This research was supported by the Industrial Technology Innovation Program (10052901), Development of highly usable nanomaterial inhalation toxicity testing system in commerce, through the Korea Evaluation Institute of Industrial Technology by the Korean Ministry of Trade, Industry &#38; Energy.

1. Numerical analysis methods
<ol>
	<li>Perform the analysis of the flow field inside the chamber according to the geometrical shape, as described in Figure 1 and Table 114. 
	NOTE: A numerical analysis of the flow field according to the geometric shape predicts the flow of the aerosol and evaluates it as a testable device.</li>
	<li>Design the chamber with 4 stages x 12 columns, 48 ports in total, where the core is divided into an inner and outer tower, as described in Figure 1B. 
	NOTE: Each stage has 12 exposure ports for placing the experimental animals. Satisfy the recommendation suggested by the OECD Guidance Document (GD) 396.</li>
	<li>For the single-concentration exposure, place the mixing plate at the top of the inner tower to mix the test material and ensure a uniform concentration across the stages. For multiconcentration exposure, separate the inner tower into four stages and exposure concentrations by a separation disk. 
	NOTE: The mixing plate</li>
</ol>
2. Preparation of the experimental evaluation
<ol>
	<li>Chamber
	<ol>
		<li>Divide the chamber into three parts: the inlet, sheath, and exhaust, as shown in the schematic diagram (Figure 2). 
		NOTE: The inlet is where the aerosol flows into the inner chamber, and the sheath is the space between the inner and outer towers for extra air flow.</li>
		<li>Supply the aerosol (or test article) to the inner tower and experimental animals, while the exhalation from the animals that contains surplus aerosol flows out through the exhaust along with the sheath air. 
		NOTE: The animal holders are closed and empty.</li>
		<li>Keep the inner pressure of the chamber constant using a blower and inverter, as the inner plenum pressure is controlled by the sheath air flow.</li>
		<li>Design equipment to measure the uniformity of the test aerosol (or article) concentration in the mixing chamber located in front of the nose-only exposure chamber in the case of single-concentration exposure. 
		NOTE: The uniformity of the test aerosol can be evaluated by its particle number concentration and size distribution. Individual chamber concentration samples should deviate from the mean chamber concentration by no more than &#177;10% for gases and vapors, and by no more than &#177;20% for liquid or solid aerosols4,5,6,7,8. Thus, when the test particles are not constant, the aerosol flow can be bypassed through the exhaust fan.</li>
		<li>Check for leakages to verify the reliability of the test and ensure safety by confirming a closed system with &#177;500 Pa that is maintained for 30 min. 
		NOTE: The leakage can be checked by soap bubbling.</li>
	</ol>
	</li>
	<li>Environmental control and monitoring
	<ol>
		<li>Set the total inflow rate of the aerosol (single/multi) and sheath air at 48 L/min or 44 L/min (single or multi, respectively) and 20 L/min, respectively, and keep the inner pressure of the chamber constant at &#8722;100 Pa in the Control settings of the User Interface.</li>
		<li>Maintain the temperature and humidity at 23 &#176;C and 45%, respectively. Use a humidifier to control the humidity of the exposure air.</li>
		<li>Carry out an experiment in an isothermal-isohumidity-controlled environment to comply with the OECD inhalation toxicity guidelines4,6,7,8.</li>
	</ol>
	</li>
	<li>Flow uniformity measurement
	<ol>
		<li>Supply 48 L/min clean air to the inhalation chamber through a clean air supply including a HEPA filter controlled by a mass flow controller (MFC). 
		NOTE: The clean air is made after filtering it with a HEPA filter.</li>
		<li>Stabilize the flow using the mixing chamber in the case of single-concentration exposure.</li>
