Lobar evenness of deposition/retention in rat lungs of inhaled silver nanoparticles: an approach for reducing animal use while maximizing endpoints.

BACKGROUND: Information on particle deposition, retention and clearance are important for the evaluation of the risk of inhaled nanomaterials to human health. Recent revised OECD inhalation toxicity test guidelines require to evaluate the lung burden of nanomaterials after rodent subacute and subchronic inhalation exposure (OECD 412, OECD 413). These revised test guidelines require additional post-exposure observation (PEO) periods that include lung burden measurements that can inform on lung clearance behavior and translocation. The latter being particularly relevant when the testing chemical is a solid poorly soluble nanomaterial. Therefore, in the spirit of 3 R's, we investigated whether measurement of retained lung burden of inhaled nanoparticles (NPs) in individual lung lobes is sufficient to determine retained lung burden in the total lung. If it is possible to use only one lobe, it will reduce animal use and maximize the number of endpoints evaluated. RESULTS: To achieve these goals, rats were exposed nose-only for 1 or 5 days (6 h/day) to an aerosol of 20 nm well-dispersed silver nanoparticles (AgNPs), which is the desired particle diameter resulting in maximum deposition in the pulmonary region when inhaled as singlets. After exposure, the five lung lobes were separated and silver concentration was measured using inductively coupled plasma-mass spectrophotometer (ICP-MS). The results showed that the retention of deposited silver nanoparticle in the different lung lobes did not show any statistically significant difference among lung lobes in terms of silver mass per gram lung lobe. This novel finding of evenness of retention/deposition of inhaled 20 nm NPs in rats for all five lobes in terms of mass per unit tissue weight contrasts with earlier studies reporting greater apical lobe deposition of inhaled micro-particles in rodents. The difference is most likely due to preferred and efficient deposition of inhaled NPs by diffusion vs. additional deposition by sedimentation and impaction for micron-sized particles. CONCLUSION: AgNPs following acute inhalation by rats are evenly retained in each lung lobe in terms of mass per unit lung tissue weight. Accordingly, we suggest sampling any of the rat lung lobes for lung burden analysis can be used to determine deposited or retained total lung burden after short-term inhalation of NPs and using the other lobes for collecting and analyzing bronchoalveolar lavage fluid (BALF) and for histopathological analysis. Therefore, by combining lung burden measurement, histopathological tissue preparation, and BALF assay in the same rat will reduce the number of animals used and maximize the number of endpoints measured.

On-Site Deployment of an Air-Liquid-Interphase Device to Assess Health Hazard Potency of Airborne Workplace Contaminants: The Case of 3-D Printers.

Biomonitoring of workers is an approach of evaluating workers' exposure to chemicals and particulate matter by measuring biomarkers of parent chemicals, their metabolites, and reaction products in workers' biospecimens. Prerequisites for biological monitoring in the workplace include permission to enter the workplace, approval of the study plan from the IRB (Institutional Review Board), and obtaining consent from workers. Because of the complex legal process involved in biomonitoring, few studies have been conducted so far on biomonitoring of workers' exposures to nanoparticles and other hazards from emerging materials and advanced nanotechnologies. We have developed a cell-based biomonitoring device that can evaluate acute cytotoxicity and various other effect biomakers, such as inflammation, at realistic workplace exposure. This device is based on air-liquid interphase (ALI) and can be used to evaluate cell toxicity and early effect biomarkers along adverse outcome pathways. Following exposure of A549 lung epithelial cells in ALI to workplace air for 1-2 h, the cells were processed to assess the induction of inflammatory and cell damage biomarkers. Initially, we estimated the deposition rate of nanoparticles in the transwell by exposing the cell-free ALI device to silver nanoparticle aerosols (AgNP 20-30 nm) for 2 h in the laboratory. Then A549 lung epithelial cells cultured on the transwell in the ALI device were exposed to AgNP nanoaerosols for 2 h and evaluated for cytotoxicity and induction of mRNAs of pro-inflammatory cytokines IL-1b, IL-6, and TNF-α. Then the cells in the ALI device were exposed to 3-D printer emissions at the workplace and evaluated for the same matched endpoints. The mRNA levels for IL-1b, IL-6, and TNF-α increased significantly at the end of 2-h exposure of A549 cells to the positive control AgNP aerosols. These mRNAs, as well as LDH and microprotein concentrations, increased even more after 24-h post-exposure incubation (p < 0.05). Cytotoxicity evaluation of 3-D printer emissions at 810 and 957 µg/m3, which was more than 80 times higher than the airborne total suspended particulate concentrations in the workplace air (9-12.5 µg/m3), suggested no significant acute cytotoxicity at the end of 2-h exposure to 3-D-printing emission, as well as at 24-h post-exposure incubation. Hyperspectral microscopic observation showed that 3-D printers emitted particles to be attached to A549 cells after 2-h exposure, and many particles were internalized by A549 cells after 24 h of post-exposure incubation. The mRNA expression of pro-inflammatory cytokine IL-1b and IL-6 increased significantly after 2-h exposure to 3-D printer emissions and after 24-h incubation (only IL-6). In contrast, the expression of TNF-α mRNA decreased significantly after 2 h of exposure to 3-D printers and decreased even more after 24-h post-exposure incubation. These results support the use of cell-based ALI devices for direct assessment of airborne hazards in the workplace, for probing toxicological properties of airborne contaminants using adverse molecular pathways, and for guiding study design for workplace biomonitoring. ALI devices can bridge conventional exposure assessment with cellular toxicity testing platforms for hazard and risk assessment.

