Toxicokinetics of Nanoparticles Deposited in Lungs Using Occupational Exposure Scenarios.

Various synthetic powders with primary particle sizes at the nanoscale and a high commercial impact have been studied using Wistar rats. The test materials were metal oxides, i.e., TiO2, ZnO and amorphous silica, and carbon black (technical soot). Dosing schemes were in the regular ranges typically used in subacute rat studies to simulate occupational exposure scenarios (mg range). Nanoscaled particle agglomerates have the potential to disintegrate and translocate as individual nanoparticles to remote locations following deposition in the lungs. The toxicokinetic fate of metal oxides post-inhalation in lungs/organs was investigated (i) by chemical analysis of the retained particulate/dissolved matter and (ii) by visualization of particles in various remote organs using transmission electron microscopy (TEM). The three titanium dioxides (NM-103, NM-104, NM-105; JRC coding) showed a very slow dissolution in lung fluids. In contrast, the coated ZnO (NM-111) dissolved quickly and was eliminated from the body within approximately 1 day. The precipitated amorphous silica (NM-200) showed a partial dissolution. Chemical analysis in lungs (particulate and soluble TiO2) and in remote organs (liver and brain) showed a small solubility effect under physiological conditions. The translocation to remote organs was negligible. This confirms that for poorly soluble TiO2 particles there was no considerable translocation to the liver and brain. The chemical analysis of zinc demonstrated a very rapid dissolution of ZnO particles after deposition in the lungs. Statistically significant increases in Zn levels in the lungs were detectable only on day 1 post-exposure (NM-111). Overall, no relevant amounts of increased NM-111 in the ionic or particulate matter were detected in any body compartment. Amorphous silica (NM-200) particles were found in the cytoplasm of intraalveolar macrophages in the lung and the cytoplasm of macrophages in the lung associated lymph node. Interestingly, these particles were found in a few animals of all treatment groups (1, 2.5, and 5 mg/m3 NM-200) even after 91 days post-exposure. In all other organs of the NM-200 treated animals such as the nasal epithelium, trachea, larynx, liver, spleen, kidney, and mesenteric lymph node no particles were found at any time point investigated. Carbon black was tagged internally ("intrinsically") with a γ tracer (7beryllium; half-time: 53.3 days). Due to limited amounts, the test item (0.3 mg per rat lung) was intratracheally instilled into the lungs. This dose avoided a particle overload effect, meaning that the toxicokinetic fate of carbon black could be followed under the approximated physiological conditions of lung clearance. Analysis of the γ labeled carbon black confirmed conclusively that there was no evidence for the translocation of carbon black beyond the lung into the blood or other body compartments. Very small amounts were only detected in lung-associated lymph nodes (LALN). On day 20 post-treatment, upon necropsy, both carbon black samples were practically exclusively found in lungs (75.1% and 91.0%, respectively) and in very small amounts in the lung-associated lymph nodes (LALN), i.e., ~0.5%. In the other organs/tissues, the test item was not significantly detectable. Separation of leukocytes and cell-free supernatant of a bronchoalveolar lavagate by centrifugation revealed that carbon black was completely located in the cell sediment, indicating total engulfment by alveolar macrophages. In conclusion, in occupational settings the nanomaterials titanium dioxide, zinc oxide, amorphous silica, and carbon black acted as microscaled agglomerates, not as individual nanoparticles. They displayed no potential to translocate beyond the lung into the blood compartment. Besides lungs, very small particulate amounts were detected only in LALN. This finding is consistent with the behavior of microscaled poorly soluble particles. Overall, there was no evidence of translocation of the nanomaterials following pulmonary exposures.

Cerium oxide and barium sulfate nanoparticle inhalation affects gene expression in alveolar epithelial cells type II.

