Whole-Body Nanoparticle Aerosol Inhalation Exposures

Summary

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.

Abstract

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 < 200 nm and a geometric standard deviation σ_g < 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 = (M_post-M_pre), where M_pre and M_post 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.

Protocol

The whole-body nanoparticle inhalation exposure step-by-step operating procedures are described as follows.

Note: 1) steps 1 and 3 should be performed in a fume hood; 2) operators must wear appropriate personal protective equipment (respirators, goggles and rubber gloves).

1. Conditioning TiO₂ Nanoparticle Dry Powders

1. Place nano-TiO₂ powders in a nontransparent container.
2. Leave the container lid open.
3. Place the container in a dry desiccator for at least 24 hr for conditioning.

2. Warming up Data Acquisition and Control System, SMPS and ELPI and All Transducers

1. Turn on the air monitoring and data acquisition system and power switches for aerosol monitoring SMPS (TSI Inc., Shoreview, MN) and ELPI (Dekati, Tampere, Finland), and warm the systems up for at least 1 hr.
2. Turn on the power switches in all transducers to warm them up for at least 1 hr.

3. Loading TiO₂ Nanoparticle Dry Powders into Aerosol Generators

1. Open the cylinder caps on the aerosol generators, and replace the filters in the aerosol generators. Note: One aerosol generator has one cylinder. The number of aerosol generators to be used depends on the desired mass concentration of the particles in exposure chamber.
2. Weigh ~4 g nano-TiO₂ powders and load them in each cylinder.
3. Replace the cylinder caps.
4. All areas suspect of TiO₂ contamination should be wet wiped.

4. Connecting Aerosol Generators to Inhalation Exposure Chamber

1. Connect all the outlets of the aerosol generators via a manifold to a cyclone separator which is at the inlet of the inhalation exposure chamber (TSE Systems GmbH, Bad Homburg, Germany).
2. Connect compressed air tubing to the Venturi dispersers in the aerosol generators.

5. Connecting Air Monitoring and Aerosol Sampling Inlets to the Inhalation Exposure Chamber

1. Connect temperature & relative humidity (RH), pressure, O₂ & CO₂ sensors supplied by TSE Systems to test atmosphere monitoring ports on the inhalation exposure chamber.
2. Connect the inlet of an aerosol dilutor to one of the aerosol sampling ports on the inhalation exposure chamber, and then connect its outlet to the inlet of the ELPI.
3. Connect SMPS to one of the aerosol sampling ports on the inhalation exposure chamber.
4. Connect inlet of a particle concentration monitor (TSE Systems) to one of the aerosol sampling ports on the exposure chamber.
5. Weigh PTFE membrane filter (P/N 66149, Pall corporation, Ann Arbor, Michigan) and load filter into a stainless steel filter holder (In-Tox products, Moriarty NM).
6. Connect the inlet of the stainless steel filter holder with a pre-weighed filter to one of the aerosol sampling ports on the inhalation exposure chamber, and connect its outlet to a sampling pump.

6. Activate Data Acquisition Systems

1. Activate ELPI data acquisition software, ELPIVI, check setup parameters, and turn on the flush pump for ~5 min and then zero the ELPI. Record pre-exposure concentration.
2. Activate SMPS data acquisition software. Record pre-exposure concentration.
3. Activate software, Daco (TSE Systems), for monitoring and controlling air flow rate, temperature and RH chamber pressure, temperature & RH, O₂ and CO₂.

7. Loading Experimental Animals into the Inhalation Exposure Chamber

1. Weigh the experimental animals.
2. Mark the experimental animals and cages so that the animals can be put back in the same cages after the exposure if needed.
3. Open the door of the inhalation exposure chamber, and load experimental animals into the wired cages.
4. Water may be provided for animals.
5. Close and secure the door of the inhalation exposure chamber.
6. Frequently observe animals through the exposure chamber observation windows for signs of distress. Animals should be relaxed and behaving normally. Stop the exposure if rapid/labored breathing, abnormal appearance, postural abnormalities or immobility are observed. Remove the animals, return them to their original cages, contact attending veterinarian and/or initiate appropriate Institutional Animal Care and Use Committee procedures.

Note: Operators must wear personal protective equipment when performing steps 8.7, 8.8 and 8.17.