		<li>Attach a supply nozzle to one port that injects fresh control air or the test aerosol (or article) in the case of multiconcentration exposure.</li>
		<li>Measure the flow velocity per port using a mass flow meter.</li>
	</ol>
	</li>
	<li>Particle generation
	<ol>
		<li>Generate NaCl nanoparticles using a five-jet atomizer to evaluate the inhalation chamber design. 
		NOTE: Use a 0.1%wt NaCl solution to generate the NaCl nanoparticles.</li>
		<li>Regulate the MFC to control the amount of production at 48 L/min of the NaCl aerosol mixed air in the single concentration and at 12 L/min of the NaCl aerosol-mixed air in the multiconcentration every four stages. 
		NOTE: Every port of the nose-only chamber receives 1 L/min (i.e., 48 ports/nose-only chamber (four-stage); 48 ports/four-stage; 12 ports/stage).</li>
		<li>Supply clean air for dilution in the bypass. 
		NOTE: The count median diameter and geometric standard deviation of NaCl nanoparticles are within 76 nm and 1.4 maintain, respectively.</li>
	</ol>
	</li>
	<li>Particle uniformity measurement
	<ol>
		<li>Measure the particle size distribution of the NaCl nanoparticles emitted from the injection nozzles using a scanning mobility particle sizer (SMPS) composed of a differential mobility analyzer (DMA) and a condensation particle counter (CPC).</li>
		<li>Use an Am aerosol neutralizer to remove the static charge of the particles and reduce the particle deposition on the walls, thereby improving the measurement efficiency18.</li>
		<li>Maintain the ratio of the aerosol and sheath air flow rate of the DMA at 1:10 to keep the aerosol flow rate and sheath air flow rate at 1 L/min and 10 L/min, respectively.</li>
	</ol>
	</li>
</ol>
3. Flow uniformity test
<ol>
	<li>Multi-concentration exposure
	<ol>
		<li>Set the flow velocity of the injection nozzles by supplying clean air at 11 L/min through the aerosol inlet. Select 11 port nozzles for each the four stages.</li>
		<li>Measure the flow rate to connect the flow meter to the selected nozzle.</li>
		<li>Repeat step 3.1.2 3x to verify the reproducibility.</li>
	</ol>
	</li>
	<li>Single-concentration exposure
	<ol>
		<li>Set the flow velocity of the injection nozzles by supplying clean air at 48 L/min through the aerosol inlet. Randomly select 24 port nozzles among the 48 ports. Measure 3x to verify the reproducibility.</li>
	</ol>
	</li>
</ol>
4. Particle uniformity test
<ol>
	<li>Multiconcentration exposure
	<ol>
		<li>Set the particle size distribution of the injection nozzles by supplying the generated particles at 11 L/min through the aerosol inlet (do this as described in section 2).</li>
		<li>Randomly selected six port nozzles among the four stages; measure 3x to verify the reproducibility.</li>
	</ol>
	</li>
	<li>Single-concentration exposure
	<ol>
		<li>Set the particle size distribution of the injection nozzles by supplying the generated particles at 20 L/min and clean air at 28 L/min, making a total of 48 L/min through the aerosol inlet (as described in 2.4 and 2.5).</li>
		<li>Randomly select six port nozzles among the four stages.</li>
		<li>Measure the particle concentration, to connect the SMPS to the selected nozzle.</li>
		<li>Repeat step 4.2.3 3x to verify the reproducibility.</li>
	</ol>
	</li>
</ol>
5. Cross-contamination test
<ol>
	<li>Set three stages in the case of multiconcentration exposure.</li>
	<li>Connect two generators with different solution concentrations and a clean air line to the three respective stages.</li>
	<li>Set the particle size distribution of the injection nozzles by supplying the generated particles and clean air at 11 L/min through the aerosol inlet (as described in 2.4 and 2.5).</li>
	<li>Randomly selected one port nozzle from all three stages.</li>
	<li>Measure the particle concentration, to connect the SMPS to the selected port.</li>
	<li>Repeat step 5.5 15x to verify the reproducibility.</li>
</ol>