BACKGROUND: Understanding the molecular mechanisms of nanomaterial interacting with cellular systems is important for appropriate risk assessment. The identification of early biomarkers for potential (sub-)chronic effects of nanoparticles provides a promising approach towards cost-intensive and animal consuming long-term studies. As part of a 90-day inhalation toxicity study with CeO2 NM-212 and BaSO4 NM-220 the present investigations on gene expression and immunohistochemistry should reveal details on underlying mechanisms of pulmonary effects. The role of alveolar epithelial cells type II (AEII cells) is focused since its contribution to defense against inhaled particles and potentially resulting adverse effects is assumed. Low dose levels should help to specify particle-related events, including inflammation and oxidative stress. RESULTS: Rats were exposed to clean air, 0.1, 0.3, 1.0, and 3.0 mg/m3 CeO2 NM-212 or 50.0 mg/m3 BaSO4 NM-220 and the expression of 391 genes was analyzed in AEII cells after one, 28 and 90 days exposure. A total number of 34 genes was regulated, most of them related to inflammatory mediators. Marked changes in gene expression were measured for Ccl2, Ccl7, Ccl17, Ccl22, Ccl3, Ccl4, Il-1α, Il-1ß, and Il-1rn (inflammation), Lpo and Noxo1 (oxidative stress), and Mmp12 (inflammation/lung cancer). Genes related to genotoxicity and apoptosis did not display marked regulation. Although gene expression was less affected by BaSO4 compared to CeO2 the gene pattern showed great overlap. Gene expression was further analyzed in liver and kidney tissue showing inflammatory responses in both organs and marked downregulation of oxidative stress related genes in the kidney. Increases in the amount of Ce were measured in liver but not in kidney tissue. Investigation of selected genes on protein level revealed increased Ccl2 in bronchoalveolar lavage of exposed animals and increased Lpo and Mmp12 in the alveolar epithelia. CONCLUSION: AEII cells contribute to CeO2 nanoparticle caused inflammatory and oxidative stress reactions in the respiratory tract by the release of related mediators. Effects of BaSO4 exposure are low. However, overlap between both substances were detected and support identification of potential early biomarkers for nanoparticle effects on the respiratory system. Signs for long-term effects need to be further evaluated by comparison to a respective exposure setting.

Biokinetics of Nanomaterials: the Role of Biopersistence.

Nanotechnology risk management strategies and environmental regulations continue to rely on hazard and exposure assessment protocols developed for bulk materials, including larger size particles, while commercial application of nanomaterials (NMs) increases. In order to support and corroborate risk assessment of NMs for workers, consumers, and the environment it is crucial to establish the impact of biopersistence of NMs at realistic doses. In the future, such data will allow a more refined future categorization of NMs. Despite many experiments on NM characterization and numerous in vitro and in vivo studies, several questions remain unanswered including the influence of biopersistence on the toxicity of NMs. It is unclear which criteria to apply to characterize a NM as biopersistent. Detection and quantification of NMs, especially determination of their state, i.e., dissolution, aggregation, and agglomeration within biological matrices and other environments are still challenging tasks; moreover mechanisms of nanoparticle (NP) translocation and persistence remain critical gaps. This review summarizes the current understanding of NM biokinetics focusing on determinants of biopersistence. Thorough particle characterization in different exposure scenarios and biological matrices requires use of suitable analytical methods and is a prerequisite to understand biopersistence and for the development of appropriate dosimetry. Analytical tools that potentially can facilitate elucidation of key NM characteristics, such as ion beam microscopy (IBM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS), are discussed in relation to their potential to advance the understanding of biopersistent NM kinetics. We conclude that a major requirement for future nanosafety research is the development and application of analytical tools to characterize NPs in different exposure scenarios and biological matrices.

Effects from a 90-day inhalation toxicity study with cerium oxide and barium sulfate nanoparticles in rats.

BACKGROUND: Nanomaterials like cerium oxide and barium sulfate are frequently processed in industrial and consumer products and exposure of humans and other organisms is likely. Generally less information is given on health effects and toxicity, especially regarding long-term exposure to low nanoparticle doses. Since inhalation is still the major route of uptake the present study focused on pulmonary effects of CeO2NM-212 (0.1, 0.3, 1.0, 3.0 mg/m3) and BaSO4NM-220 nanoparticles (50.0 mg/m3) in a 90-day exposure setup. To define particle-related effects and potential mechanisms of action, observations in histopathology, bronchoalveolar lavage and immunohistochemistry were linked to pulmonary deposition and clearance rates. This further allows evaluation of potential overload related effects. RESULTS: Lung burden values increased with increasing nanoparticle dose levels and ongoing exposure. At higher doses, cerium clearance was impaired, suggesting lung overload. Barium elimination was extremely rapid and without any signs of overload. Bronchoalveolar lavage fluid analysis and histopathology revealed lung tissue inflammation with increasing severity and post-exposure persistency for CeO2. Also, marker levels for genotoxicity and cell proliferation were significantly increased. BaSO4 showed less inflammation or persistency of effects and particularly affected the nasal cavity. CONCLUSION: CeO2 nanoparticles penetrate the alveolar space and affect the respiratory tract after inhalation mainly in terms of inflammation. Effects at low dose levels and post-exposure persistency suggest potential long-term effects and a notable relevance for human health. The generated data might be useful to improve nanoparticle risk assessment and threshold value generation. Mechanistic investigations at conditions of non-overload and absent inflammation should be further investigated in future studies.