8. Exposing Small Animals to Nanoparticle Aerosols

1. Turn on the exhaust vacuum pump of the inhalation exposure chamber.
2. Run data acquisition software, Daco, to: a) supply filtered dry air to the exposure chamber, b) control the pressure in the exposure chamber, and c) collect the data of the exposure environment, such as pressure, temperature, RH, O₂ and CO₂.
3. Establish a slightly negative pressure (set point = -0.2 mbar) in the chamber pressure.
4. Turn on the aerosol generators.
5. Run ELPI and SMPS data acquisition software to continuously monitor particle size and relative mass concentration in the inhalation exposure chamber.
6. When the aerosol concentration is stable, i.e. the concentration profile on ELPI monitor reached plateau (Normally: this takes 20 min after the aerosol generators are in operation), set up the sampling time (for example, 1 hr) and turn on the aerosol sampling pump to collect representative sample of nanoparticles with filters.
7. Once the sampling time is reached, remove the filters and plug the sampling ports with rubber plugs to prevent test materials from escaping the exposure chamber.
8. Weigh the filters, and calculate the mean mass concentration in the exposure chamber as described above.
9. If the mean concentration is off the targeted concentration, manually adjust the airflow in the generators to ensure the targeted concentration is attained.
10. Calculate particle deposition in the animal lungs as D = C x V_m x t x F_r, where D = Dose, C = mean mass concentration of test material, V_m= minute volume, t = exposure duration, and F_r = fraction of material that is deposited or absorbed.
11. Replace the filters in the filter holders with clean, pre-weighted filters, and repeat steps 8.6 and 8.8.
12. Based upon the real mass concentration in the exposure chamber and targeted particle deposition in the animal lungs, estimate the remaining exposure time as, t_remain = (D_targeted -D) / (C x V_m x F_r), where t_remain = remain exposure duration, D_targeted = targeted dose, C = mean mass concentration of test material, V_m = minute volume, F_r = fraction of material that is deposited or absorbed.
13. Turn off the aerosol generator when t_remain is reached.
14. Before removing the animals from the exposure chamber, flush the inhalation exposure chamber with the filtered air until the particle concentration indicated in the monitor is close to the pre-exposure particle concentration in the chamber.
15. Turn off the chamber exhaust vacuum pump.
16. Stop data acquisition software, Daco.
17. After exposure, observe animals to verify normal respiration and behavior, and document that no other study complications exist. If nasal discharge, respiratory distress or any other animal welfare complications are observed, contact attending veterinarian and/or initiate appropriate Institutional Animal Care and Use Committee procedures.
18. Stop ELPI and SMPS data acquisition software.

9. Creating Test Report

9.1 Test conditions include

1. Description of the aerosol generation system and its operating parameters used in this test.
2. Description of the exposure apparatus including design, type, dimensions and its operating parameters used during the exposure.
3. Equipment for measuring temperature, humidity, particle size, and actual concentration.
4. Treatment of exhaust air and the method of housing the animals in the test chamber when used.

9.2 Exposure atmosphere data include

1. Airflow rates through the inhalation equipment.
2. Temperature and humidity of the air.
3. Actual (analytical or gravimetric) concentration in the aerosol sampling zone which is near the animal cages.
4. Particle size distribution, and calculated count median aerodynamic diameter and geometric standard deviation.
5. Explanation as to why the desired chamber concentration and/or particle size could not be achieved (if applicable), and the efforts taken to comply with these aspects of the guidelines.

9.3 Other

1. Slightly negative pressure in the room containing inhalation facility should be maintained to prevent test materials from escaping inhalation exposure lab.
2. Clean the exposure chamber daily to eliminate the influences of the animal wastes.
3. ELPI, SMPS and other instruments should be cleaned and calibrated based on the user manuals.

Results

An inhalation exposure study typically involves maintaining an experimental animal in a known and constant test environment while exposing the experimental animal to a defined concentration of a test material ^8,9. The whole-body nanoparticle inhalation exposure system is shown in Figure 1. The whole-body chamber was operated on a dynamic flow basis where there was a 90 LPM continuous flow of air through the chamber. This air flow provided 10.8 air changes/hr which exceeds the minimum number of...

Discussion

We have assembled and described here in a whole-body nanoparticle aerosol inhalation exposure system. The system functionality was validated with state-of-the-art nanoparticle aerosol characterization techniques. With a novel nanoparticle aerosol generation system, this inhalation exposure system can provide a well characterized, controlled and uniform nanoparticle aerosol test atmosphere with relatively consistent temperature, humidity, air flow, and oxygen content for experimental animals. The exposure system is most e...

Materials

Name	Company	Catalog Number	Comments
Name of Reagent/Material	Company	Catalog Number	Comments
Inhalation exposure system	TSE Systems GmbH, Bad Homburg, Germany
Air monitoring system	TSE Systems GmbH, Bad Homburg, Germany
Titanium dioxide Aeroxide P25	Evonik, Germany
Scanning mobility particle sizer-3936L75	TSI Inc., Shoreview, MN
Electric low pressure impactor, Standard 10 LPM	Dekati, Tampere, Finland
Ultra Micro Balance, XP2U	METTLER TOLEDO, Switzerland
Field Emission Scanning Electron Microscope-S-4800	Hitachi, Japan
Energy dispersive X-ray analysis	Princeton Gamma-Tech, Rocky Hill, N.J.
Nuclepore polycarbonate filters	Whatman, Clinton, PA
PTFE membrane filters	Pall corporation, Ann Arbor, Michigan