<ol>
	<li>Phalen, R. F., Phalen, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Inhalation+Toxicology.">Methods in Inhalation Toxicology.</a> Inhalation Exposure Methods. , 69-84 (1997).</li><li>Moss, O. R., James, R. A., Asgharian, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+exhaled+air+on+inhalation+exposure+delivered+through+a+directed-flow+nose-only+exposure+system.">Influence of exhaled air on inhalation exposure delivered through a directed-flow nose-only exposure system.</a> Inhalation Toxicology. 18, 45-51 (2006).</li><li>White, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluid Mechanics. , (2004).</li><li>OECD TG 403. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD guideline of the testing of chemicals 403: Acute inhalation toxicity testing. , (2009).</li><li>OECD TG 436. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD guideline of the testing of chemicals 436: Acute inhalation toxicity - Acute Toxic Class Method. , (2009).</li><li>OECD GD 39. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Series on testing and assessment Number 39: Guidance document on acute Inhalation toxicity testing. , (2009).</li><li>OECD TG 412. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD guideline of the testing of chemicals 412: Subacute inhalation toxicity testing. , (2018).</li><li>OECD TG 413. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> OECD guideline of the testing of chemicals 413: Subchronic inhalation toxicity testing. , (2018).</li><li>Cannon, W. C., Blanton, E. F., McDonald, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flow-past+chamber:+an+improved+nose-only+exposure+system+for+rodents.">The flow-past chamber: an improved nose-only exposure system for rodents.</a> American Industrial Hygiene Association Journal. 44, 923-928 (1983).</li><li>Oldham, M. J., Phalen, R. F., Robinson, R. J., Kleinman, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Performance+of+a+portable+whole-body+mouse+exposure+system.">Performance of a portable whole-body mouse exposure system.</a> Inhalation Toxicology. 16, 657-662 (2004).</li><li>Oldham, M. J., Phalen, R. F., Budiman, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Predicted+and+Experimentally+Measured+Aerosol+Deposition+Efficiency+in+BALB/C+Mice+in+a+New+Nose-Only+Exposure+System.">Comparison of Predicted and Experimentally Measured Aerosol Deposition Efficiency in BALB/C Mice in a New Nose-Only Exposure System.</a> Aerosol Science and Technology. 43, 970-997 (2009).</li><li>Tuttle, R. S., Sosna, W. A., Daniels, D. E., Hamilton, S. B., Lednicky, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+assembly,+and+validation+of+a+nose-only+inhalation+exposure+system+for+studies+of+aerosolized+viable+influenza+H5N1virus+in+ferrets.">Design, assembly, and validation of a nose-only inhalation exposure system for studies of aerosolized viable influenza H5N1virus in ferrets.</a> Virology Journal. 7, 135 (2010).</li><li>Jeon, K., Yu, I. J., Ahn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+newly+developed+nose-only+inhalation+exposure+chamber+for+nanoparticles.">Evaluation of newly developed nose-only inhalation exposure chamber for nanoparticles.</a> Inhalation Toxicology. 24 (9), 550-556 (2012).</li><li>Ji, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twenty-Eight-Day+Inhalation+Toxicity+Study+of+Silver+Nanoparticles+in+Sprague-Dawley+Rats.">Twenty-Eight-Day Inhalation Toxicity Study of Silver Nanoparticles in Sprague-Dawley Rats.</a> Inhalation Toxicology. 19, 857-871 (2007).</li><li>Ostraat, M. L., Swain, K. A., Krajewski, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SiO2+Aerosol+Nanoparticle+Reactor+for+Occupational+Health+and+Safety+Studies.">SiO2 Aerosol Nanoparticle Reactor for Occupational Health and Safety Studies.</a> Journal of Occupational and Environmental Hygiene. 5, 390-398 (2008).</li><li>Pauluhn, J., Thiel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+approach+to+validation+of+directed-flow+nose-only+inhalation+chambers.">A simple approach to validation of directed-flow nose-only inhalation chambers.</a> Journal of Applied Toxicology. 27, 160-167 (2007).</li></ol>

The authors have nothing to disclose.