Translocation and biokinetic behavior of nanoscaled europium oxide particles within 5 days following an acute inhalation in rats.

Nanoscaled europium oxide (Eu2O3) particles were inhaled by rats after acute exposure and the potential translocation of particles followed by chemical analysis and transmission electron microscopy (TEM) was investigated. An aqueous dispersion (phosphate buffer/bovine serum albumin) of a commercially available Eu2O3 particle fraction consisting partially of nanoscaled particles was aerosolized with pressurized air. After rapid evaporation, rats inhaled the dry aerosol for 6 h in a single exposure resulting in an alveolar calculated dose of approximately 39.5 µg Eu2O3. Using chemical analysis, 36.8 µg Eu2O3 was detected 1 h after lung inhalation. The amount declined slightly to 34.5 µg after 1 day and 35.0 µg after 5 days. The liver showed an increase of Eu2O3 from 32.3 ng 1 h up to 294 ng 5 days after inhalation. Additionally, lung-associated lymph nodes, thymus, kidneys, heart and testis exhibited an increase of europium over the period investigated. In the blood, the highest amount of europium was found 1 h after treatment whereas feces, urine and mesenteric lymph nodes revealed the highest amount 1 day after treatment. Using TEM analysis, particles could be detected only in lungs, and in the liver, no particles were detectable. In conclusion, the translocation of Eu2O3 within 5 days following inhalation could be determined very precisely by chemical analysis. A translocation of Eu2O3 particulate matter to liver was not detectable by TEM analysis; thus, the overproportional level of 0.8% of the lung load observed in the liver after 5 days suggests a filtering effect of dissolved europium with accumulation.

In vivo evaluation of a magnesium-based degradable intramedullary nailing system in a sheep model.

The biocompatibility and the degradation behavior of the LAE442 magnesium-based intramedullary interlocked nailing system (IM-NS) was assessed in vivo in a comparative study (stainless austenitic steel 1.4441LA) for the first time. IM-NS was implanted into the right tibia (24-week investigation period; nails/screws diameter: 9 mm/3.5 mm, length: 130 mm/15-40 mm) of 10 adult sheep (LAE442, stainless steel, n=5 each group). Clinical and radiographic examinations, in vivo computed tomography (CT), ex vivo micro-computed tomography (µCT), mechanical and histological examinations and element analyses of alloying elements in inner organs were performed. The mechanical examinations (four-point bending) revealed a significant decrease of LAE442 implant stiffness, force at 0.2% offset yield point and maximum force. Periosteal (new bone formation) and endosteal (bone decline) located bone alterations occurred in both groups (LAE442 alloy more pronounced). Moderate gas formation was observed within the LAE442 alloy group. The CT-measured implant volume decreased slightly (not significant). Histologically a predominantly direct bone-to-implant interface existed within the LAE442 alloy group. Formation of a fibrous tissue capsule around the nail occurred in the steel group. Minor inflammatory infiltration was observed in the LAE442 alloy group. Significantly increased quantities of rare earth elements were detected in the LAE442 alloy group. µCT examination showed the beginning of corrosion in dependence of the surrounding tissue. After 24 weeks the local biocompatibility of LAE442 can be considered as suitable for a degradable implant material. STATEMENT OF SIGNIFICANCE: An application oriented interlocked intramedullary nailing system in a comparative study (degradable magnesium-based LAE442 alloy vs. steel alloy) was examined in a sheep model for the first time. We focused in particular on the examination of implant degradation by means of (µ-)CT, mechanical properties (four-point bending), clinical compatibility, local bone reactions (X-ray and histology) and possible systemic toxicity (histology and element analyses of inner organs). A significant decrease of magnesium (LAE442 alloy) implant stiffness and maximum force occurred. Moderate not clinically relevant gas accumulation was determined. A predominantly direct bone-to-implant contact existed within the magnesium (LAE442 alloy) group compared to an indirect contact in the steel group. Rare earth element accumulation could be observed in inner organs but H&E staining was inconspicuous.

