation was created, using the human cell line Calu-3 and primary rat type II pneumocytes. Cells were seeded on permeable membranes with a 0.4 microm or 3 microm pore size, utilizing different culture conditions such as medium formulation and cell density. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. When seeded on inserts with 0.4 microm pore size, the Calu-3 cells and primary rat type II pneumocytes created high TEER values of 949+/-182 Omega cm(2) and 400+/-257 Omega cm(2), respectively. On membranes with 3 microm pores, Calu-3 cells achieved a high TEER value of 500+/-95 Omega cm(2). Our experiments indicate that the culture medium was more critical than the cell density, regarding the influence on TEER values. For both cell types a reduction of serum in the medium resulted in a decrease in TEER value. We established a good ('tight') monolayer of primary type II pneumocytes in Waymouth medium at a cell density of 0.9x10(6) cells/cm(2); the Calu-3 cells should be grown in DMEM medium containing Hepes at 0.75x10(6) cells/cm(2).

Enhanced peripheral thrombogenicity after lung inflammation is mediated by platelet-leukocyte activation: role of P-selectin.

BACKGROUND: Inhaled ultrafine particles trigger peripheral thrombotic complications. METHODS: We have analyzed the systemic prothrombotic risk following lung inflammation induced by pulmonary carbon nanotubes (CNTs). RESULTS: Intratracheal instillation in Swiss mice of 200 and 400 microg of multiwall ground CNTs triggered substantial lung neutrophil, but not macrophage influx, 24 h later. The detection of circulating platelet-leukocyte conjugates exclusively 6 h after CNT instillation pointed to early but transient activation of circulating platelets. At 24 h, elevated plasma procoagulant microvesicular tissue factor activity was found in CNT-exposed but not in saline-exposed mice. However, at 24 h, both the tail and jugular vein bleeding times were prolonged in CNT-exposed but not in saline-exposed mice, arguing against strong CNT-induced platelet activation at this point. Nevertheless, at 24 h, enhanced peripheral thrombogenicity was detected in CNT-exposed but not in saline-exposed mice, via quantitative photochemically induced carotid artery thrombosis measurements. P-selectin neutralization abrogated platelet-leukocyte conjugate formation and microvesicular tissue factor generation, and abolished the CNT-induced thrombogenicity amplification. In contrast, the weak vascular injury-triggered thrombus formation in saline-treated mice was not affected by P-selectin neutralization at 24 h. CONCLUSIONS: The mild CNT-induced lung inflammation translates via rapid but mild and transient activation of platelets into P-selectin-mediated systemic inflammation. Leukocyte activation leads to tissue factor release, in turn eliciting inflammation-induced procoagulant activity and an associated prothrombotic risk.

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Concentrations of domestic mite and pet allergens and endotoxin in Palestine.

BACKGROUND: A few studies have compared indoor allergens and endotoxin levels between urban and rural settings as important determinants for asthma and atopy in children. However, no study was done in the Middle East or investigated refugee camps. METHODS: As part of a nested case-control study in Ramallah in 2001, we measured house dust mite and pet allergens, as well as endotoxin in dust collected from 110 children's mattresses and living room floors. RESULTS: Geometric mean (GM) concentrations of Dermatophagoides pteronyssinus (Der p1) antigen were 4.48 microg/g in mattress dust and 1.23 microg/g floor dust. The highest Der p1 levels were seen in refugee camps. Concentrations of Dermatophagoides farinae antigen (Der f1) were much lower (<0.08 microg/g dust). Concentrations of cat allergen (Fel d1) were highest in villages, and those of dog allergen (Can f1) were highest in mattresses from cities and in floor dust from refugee camps. GM of endotoxin levels were 25.7 EU/mg in mattress dust and 49 EU/mg dust in floor dust. CONCLUSIONS: Concentrations of Der p1 were high compared to Western European countries, but were lower compared to UK and Australia. Levels of pet allergens were lower than in Western Europe. Endotoxin levels were higher compared to developed countries. Indoor environmental factors such as dampness seemed to be important determinants for allergen and endotoxin, but living habits such as lack of mattress cover appeared unimportant.

Kinetics of an intratracheally administered chromium catalyst in rats.

