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

Summary

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.

Abstract

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.

Introduction

Inhalation toxicity testing is the most reliable method for assessing the risks of chemical agents, particles, fibers, and nanomaterials^1,2,3. Thus, most regulatory agencies require the submission of inhalation toxicity testing data when the exposure to chemicals, particles, fibers, and nanomaterials is via inhalation^4,5,6,7,8. 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 concentrations^7,8. 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 response^7,8. 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 results^7,8. 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 concentrations^7,8. The low concentration level, which would preferably be a NOAEC (no-observed-adverse-effect concentration), should produce little or no sign of toxicity^7,8. 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 systems^4,5,6,7,8.

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 uniformity^{9,10,11,12,13}. 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.

Protocol

1. Numerical analysis methods

1. 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.
2. 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.
3. 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

2. Preparation of the experimental evaluation

1. Chamber
   1. 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.
   2. 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.
   3. 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.
   4. 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 ±10% for gases and vapors, and by no more than ±20% for liquid or solid aerosols^4,5,6,7,8. Thus, when the test particles are not constant, the aerosol flow can be bypassed through the exhaust fan.
   5. Check for leakages to verify the reliability of the test and ensure safety by confirming a closed system with ±500 Pa that is maintained for 30 min.
      NOTE: The leakage can be checked by soap bubbling.
2. Environmental control and monitoring
   1. 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 −100 Pa in the Control settings of the User Interface.
   2. Maintain the temperature and humidity at 23 °C and 45%, respectively. Use a humidifier to control the humidity of the exposure air.
   3. Carry out an experiment in an isothermal-isohumidity-controlled environment to comply with the OECD inhalation toxicity guidelines^4,6,7,8.
3. Flow uniformity measurement
   1. 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.
   2. Stabilize the flow using the mixing chamber in the case of single-concentration exposure.
   3. Attach a supply nozzle to one port that injects fresh control air or the test aerosol (or article) in the case of multiconcentration exposure.
   4. Measure the flow velocity per port using a mass flow meter.
4. Particle generation
   1. 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.
   2. 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).
   3. 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.
5. Particle uniformity measurement
   1. 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).
   2. 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.
   3. 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.

3. Flow uniformity test

1. Multi-concentration exposure
   1. 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.
   2. Measure the flow rate to connect the flow meter to the selected nozzle.
   3. Repeat step 3.1.2 3x to verify the reproducibility.
2. Single-concentration exposure
   1. 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.

4. Particle uniformity test

1. Multiconcentration exposure
   1. 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).
   2. Randomly selected six port nozzles among the four stages; measure 3x to verify the reproducibility.
2. Single-concentration exposure
   1. 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).
   2. Randomly select six port nozzles among the four stages.
   3. Measure the particle concentration, to connect the SMPS to the selected nozzle.
   4. Repeat step 4.2.3 3x to verify the reproducibility.

5. Cross-contamination test

1. Set three stages in the case of multiconcentration exposure.
2. Connect two generators with different solution concentrations and a clean air line to the three respective stages.
3. 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).
4. Randomly selected one port nozzle from all three stages.
5. Measure the particle concentration, to connect the SMPS to the selected port.
6. Repeat step 5.5 15x to verify the reproducibility.

Results

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

Discussion

Inhalation toxicity testing is currently the best method for evaluating aerosolized materials (particles and fibers), vapors, and gases inhaled by the human respiratory system^14,15. 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FLUENT V.17.2	ANSYS		Software
mass flow meter (MFM)	TSI	4043
SMPS (scanning mobility particle sizer)	Grimm	SMPS+C
5-Jet atomizer	HCTM	5JA-1000
Mass flow controller (MFC)	Horiba	S48-32