Beryllium Concentrations at European Workplaces: Comparison of 'Total' and Inhalable Particulate Measurements.

A field study was carried out in order to derive a factor for the conversion of historic worker exposure data on airborne beryllium (Be) obtained by sampling according to the 37-mm closed faced filter cassette (CFC) 'total' particulate method into exposure concentration values to be expected when sampling using the 'Gesamtstaubprobenahmesystem' (GSP) inhalable sampling convention. Workplaces selected to represent the different copper Be work processing operations that typically occur in Germany and the EU were monitored revealing a broad spectrum of prevailing Be size distributions. In total, 39 personal samples were taken using a 37-mm CFC and a GSP worn side by side for simultaneous collection of the 'total' dust and the inhalable particulates, respectively. In addition, 20 static general area measurements were carried out using GSP, CFC, and Respicon samplers in parallel, the latter one providing information on the extra-thoracic fraction of the workplace aerosol. The study showed that there is a linear relationship between the concentrations measured with the CFC and those measured with the GSP sampler. The geometric mean value of the ratios of time-weighted average concentrations determined from GSP and CFC samples of all personal samples was 2.88. The individual values covered a range between 1 and 17 related to differences in size distributions of the Be-containing particulates. This was supported by the area measurements showing that the conversion factor increases with increasing values of the extra-thoracic fraction covering a range between 0 and 79%.

Particle deposition in the lung of the Göttingen minipig.

We report on particle deposition in the tracheobronchial and pulmonary regions of the respiratory tract of the minipig and its dependence on particle size. Four animals breathing spontaneously via the nose were exposed for 1 h to known concentrations of three different polydisperse dry aerosols composed of bovine serum albumin (BSA) and an oxide of a rare earth element: Y2O3, Sm2O3, and Er2O3. The mass size distributions of the rare earth elements of the three test aerosols have mass median aerodynamic diameters of 0.9, 2.5, and, 4.3 microm, and geometric standard deviations of sigma(g) = 2.0, 1.8, and, 1.7. The extrathoracic, tracheobronchial, and pulmonary regions of the respiratory tract were dissected, separately lyophilized, and chemically digested by microwave-assisted high pressure digestion. The tracer element in each compartment was determined by inductively coupled plasma mass spectrometry. A mass balance equation relating the tracer mass found in the lung compartments to the tracer mass inhaled was solved by linear regression to obtain the deposition fraction as function of particle sizes for the tracheobronchial and the pulmonary lung region. Estimated values for the respiratory minute volume were used in this context. For coarse particles > 6 microm, the deposition fraction is < 5% for both compartments. The deposition fraction for particles with aerodynamic diameter of approximately 3 microm is 21% in the tracheobronchial airways and 40% in the pulmonary airways.

Use of biocidal products (insect sprays and electro-vaporizer) in indoor areas--exposure scenarios and exposure modeling.

Five commercially available insect sprays were applied in a model room. Spraying was performed in accordance with the manufacturers' instructions and in an overdosed manner in order to simulate worst-case conditions or an unforeseeable misuse. In addition, we examined electro-vaporizers. The Respicon aerosol monitoring system was applied to determine inhalation exposure. During normal spraying (10 seconds) and during the following 2-3 minutes, exposure concentrations ranged from 70 to 590 microg/m3 for the pyrethroids tetramethrin, d-phenothrin, cyfluthrin, bioallethrin, and the pyrethrins. Calculated inhalable doses were 2-16 microg. A concentration of approximately 850 microg chlorpyrifos/m(3) (inhalable dose: approximately 20 microg) was determined when the "Contra insect fly spray" was applied. Highest exposure concentrations (1100-2100 microg/m3) were measured for piperonyl butoxide (PBO), corresponding to an inhalation intake of 30-60microg. When simulating worst-case conditions, exposure concentrations of 200-3400microg/m3 and inhalable doses of 10-210microg were determined for the various active substances. Highest concentrations (4800-8000 microg/m3) were measured for PBO (inhalable: 290-480 microg). By applying the electro-vaporizer "Nexa Lotte" plug-in mosquito killer concentrations for d-allethrin were in the range of 5-12microg/m3 and 0.5-2 microg/m3 for PBO while with the "Paral" plug-in mosquito killer concentrations of 0.4-5microg/m3 for pyrethrins and 1-7 microg/m3 for PBO were measured. Potential dermal exposures were determined using exposure pads. Between 80 and 1000microg active substance (tetramethrin, phenothrin, cyfluthrin, bioallethrin, pyrethrins, chlorpyrifos) were deposited on the clothing of the total body surface area of the spray user. Highest levels (up to 3000 microg) were determined for PBO. Worst-case uses of the sprays led to 5-9 times higher concentrations. Also a 2-hour stay nearby an operating electro-vaporizer led to a contamination of the clothing (total amounts on the whole body were 450 microg d-allethrin and 50 microg PBO for "Nexa Lotte" plug-in mosquito killer and 80 microg pyrethrins and 190 microg PBO for "Paral" plug-in mosquito killer). Human biomonitoring data revealed urine concentrations of the metabolite (E)-trans-chrysanthemum dicarboxylic acid ((E)-trans-CDCA) between 1.7 microg/l and 7.1 microg/l after 5 minutes of exposure to the different sprays. Also the use of electro-vaporizers led to (E)-trans-CDCA concentrations in the urine in the range of 1.0 microg/l to 6.2 microg/l (1-3 hours exposure period). The exposure data presented can be used for performing human risk assessment when these biocidal products were applied indoors. The airborne concentrations of the non-volatile active chemical compounds could be predicted from first principles using a deterministic exposure model (SprayExpo).