Inhalation toxicity testing is currently the best method for evaluating aerosolized materials (particles and fibers), vapors, and gases inhaled by the human respiratory system14,15. There are two inhalation exposure methods: whole-body and nose-only. However, a nose-only system minimizes exposure by noninhalation routes, such as skin and eyes, and allows testing with minimal quantities of the test article, making it the preferred exposure method recommended by th...

Inhalation toxicity testing is the most reliable method for assessing the risks of chemical agents, particles, fibers, and nanomaterials1,2,3. Thus, most regulatory agencies require the submission of inhalation toxicity testing data when the exposure to chemicals, particles, fibers, and nanomaterials is via inhalation4,5,6,7,8. Currently, there are two types of inhalation toxicity systems: whole-body and nose-only exposure systems. A standard inhalation toxicity test system, either whole-body or nose-only, requires at least four chambers to expose animals such as rats and mice to four different concentrations, namely fresh air control and low, moderate, and high concentrations7,8. The Organization for Economic Co-operation and Development (OECD) test guidelines suggest that the selected target concentration should allow the identification of the target organ(s) and demonstration of a clear concentration response7,8. The high concentration level should result in a clear level of toxicity but not cause mortality or persistent signs that might lead to death or prevent a meaningful evaluation of the results7,8. The maximum achievable level or high concentration of the aerosols can be reached while meeting the particle size distribution standard. The moderate concentration level(s) should be spaced to produce a gradation of toxic effects between that of the low and high concentrations7,8. The low concentration level, which would preferably be a NOAEC (no-observed-adverse-effect concentration), should produce little or no sign of toxicity7,8. The whole-body chamber exposes animals in an unrestrained condition in wired cages, while the nose-only chamber exposes an animal in a restrained condition in the confined tube. The restraint prevents loss of aerosol by leakage around the animal. Due to the high volume of the whole-body chamber, it requires a large number of test articles to be exposed to experimental animals, while the restraint of the tube in the nose-only exposure system hinders animal movement and may cause discomfort or suffocation. Nevertheless, the regulatory OECD inhalation toxicity test guidelines prefer the use of nose-only inhalation systems4,5,6,7,8.
However, accommodating a four-chamber system, either whole-body or nose-only, is expensive, space-consuming, and requires a built-in air cleaning and circulation system. Furthermore, a four-chamber system can also require separate test article generators to expose animals to the desired concentrations, and a separate measurement apparatus to monitor the test article concentrations. Therefore, since standard inhalation toxicity testing involves significant investment, a more convenient and economical whole-body or nose-only exposure system needs to be developed for use in small research facilities. When designing an inhalation chamber, computational fluid dynamics (CFD) modeling is also frequently used to achieve particle, gas, or vapor uniformity9,10,11,12,13. Evaluation by numerical analyses and validation by experimental results has already been performed for the whole-body exposure chamber for mice10. For example, the air flow and particle trajectory have been modeled using CFD, and the uniformity of particle distribution has been measured in nine parts of the whole-body chamber10. Also, the nose-only chamber has been evaluated by numerical analysis by CFD13. After that, evaluation for the nose-only exposure chamber was performed by comparing the numerical analysis results with an experimental study using nanoparticles13.
This study presents a nose-only inhalation chamber system that can expose experimental animals to four different concentrations in one chamber. Initially designed using CFD and a numerical analysis, the proposed system is then compared with an experimental study using nanoscale sodium chloride particles to validate the uniformity and cross-contamination. The results presented here indicate that the presented nose-only chamber that can expose animals to four different concentrations can be used for animal exposure studies in small-scale academic and research facilities. The numerical analysis is set as follows, in the same manner as the experiment setting. For single-concentration exposure, the aerosol flow to the inner tower is set to 48 L/min and the sheath flow to the outer tower is set to 20 L/min. For multiconcentration exposure, the aerosol flow to the inner tower input is 11 L/min for each stage. The outlet differential pressure keeps at -100 Pa to maintain a smooth exhaust flow and prevent leakage. Assume the animal holders are closed and empty.