Assessment and Mitigation of Exposure of 3-D Printer Emissions.

This study monitored particulates, and volatile organic compounds (VOCs) emitted from 3-D printers using acrylonitrile-butadiene-styrene copolymer (ABS) filaments at a workplace to assess exposure before and after introducing exposure mitigation measures. Air samples were collected in the printing room and adjacent corridor, and real-time measurements of ultrafine and fine particle were also conducted. Extensive physicochemical characterizations of 3-D printer emissions were performed, including real-time (size distribution, number concentration) nanoparticle characterization, size-fractionated mass distribution and concentration, as well as chemical composition for metals by ICP-MS and VOCs by GC-FID, real-time VOC monitors, and proton-transfer-reaction time-of-flight mass spectrometer (PTR-TOF-MS). Air sampling showed low levels of total suspended particulates (TSP, 9-12.5/m3), minimal levels (1.93-4 ppm) of total volatile organic chemicals (TVOC), and formaldehyde (2.5-21.7 ppb). Various harmful gases, such as formaldehyde, acrolein, acetone, hexane, styrene, toluene, and trimethylamine, were detected at concentrations in the 1-100 ppb by PTR-TOF-MS when air sample was collected into the Tedlar bag from the front of the 3-D printer. Ultrafine particles having an average particle size (30 nm count median diameter and 71 nm mass median diameter) increased during the 3-D printing operation. They decreased to the background level after the 3-D printing operation, while fine particles continually increased after the termination of 3-D printing to the next day morning. The exposure to 3-D printer emissions was greatly reduced after isolating 3-D printers in the enclosed space. Particle number concentration measured by real-time particle counters (DMAS and OPC) were greatly reduced after isolating 3-D printers to the isolated place.

28-Day inhalation toxicity study with evaluation of lung deposition and retention of tangled multi-walled carbon nanotubes.

Lung deposition and retention measurements are now required by the newly revised OECD inhalation toxicity testing guidelines 412 and 413 when evaluating the clearance and biopersistence of poorly soluble nanomaterials, such as multi-walled carbon nanotubes (MWCNTs). However, evaluating the lung deposition concentration is challenging with certain nanomaterials, such as carbon-based and iron-based nanomaterials, as it is difficult to differentiate them from endogenous elements. Therefore, the current 28-day inhalation toxicity study investigated the lung retention kinetics of tangled MWCNTs. Male Sprague Dawley rats were exposed to MWCNTs at 0, 0.257, 1.439, and 4.253 mg/m3 for 28 days (6 h/day, 5 days/week, 4 weeks). Thereafter, the rats were sacrificed at day 1, 7, and 28 post-exposure and the pulmonary inflammatory response evaluated by analyzing the bronchoalveolar lavage fluid. Plus, the blood biochemistry, hematology, and histopathology of the lungs were also examined. The lung deposition and retention of MWCNTs were determined based on the elemental carbon content in the lungs after tissue digestion. The number of polymorphonuclear cells and LDH concentration were both found to be significantly higher with the medium and high concentrations (1.439 and 4.253 mg/m3) and dose dependent. The estimated retention half-life for the high concentration (4.253 mg/m3) was about 35 days. The results of this study indicate that tangled MWCNTs seem to have a relatively shorter retention half-life when compared to previous reports on rigid MWCNTs, and the no-observed adverse effect level (NOAEL) for the tested tangled MWCNTs was 0.257 mg/m3 in a previous rat 28-day subacute inhalation toxicity study.