Aircraft disinsection: exposure assessment and evaluation of a new pre-embarkation method.

A new "pre-embarkation" method for aircraft disinsection was investigated using two different 2% d-phenothrin containing aerosols. Five experiments in aircrafts of the type Airbus 310 (4x) and Boeing 747-400 (1x) were performed. In the absence of passengers and crew the d-phenothrin aerosol was sprayed under the seat rows and in a second step at the height of approximately 1.60 m by moving from one end of the cabin to the other. Concentration levels of d-phenothrin were determined at different time periods after application of the aerosol spray. In a B 747-400 with the air conditioning system operating the concentrations ranged between 853 and 1753 microg/m3 during and till 5 min after the beginning of spraying at different locations in the cabin. Within 5-20min after the end of the spraying concentrations of 36-205 microg/m3 and 20-40 min thereafter only ca. 1 microg d-phenothrin/m3 were detectable (average values in relation to each period of measurement). On cabin interior surfaces the median values for mainly horizontal areas ranged from 100 to 1160 ng d-phenothrin/cm2. d-Phenothrin concentrations in the air were sufficient to kill flying insects like house flies and mosquitoes within 20 min. Horizontal surfaces were 100% effective against insects up to 24 h after spraying. Doses inhaled by sprayers determined by personal measurements were calculated to be 30-235 microg d-phenothrin per 100 g spray applied (30% in the respirable fraction for Arrow Aircraft Disinsectant; 10% for Aircraft Disinsectant Denka). If passengers will board, e.g., 20 min after the end of the disinsection operation, inhalation exposure is estimated to be practically negligible. Also possible dermal exposure from residues in seats and headrests is very low for passengers during the flight. Therefore any health effects for passengers and crew members are very unlikely.

In-flight spraying in aircrafts: determination of the exposure scenario.

Exposure measurements were carried out in parked aircrafts during and after application of a biocide aerosol spray (simulated in-flight spraying). The aerosol product SRA spray (Standard Reference Aerosol Spray) was used for spraying. Concentrations of the pyrethrins--the active ingredients--in the air of the passenger cabin (airborne particles, measured during spray application and 40 minutes afterwards) varied from 11 to 65 microg/m3; those of the synergist piperonyl butoxide were 200-485 microg/m3. The concentrations on surfaces of the cabin furniture differed widely. Low concentrations were determined on surfaces in vertical positions (median values: pyrethrins < or =2 ng/cm2; piperonyl butoxide < or =17 ng/cm2), while under seats, on seats and on headrests the concentrations were up to 55.5 ng/cm2 for pyrethrins and 1162.5 ng/cm2 for piperonyl butoxide (median values). The inhaled doses for sprayers (using 100 g of spray) and persons sitting in the passenger cabin were calculated to be 17 microg for pyrethrins and 200 microg for piperonyl butoxide (maximum values). Maximum total external body doses for the applicators during spraying were 830 microg for pyrethrins and 8840 microg for piperonyl butoxide. The potential dermal dose for persons sitting in the passenger cabin was about a factor of two lower.