Chromium-based catalysts are used for the synthesis of polyethylene, but little is known about the hazard and biomonitoring possibilities of this type of chromium for workers who may be occupationally exposed to such compounds. Therefore, the bioavailability and toxicokinetics of chromium were studied in male Wistar rats after a single intratracheal instillation (2 ml/kg body weight) of various doses (1, 5, or 25 mg/kg body weight) of the catalyst (approximately 1% chromium bound to an amorphous silica matrix), either before (CAT-Cr[III]) or after (CAT-Cr[VI]) heat treatment. The results were compared with those of equivalent amounts of two chromium salts (CrCl(3) and K(2) Cr(2) O (7). Each dose group was composed of three rats. The concentration of chromium was determined by atomic absorption spectrometry in urine (collected daily for 7 d) and in plasma, erythrocytes, lung, and liver tissue obtained 2 d (only highest concentration) and 7 d after dosing. On d 2, a significant increase in lung weight was found in the animals treated with the highest dose of the hexavalent Cr products. On d 7, on the basis of body weights, lung weights, and lung histology, there was no overt toxicity, except after the highest dose of CAT-Cr(VI). The elimination of all forms of chromium was apparently monoexponential, with calculated half-life elimination times in urine of 4-11 h for Cr(III) (CAT-Cr[III] and CrCl3 ) and 8-21 h for Cr(VI) (CAT-Cr[VI] and K(2) Cr(2) O(7). On d 2, the erythro-cytes Cr concentrations were significantly higher for the hexavalent Cr products than for the trivalent Cr products. After 7 d, the erythrocytes Cr concentrations were significantly increased above control values (3 microg/L) only in rats treated with the 2 highest doses of Cr( VI) compounds (12 and 64 microg/L for K(2) Cr(2) O(7), and 14 and 79 microg/L for CAT-Cr[VI]). The present study shows that intratracheally instilled Cr(VI) and Cr(III) have different toxicokinetic profiles and that the Cr(VI) catalyst has the same bioavailability and excretion kinetics as a water-soluble Cr(VI) salt. Exposure to chromium compounds could be monitored by measuring Cr concen-trations in urine (shortly after exposure) and in erythrocytes (also at later time points after high Cr[VI] exposure).

Increased HLA-DR expression after exposure of human monocytic cells to air particulates.

BACKGROUND: The expression of HLA-DR on the cell membrane of antigen-presenting cells is of major importance for the induction of an allergic response in the airways. Environmental particulates are thought to play an important role in inducing or enhancing allergic sensitization, possibly by increasing the expression of HLA-DR on the cell membrane of antigen-presenting cells. In addition, these particulates may synergize with common sensitizing agents in inducing or enhancing HLA-DR and thus antigen presentation. OBJECTIVE: In this study, we investigated the potential of three particle types, namely carbon black, diesel exhaust particles and urban air particulates (0.1-1000 ng/cm(2)), to induce the expression of HLA-DR on differentiated THP-1 cells, taken as a model for alveolar macrophages. We also assessed the "adjuvant" potential of the particles on interferon (IFN)-gamma, a known enhancer of HLA-DR. RESULTS: By themselves, the particles (0.1-1000 ng/cm(2)) were not able to induce HLA-DR on the THP-1 cells after an incubation of 48 h. However, even at very low concentrations, carbon black (from 1 ng/cm(2) on) and diesel exhaust particles (from 0.1 ng/cm(2) on), interacted with IFN-gamma (100 U/mL) to enhance HLA-DR expression (up to 2.5-fold increase). CONCLUSION: This finding may reflect in vitro one of the mechanisms by which pollutant particles exert an "adjuvant" activity and may partially explain how exposure to particles can be related to the enhancement of allergic sensitization.

Passage of inhaled particles into the blood circulation in humans.

BACKGROUND: Pollution by particulates has been consistently associated with increased cardiovascular morbidity and mortality. However, the mechanisms responsible for these effects are not well-elucidated. METHODS AND RESULTS: To assess to what extent and how rapidly inhaled pollutant particles pass into the systemic circulation, we measured, in 5 healthy volunteers, the distribution of radioactivity after the inhalation of "Technegas," an aerosol consisting mainly of ultrafine (99m)Technetium-labeled carbon particles (<100 nm). Radioactivity was detected in blood already at 1 minute, reached a maximum between 10 and 20 minutes, and remained at this level up to 60 minutes. Thin layer chromatography of blood showed that in addition to a species corresponding to oxidized (99m)Tc, ie, pertechnetate, there was also a species corresponding to particle-bound (99m)Tc. Gamma camera images showed substantial radioactivity over the liver and other areas of the body. CONCLUSIONS: We conclude that inhaled (99m)Tc-labeled ultrafine carbon particles pass rapidly into the systemic circulation, and this process could account for the well-established, but poorly understood, extrapulmonary effects of air pollution.