<table><tbody><tr><td>FLUENT V.17.2&amp;nbsp;</td><td>ANSYS</td><td></td><td>Software</td></tr><tr><td>mass flow meter (MFM)</td><td>TSI</td><td>4043</td><td></td></tr><tr><td>SMPS (scanning mobility particle sizer)</td><td>Grimm&amp;nbsp;</td><td>SMPS+C</td><td></td></tr><tr><td>5-Jet atomizer&amp;nbsp;</td><td>HCTM</td><td>5JA-1000</td><td></td></tr><tr><td>Mass flow controller (MFC)</td><td>Horiba</td><td>S48-32</td><td></td></tr></tbody></table>

development of a nose-only inhalation toxicity test chamber that provides four exposure concentrations of nano-sized particles

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is designed and validated for flow field uniformity and cross-contamination among the exposure ports for each concentration. The designed flow field values are compared with the measured values from exposure ports located horizontally and vertically. For this purpose, nanoscale sodium chloride particles are generated as test particles and introduced to the inhalation chamber to evaluate the cross-contamination and concentration maintenance among the chambers, for each concentration group. The results indicate that the designed multiconcentration inhalation chamber can be used in animal inhalation toxicity testing without cross-contamination among concentration groups. Moreover, the designed multiconcentration inhalation toxicity chamber can also be converted to a single-concentration inhalation chamber. Further testing with gas, organic vapor, or non-nanoscale particles will ensure the use of the chamber in the inhalation testing of other test articles.

Experimental set-up
Figure 1 shows a schematic diagram of a nose-only inhalation chamber system, including a particle generator with an MFC, nose-only chamber, and particle measurement instrument for monitoring the air quality, controller, and exhaust module, based on section 2 of the protocol.
Numerical analysis design
<p class="jove_conten...

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Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

A nose-only inhalation toxicity chamber capable of testing inhalation toxicity at four different exposure concentrations was designed and validated for flow field uniformity and cross-contamination between the exposure ports for each concentration. Here, we present a protocol to confirm that the designed chamber is effective for inhalation toxicity testing.

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is ...

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6.3K Views.  Inha University. A nose-only inhalation toxicity chamber capable of testing inhalation toxicity at four different exposure concentrations was designed and validated for flow field uniformity and cross-contamination between the exposure ports for each concentration. Here, we present a protocol to confirm that the designed chamber is effective for inhalation toxicity testing.

Video: Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

A System to Create Stable Nanoparticle Aerosols from Nanopowders

Nanoparticle aerosols released from nanopowders in workplaces are associated with human exposure and health risks. We developed a novel system, requiring minimal amounts of test materials (min. 200 mg), for studying powder aerosolization behavior and aerosol properties. The aerosolization procedure follows the concept of the fluidized-bed process, but occurs in the modified volume of a V-shaped aerosol generator. The airborne particle number concentration is adjustable by controlling the air flow rate. The system supplied stable aerosol generation rates and particle size distributions over long periods (0.5-2 hr and possibly longer), which are important, for example, to study aerosol behavior, but also for toxicological studies. Strict adherence to the operating procedures during the aerosolization experiments ensures the generation of reproducible test results. The critical steps in the standard protocol are the preparation of the material and setup, and the aerosolization operations themselves. The system can be used for experiments requiring stable aerosol concentrations and may also be an alternative method for testing dustiness. The controlled aerosolization made possible with this setup occurs using energy inputs (may be characterized by aerosolization air velocity) that are within the ranges commonly found in occupational environments where nanomaterial powders are handled. This setup and its operating protocol are thus helpful for human exposure and risk assessment.

