Name: development of a nose-only inhalation toxicity test chamber that provides four exposure concentrations of nano-sized particles
Uploaded: 2019-03-18T12:30:00
Description: Watch this Scientific Journal Video about Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles at JoVE.com

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 ou...

<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>

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.

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			<td height="21" style="height:21px;width:216px;">FLUENT V.17.2&#160;</td>
			<td style="width:71px;">ANSYS</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mass flow meter (MFM)</td>
			<td style="width:71px;">TSI</td>
			<td align="right" style="width:119px;">4043</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SMPS (scanning mobility particle sizer)</td>
			<td style="width:71px;">Grimm&#160;</td>
			<td style="width:119px;">SMPS+C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">5-Jet atomizer&#160;</td>
			<td style="width:71px;">HCTM</td>
			<td style="width:119px;">5JA-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mass flow controller (MFC)</td>
			<td style="width:71px;">Horiba</td>
			<td style="width:119px;">S48-32</td>
			<td></td>
		</tr>
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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
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Watch this Scientific Journal Video about Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles at JoVE.com

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 ...

We present a test protocol that complies with the OECD Guidelines for inhalation toxicity testing for single-and multiple-concentration exposure. The multiple-concentration inhalation chamber, which can test up to four exposure concentrations at once, will be more economical for small research institutes. To begin testing a single-concentration chamber, seal the chamber and check it for leaks at 500 and negative 500 pascals gauge for 30 minutes each.Then, set the clean air temperature to 23 degrees Celsius and 45%relative humidity. Start flowing clean air through the aerosol inlet and the sheath at 48 and 20 liters per minute, respectively. Set the exhaust pressure differential to negative 100 pascals.Once the flow has stabilized, measure the flow velocities at three randomly selected ports from each level to confirm that the flow is uniform. The flow rate should be about one liter per minute. Repeat the measurements twice more.Next, stop the air flow, and load a 0.1%by weight sodium chloride solution into the aerosol generation system. Start aerosol generation at 20 liters per minute with clean air as the carrier gas. Dilute the aerosol with 28 liters per minute of clean air.Set up a particle size measurement system, and randomly select three ports from each level of the chamber. Measure the particle size distribution at each selected port three times. To begin setting up a multi-concentration chamber, check it for leaks and set the clean air supply to 23 degrees Celsius and 45%relative humidity.Set the exhaust to maintain a differential pressure of negative 100 pascals. Then, connect a clean air supply to a port in the top compartment, and begin flowing clean air through the compartment at 11 liters per minute. Once the flow has stabilized, measure the flow velocity at each available port of the top compartment three times.Repeat this process for the other three compartments. Then, load a 0.1%by weight sodium chloride solution into the aerosol generator. Configure the aerosol dilution system to produce an aerosol flow at 11 liters per minute.Connect the aerosol inlet to the top compartment, wait for the flow to stabilize, and randomly select six ports from that compartment. Measure the particle size distribution at each of those ports three times using the particle size measurement system. Repeat this process for each compartment.Flush the compartments with clean air for 30 minutes when complete. Then, connect a clean air inlet to the top compartment, and flow clean air through it at 11 liters per minute. Configure the aerosol dilution system for low-concentration and high-concentration aerosol flows at 11 liters per minute.Connect the low-concentration line to the second highest compartment and the high-concentration line to the bottom compartment. Wait 10 minutes for the flows to stabilize. Then, randomly select one port from each of the three compartments, and measure the aerosol concentration at the selected ports 15 times each to check for cross-contamination.Both the single-concentration and multi-concentration exposure chambers showed good flow uniformity at each horizontal level of the chamber. The normalized flows were close to the values calculated during modeling of the system. Good flow uniformity was also observed vertically in the single-concentration chamber.The multi-concentration chamber has only one level of ports per compartment, so direct vertical uniformity measurements were not applicable. The particle concentration was also uniform horizontally for both chambers, and was uniform vertically for the single-concentration chamber. The aerosol concentrations in different compartments of the multi-concentration chamber showed relatively little variation across 15 measurements, indicating minimal cross-contamination between compartments.It is important to check for reproducibility throughout the procedure, especially during the experimental cross-contamination test performance evaluation test. Performance evaluation tests can be extended by comparing the experimental result to computational fluid dynamics modeling of the chamber system.

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