We designed and developed an effective nanopowder aerosolization setup and operating protocol. The system generated nanoparticle aerosols with stable number concentrations and size distributions for long durations, requiring only small quantities of test material (min. 200 mg).

Nanoparticle aerosols released from nanopowders in workplaces are associated with human exposure and health risks. We developed a novel system, requiring ...

Testing of Nanoparticle Release from a Composite Containing Nanomaterial Using a Chamber System

With the rapid development of nanotechnology as one of the most important technologies in the 21st century, interest in the safety of consumer products containing nanomaterials is also increasing. Evaluating the nanomaterial release from products containing nanomaterials is a crucial step in assessing the safety of these products, and has resulted in several international efforts to develop consistent and reliable technologies for standardizing the evaluation of nanomaterial release. In this study, the release of nanomaterials from products containing nanomaterials is evaluated using a chamber system that includes a condensation particle counter, optical particle counter, and sampling ports to collect filter samples for electron microscopy analysis. The proposed chamber system is tested using an abrasor and disc-type nanocomposite material specimens to determine whether the nanomaterial release is repeatable and consistent within an acceptable range. The test results indicate that the total number of particles in each test is within 20% from the average after several trials. The release trends are similar and they show very good repeatability. Therefore, the proposed chamber system can be effectively used for nanomaterial release testing of products containing nanomaterials.

Nanoparticle release is tested using a chamber system that includes a condensation particle counter, an optical particle counter and sampling ports to collect filter samples for microscopy analysis. The proposed chamber system can be effectively used for nanomaterial release testing with a repeatable and consistent data range.

With the rapid development of nanotechnology as one of the most important technologies in the 21st century, interest in the safety of consumer products ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

Environment

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Bioengineering

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

Chemistry

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Medicine

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Biology

Symptom Assessment of Patients with Allergic Rhinitis Using an Allergen Exposure Chamber

Allergen exposure chambers (AECs) are clinical facilities that allow the exposure of participants to allergenic and non-allergenic airborne particles. They provide stable particle concentrations under controlled environmental conditions. This is of great importance both for diagnostic purposes and for the monitoring of treatment effects. 
Here, a protocol and the technical prerequisites for performing a safe and effective allergen challenge in subjects sensitized to airborne allergens (i.e., house dust mite [HDM]) in the ALL-MED AEC are presented. With this method, triggering allergic symptoms corresponds to natural exposure. This can be used for an allergy diagnosis or as a plausible endpoint in clinical trials, particularly for allergen immunotherapy (AIT). A controlled environment (temperature, humidity, and carbon dioxide [CO2]) in the chamber must be maintained. Allergen particles must be dispersed evenly within the AEC at stable levels throughout the challenge. For this presentation, allergic rhinitis (AR) patients sensitive to HDM allergens were enrolled. AR symptoms were assessed by the following parameters: total nasal symptom score (TNSS), acoustic rhinometry (ARM), peak nasal inspiratory flow (PNIF), and nasal secretion weight. The safety of the procedure was assessed by the peak expiratory flow rate (PEFR) and the forced expiratory volume in the first second (FEV1). The allergic subjects developed symptoms within 120 min of the trial. On average, the most intense symptoms appeared after 60-90 min and, after reaching a plateau, remained stable until the end of the trial.

A protocol for conducting a challenge in an allergen exposure chamber (AEC) facility is presented. AECs have proven to be safe and effective tools for the induction of allergic symptoms or as an endpoint in the efficacy testing of allergen immunotherapy due to their ability to maintain stable particle concentrations and environmental conditions.

Allergen exposure chambers (AECs) are clinical facilities that allow the exposure of participants to allergenic and non-allergenic airborne particles. ...

Immunology and Infection

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Summary

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Chapters in this video

A System to Create Stable Nanoparticle Aerosols from Nanopowders

Testing of Nanoparticle Release from a Composite Containing Nanomaterial Using a Chamber System

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Symptom Assessment of Patients with Allergic Rhinitis Using an Allergen Exposure Chamber

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing