Abstract
The respiratory system, the major route for entry of oxygen into the body, provides entry for external compounds, including pharmaceutic and toxic materials. These compounds (that might be inhaled under environmental, occupational, medical, or other situations) can be administered under controlled conditions during laboratory inhalation studies. Inhalation study results may be controlled or adversely affected by variability in four key factors: animal environment; exposure atmosphere; inhaled dose; and individual animal biological response. Three of these four factors can be managed through engineering processes. Variability in the animal environment is reduced by engineering control of temperature, humidity, oxygen content, waste gas content, and noise in the exposure facility. Exposure atmospheres are monitored and adjusted to assure a consistent and known exposure for each animal dose group. The inhaled dose, affected by changes in respiration physiology, may be controlled by exposure-specific monitoring of respiration. Selection of techniques and methods for the three factors affected by engineering allows the toxicologic pathologist to study the reproducibility of the fourth factor, the biological response of the animal.
Introduction
The primary function of the respiratory system is to transport oxygen into, and carbon dioxide from, the blood stream. The respiratory system also warms and humidifies the incoming air. These functions are facilitated by the structure of lung tissues as a thin membrane with a large surface area to aid the transfer of gases between air on one side and blood circulatory system on the other side. The outside air then has an extensive area of contact that puts the respiratory system at risk and provides access to the blood circulation for materials and foreign compounds carried in with inspired air. Inhalation toxicologists assess the biological effects of these inhaled compounds by presenting an atmosphere containing specified concentrations or doses of the compound of interest to the test subject for inhalation.
A fundamental concept of toxicology is “the dose makes the poison.” For toxic materials or pharmaceutics administered directly into the body, such as by intravenous, intramuscular, or intraperitoneal injection, or by gavage, dose is a direct calculation of the amount of test compound applied per subject or per body mass. Dosage application via the dermal route is also readily determinable as amount per square area. Administration of doses by indirect methods may require more information. Dosage from administration by food or water may be calculated by measuring the quantity of food or water consumed.
Determination of dose by inhalation is imprecise due to the difficulty of measuring the amount of air inhaled by the individual animal. Breathing frequency and tidal volume determine minute volume, the amount of air inhaled in a minute. Estimates for the average minute ventilation as a function of body weight and activity exist. However, for individual animals, actual minute ventilation can vary greatly during an exposure, depending on the activity of the animal, and alterations in breathing pattern in response to the inhaled material.
Not only does the inhaled dose depend on the amount of the exposure atmosphere inhaled into the respiratory system, but also on the amount of the test material that is taken up or deposited in the respiratory system from the inhaled atmosphere. For gases and vapors, the uptake is influenced by the solubility, reactivity, and metabolism of the inhaled material. For aerosols, the uptake or deposition depends on aerodynamic properties that include the particle size, respiratory system geometry, and respiratory physiology (air flow). Primary mechanisms of deposition in the respiratory tract include sedimentation, impaction, and diffusion. Other factors such as electrical charge, interception, and hygroscopicity will also influence the deposition of particles. Thus, the exposure concentration is modified by breathing physiology and uptake factors to give the inhaled or applied dose to the respiratory system.
An accurate determination of the applied dose is needed in order to assess the pathologic effects from the administration of a foreign compound. When the compound is administered by inhalation, biologic results are subject to variability in several areas: the overall animal environment and surroundings; the exposure atmosphere of the compound of interest and other materials; the applied dose; and individual animal biological sensitivity. This article discusses methods and techniques to control these various factors in order to make a more precise determination of the dose and resultant biological results.
Exposure Environment
The exposure environment, including the animal housing conditions and the exposure systems are 2 of the major factors that should be controlled.
Housing Environment
The overall environment and surroundings including factors such as temperature, humidity, oxygen, light and noise levels, presence of other animals and other general environmental conditions can vary widely. Extremes or changes in these factors may affect an animal’s metabolism, eating and drinking behavior, and stress levels which, in turn could alter the biologic response to the administered compound. Variability in the environment and surroundings can be managed by using engineering processes and strict building management to tightly control temperature, humidity, oxygen levels to the recommended ranges (National Research Council, 1996). Lighting is usually set for 12-hour on, 12-hour off cycle to simulate the normal diurnal pattern. Depending on the study, there may be other specific requirements for the environment such as (1) avoiding loud, sudden noises, or excessive vibrations, (2) providing environmental enrichment, or (3) space for the separation of species. All groups of animals on a study should be housed under similar environmental conditions.
Exposure Systems
Various systems have been developed to house or restrain experimental subjects and provide a controlled atmosphere for exposure. These systems fall into the broad categories of whole-body chambers, nose or head-only chambers, and specialized exposure methods. The specialized exposure methods will be discussed in a separate section under controlling inhaled or applied dose.
Whole-Body Exposure Chamber
In a whole-body exposure chamber, the subject is immersed in the atmosphere contained in the chamber, simulating environmental or work-place exposures (Table 1). Whole-body exposure chambers come in a wide variety of sizes, from very small chambers that hold 1 animal, to very large, room-sized chambers. Small chambers may be constructed from readily available containers such as dessicator jars, aquaria, and other types of containers (Cheng and Moss, 1995). Small, single animal holders, developed for individual exposures (Leavens et al., 1996), have also been used to house a dam and pups for reproductive and developmental studies to inhaled materials (Dorman et al., 1996, Vitarella et al., 1998).
Large whole-body chambers (Figure 1) are most commonly used for long-duration exposure studies and for large numbers of test subjects. The large chambers are designed to house the test subjects during exposure and nonexposure periods. These large chambers are operated on a dynamic flow basis where there is a continuous flow of air through the chamber. Some chambers are mobile (Hinners et al., 1968) and may be moved in and out of place.
Inside a whole-body chamber, animals may be housed individually or in groups. Group-housed animals may huddle leading to the potential that an individual may inhale less of the test compound due to the filtration or reaction with surrounding animals’ fur. Also, an individual might inhale air that has been exhaled and cleaned of the test compound by surrounding animals. Both of these possibilities could result in varying uptake of the compound among animals. Also, during preening, animals may ingest material that has deposited on the fur.
To minimize the influence of the animals’ surface (fur) compared with the internal chamber surface Silver (1946) recommended that the volume of animals not exceed 5% of the total volume of the chamber. Some authors (Snellings and Dodd, 1990) have suggested that an animal volume of 1 to 2% is more desirable based on heat generation from the animals and temperature control considerations.
Head and Nose-Only Exposure Systems
Head and nose-only exposure systems are designed to expose test subjects while minimizing skin or fur contamination and also minimizing the amount of compound used (Table 1). The test subject is confined so that only the head or nose is exposed to the test atmosphere. A continuous flow of air is supplied to the system. In some designs, the test subjects inhale from the plenum containing the test atmosphere and exhale back into the same plenum (Smith et al., 1981). In this type of system, the downstream test subjects may inhale air that was exhaled from the upstream test subjects. If the airflow is too low, downstream test subjects may get a lower dose, or the dose to each test subject may be more variable (Cannon et al., 1983). This potential problem has been addressed by a system design in which the test atmosphere flows into an inlet manifold that directs the flow towards the nose or head of each test subject. The exhaled air is then carried along with the excess airflow into an exhaust plenum (Figure 2). Thus all the test subjects are exposed to the same atmosphere (Raabe et al., 1973; Cannon et al., 1983; Prasad et al., 1988; Pauluhn, 1994).
Smaller animals such as rodents are held in tubes (Phalen, 1997). The tube is attached to the chamber so that a hole or extension from the inlet manifold directs the atmosphere flow towards the animal’s nose. An adjustable back restraint is used to prevent the animal from backing out. A restraint that is open to the atmosphere can allow heat and humidity to escape. However the test atmosphere can leak around the animal. Leaks may be prevented by using a restraint system that seals the tube, though heat and moisture buildup in the tube is a concern (Phalen, 1997). A sealed restraint system is desirable if the test compound is particularly toxic or hazardous.
Animals inside the restraint tubes do not have access to food or water. While loading the animals in the tubes, care must be taken to position them correctly. Animals, particularly the younger or smaller ones, may attempt to turn around inside the tubes with the risk of suffocation. The airflow through the nose-only chamber may be reduced to minimize the amount of test compound used. However, if the flow through each port approaches the minute ventilation rate of the animal, the flow may be insufficient to clear the exhaled atmosphere away from the animal. The animal will begin to rebreathe its exhaled atmosphere, reducing the delivered dose and oxygen concentration while increasing the carbon dioxide concentration. If the flow is too low, the animals may suffocate. Various minimum flow recommendations have been made for nose-only exposure chambers.
A flow of 1.5 times the minute volume (Barr et al., 1987; Cheng and Moss, 1995) has been recommended based on oxygen depletion. A flow of 2.5 to 4 times the animal minute ventilation has been recommended based on minimizing compound consumption while maintaining the concentration at 90% of target (Moss et al, 2006). Finally, 10 times the minute volume (Phalen, 1997) per animal has been recommended as a minimum flow through a nose-only chamber to prevent rebreathing of exhaled air, especially if the animal becomes active. The initial flow rate for a nose-only inhalation study design should start at 2.5 times the animal minute ventilation. If test material is rare and unavailable, flow may be decreased to 1.5 times, and if test material is readily available, flow rates should be increased to 5 or more times the animal minute ventilation.
The environmental conditions listed in Table 2 are guidelines for acute inhalation exposures, with similar conditions for longer-term exposures. While the researcher factor strives to maintain very uniform housing and environmental conditions for the exposure subject, the exposure atmosphere concentration is the primary factor designed to be varied. The exposure concentration is controlled by the test compound generation and monitoring methods, airflow, and mixing in the systems used to administer the atmosphere to the animal. Methods used to generate and characterize the test atmosphere are described next.
Generation and Characterization of the Test Atmosphere
A test compound is classified as an aerosol, a vapor, or a gas. The behavior of these forms is different which necessitates different methods of generation and monitoring. The investigator’s goal is to produce the test compound at the target concentration in as steady and reproducible a manner as possible for the duration of the experiment. The distinction between a gas and vapor is that at room temperature, the gas test compound exists as a gas, while the vapor test compound is in equilibrium with its liquid. For both a gas and a vapor test compound, the exposure atmosphere delivered to the animal is gaseous (individual molecules dispersed in the air).
Generation of Gases
Compounds may be commercially available under pressure in a gas cylinder (Figure 3). A pressure regulator is used to reduce and regulate the pressure. A flow controller controls the flow of gas into the chamber air supply (Wong, 1995). A gas may be generated by a chemical reaction from precursor compounds. Ozone is a highly reactive gas and cannot be stored for any length of time. Ozone is generated as required for use in studies by passing oxygen through an ultraviolet generator or an electric discharge system (Nelson, 1992).
Formaldehyde gas is directly generated from the thermal decomposition and sublimation of paraformaldehyde, a solid polymer of formaldehyde. Paraformaldehyde is held in stainless steel container and heated in a well-regulated oven. Nitrogen gas passing through the stainless steel container mixes with formaldehyde vapors and carries the vapors out to the chamber. Formaldehyde concentration is controlled by varying oven temperature, container size, and chamber air flow (Chang et al., 1983).
Generation of Vapors
A test atmosphere of a vapor is generated by conducting the gas portion of the liquid-vapor equilibrium into the chamber air supply. A carrier gas such as air or nitrogen may be mixed with the vapors to carry them out to the chamber air supply. In order to replenish the vapor or to increase the amount of vapor, the liquid may be heated. If all of the liquid entering the generation system is vaporized, then the liquid flow rate controls the vaporization rate and gas concentration can be controlled by metering the flow of liquid. For example, the J-tube generation system uses a liquid metering pump to introduce the liquid into the long end of a J-shaped tube (Figure 4).
The J-tube is filled with glass beads to increase the evaporative surface area. Carrier gas is introduced into the short end of the J-tube to pick up vapors that are carried to the chamber air supply. The J-tube or the carrier gas can be heated to aid evaporation (Miller et al., 1980; Tilbury et al., 1993; Wong, 1995). Many other techniques use variations on heating or increasing evaporative surface area to generate vapors for inhalation studies (Nelson, 1992).
Characterization of Gas Concentration
Instruments commonly used to characterize the exposure concentration in inhalation atmospheres are the gas chromatograph (GC) and the infrared spectrophotometer (IR). A wide range of gases and vapors can be analyzed by both instruments. Infrared spectrophotometry and gas chromatography have the potential to provide atmosphere concentration in near real time, and have been used to provide data for automated feedback loops for controlling chamber atmosphere concentration (Wong and Moss, 1996; Wong, 2003).
Infrared Spectrophotometry
An organic molecule will absorb infrared radiation at wavelengths that are characteristic of the bonds between its constituent atoms. The concentration of a gas can be determined by measuring the amount of absorption of a selected wavelength of light as it passes through a cell containing the gas (Wong, 1995).
Gas Chromatograph
Gases can be injected into a carrier gas stream flowing through a column containing a packing material with varying affinity for the gases. The gases will require different times to pass through the column and are identified by their retention times within the column. A detector at the end of the column provides a measure of the amount of gas that is present (Wong, 1995).
Batch Methods
A batch of the exposure atmosphere is sampled and held in a storage system for later analysis. A sample of the exposure atmosphere can be drawn through an impinger or bubbler at a known flow rate for a certain period of time. The gas dissolves in the impinger fluid, or can react with a chemical in the fluid. The impinger fluid is later analyzed by appropriate methods for the total gas content (Wong, 1995).
The Aerosol Test Atmosphere
An aerosol is defined as liquid or solid particles suspended in a gas. An important factor to consider when generating an aerosol for inhalation studies is the size and respirability of the particles. The size and shape of a particle determines aerodynamic properties that govern entry into the lung, depth of penetration into the lung, and deposition in the lung. The deposition efficiency as a function of particle diameter has been extensively studied. Representative curves for the human and rat show how different size particles will deposit in different regions of the respective respiratory tracts (Figure 5). Most aerosols contain particles that span a range of sizes. Some conventions are in common usage to describe the particle size distribution for inhalation studies.
Particle Size Distribution
An aerosol used for inhalation studies is commonly characterized by two parameters that describe the size distribution function and a concentration parameter. An aerosol size distribution can be described by a lognormal distribution in which the logarithm of the particle diameters is distributed normally. The mass median diameter (MMD) is defined as the diameter for which the particles larger than that diameter contribute one-half total mass and particles smaller than that diameter contribute one-half of the total mass. If the particle diameter is given in reference to a unit density sphere, then the diameter is called a mass median aerodynamic diameter (MMAD). The spread of the distribution can be described by the geometric standard deviation (GSD), analogously to how the standard deviation describes the spread in a normal distribution.
Aerosol Concentration
The concentration of the aerosol may be specified in mass per volume units such as micrograms per cubic meter (μg/m3). Use of other properties of the aerosol, such as the surface area or number of particles, may provide a metric that better correlates with observed toxicity. Hence, it is important to characterize the aerosol appropriately for the biological responses being studied.
The nature of the study and the test material determines the particle size distribution that should be generated. The U.S. EPA TSCA acute inhalation toxicity test guidelines recommend an MMAD between 1 and 4 μm for the test aerosol (U.S. EPA, 1997; Commentary, 1992). Other inhalation studies may specifically target other particle sizes. Research is currently being conducted with submicron or nanometer particles, in part due to recent studies linking environmental particulate matter (PM) pollution to increased morbidity and mortality (Schwartz, 1994; Oberdorster et al., 1995) and due to recent developments in nanotechnology and nano-sized materials (Colvin, 2003; Oberdorster et al., 2005).
Generation of an Aerosol
Liquid Aerosol Generation
Nebulization is used to produce an aerosol of liquid particles by pushing air at high pressure through a nozzle (Figure 6). The air flow through the nozzle induces liquid to flow into the air stream. The liquid is broken up into a distribution of droplet sizes. The air jet may be aimed at a surface so that droplets impact and break up. Larger droplets drain back down into the liquid reservoir while the smaller droplets are carried out the air stream. A wide variety of nebulizers are available, with most producing liquid drops in the 2- to 7-μm range (Chen and John, 2001).
Soluble Compounds
Methods used to generate liquid aerosols can also be used to generate solid particles composed of a compound that is soluble in water or other solvent. Upon nebulization, the liquid droplets dry, leaving behind solid particles.
Solid Aerosol Generation
Bulk solid compounds need to be broken up by mechanical grinding or pulverizing to produce particles in the respirable size range. Alternatively, the compound can be produced as a respirable powder during the chemical synthesis process. Once a respirable powder has been produced, dry powder generators are designed to entrain the powder into an air stream. Most aerosol generators use some method to feed either loose or compacted powder into a mechanical or turbulent air system to break up and aerosolize the powder (Figure 7).
Wright Dust Feed
The Wright Dust feeder is a dry powder disperser where the powder is packed into a cup. The cup slowly rotates down across a scraper blade such that an amount of packed powder is slowly scraped off. An airstream moving along the scraper blade entrains the powder and carries it out of the generator. The generator works best with nonsticky powders that have a significant portion of the particle size distribution within the respirable size range (Hinds, 1999).
Rotating Brush
Instead of a blade, some powder dispersers use a rotating brush to remove powder from a plug of powder that is slowly being pushed into the brush. The removed powder is entrained into an air stream. This method has also been used to generate fibrous particulates (Bernstein et al., 1994).
The previously described generators compress or pack the powder. Another type of generator feeds a free-flowing powder into a turbulent air stream to break up the particles. A commercially available powder feeder that uses a spiral screw feeder slowly dispenses the powder. The feed rate is controlled by the rotation rate of the screw. Other bulk powder feeders use a conveyer belt system (Moss and Cheng, 1995).
Venturi
Commercial feeders have been used to dispense powders into a venturi, in which air is pushed though a narrow opening. The venturi effect at the narrow opening causes a low pressure that entrains the powder. As the constriction opens out again, turbulence is created that helps to break apart the powder (Cheng et al., 1989). In another disperser, the dispensed powder falls into high-velocity air jets blowing horizontally, that break up the powder (Koch et al., 1986).
Turntable Disperser
The venturi effect has also been used to aspirate particles up from a rotating turntable. The rate of rotation controls the generation rate (Chen and John, 2001). The dry powder dispersers generally do not break up a particle smaller than its initial size. If a smaller size distribution is required for an inhalation study, the size distribution of the parent powder must be reduced, or the larger particles must be removed from the test atmosphere. The separation can be accomplished by first passing the aerosol through a settling chamber where the larger particles settle out under the influence of gravity. The large particles can also be separated out of an aerosol stream by using a cyclone or virtual impactor (Sioutas et al., 1994; Sioutas and Koutrakis, 1996).
Aerosols of Nanomaterials
Nanotechnology is currently undergoing intense investment and development (Colvin, 2003) including the production of nanomaterials or nanoparticles. While definitions differ, one common definition for nanoparticles is highly purified and engineered materials with at least one dimension less than 100 nm. These engineered materials are of interest to the inhalation toxicologist because they may be composed of potentially toxic materials and because they are in a respirable size range that, regardless of chemical makeup, is hypothesized to cause toxicity.
Production of Submicron and Nanometer Aerosols
Commercially available powders that are produced in sub-micron and nanometer size ranges may be aerosolized using existing methods of powder dispersion. These methods may not aerosolize the powder into individual nanometer-sized particles, but may instead produce agglomerates. Aerosols of separate nanoparticles are more effectively produced by condensation from a vapor. A compound can be heated, vaporized, and transported to a cooling section. As the vapor cools, it condenses either with itself (homogeneous nucleation) or onto existing (seed) particles (heterogeneous nucleation). The vapor condensation method has been used to produce a relatively large number concentration of monodisperse particles (Tomaides et al., 1971). Other methods have used the combustion or high-temperature reactions of precursor compounds to produce submicron particles of various metal oxides (Kanapilly et al., 1970; Kanapilly et al., 1978; Crisp et al, 1981; Roth et al., 2004).
Characterizing an Aerosol Test Atmosphere
Filter Samples
The mass concentration of an aerosol is the primary parameter for characterizing a test atmosphere containing an aerosol. The most basic way to accomplish this is to pull a sample of the atmosphere through a filter at a known flow rate for a known period of time (Lee and Mukund, 2001) without altering the concentration within the chamber (Moss, 2001). The filter should be located as close as practicable to the breathing zone of the test subjects.
Optical Aerosol Samplers (Nephelometry)
An optical aerosol sensor measures the light scattered from a continuous flow of aerosol through a sampling volume. The scattered light is proportional to the concentration of aerosol particles. One problem with an optical light scattering instrument is that the response is dependent on the optical properties of the aerosol. It generally must be calibrated with a mass filter sample (Cheng et al., 1988).
Cascade Impactor
The cascade impactor is the most commonly used instrument to measure the size distribution of a aerosol. In an impactor, the aerosol is accelerated through a nozzle towards a collection surface. Larger particles with greater inertia impact on the surface and are collected, while smaller particles turn with the air around the collection surface. The cascade impactor has several stages, each collecting a smaller size cut of particle. From the mass of particles on each stage, the MMAD and GSD can be determined (Hinds, 1999; Marple et al., 2001).
Optical Light Scattering (Individual Particles)
Some instrument use light scattering to measure particle size. The intensity of scattered light is proportional to the particle size. Again, this property is dependent on optical properties of the particle and requires calibration against the specific particle compound (Gebhart, 2001).
Other instruments use optical techniques to measure the time of flight of a decelerating aerosol particle. The time is directly proportional to the aerodynamic size of the particle. In these instruments, sizing is not dependent on the optical properties of the compound (Baron et al., 2001).
Bioaerosols
Another area of current concern is the exposure to bioaerosols, related, in part, to terrorism scenarios. An aerosol of biological origin may contain microorganisms such as viruses, bacteria, fungi, spores or pollen. The microorganisms can be viable or nonviable. The products of microorganisms can include toxins, endotoxins, mycotoxins, fecal material, allergens, or other biological components that have been broken up into inhalable sizes (Reponen et al., 2001).
Generation of Bioaerosols
A bioaerosol can be generated using many of the same techniques as for nonbiological aerosols. The generation technique needs to be selected carefully, since the stress of aerosolization can cause injury or loss of viability of a microorganism.
Nebulization
Aerosols of microorganisms may be produced by nebulizing the liquid growth media containing suspensions of the microorganism (Gardner, 1982; Thompson et al., 1994). In a nebulizer, however, injection of the liquid into a high-velocity air stream can produce shear forces that injure and recirculation of the liquid increases the possibility of injury of the microorganisms. An aerosol disperser was developed to produce an aerosol of microorganisms from droplets formed from the bursting of bubbles (Ulevicius, 1997; Mainelis et al., 2005). A standard nebulizer was shown to produce a significant number of injured bacteria that increased over time, while the bubbling aerosol disperser had a low number of injured bacteria that remained stable (Reponen et al., 1997).
Dry Powder Dispersers
Standard dry powder dispersion techniques described previously can be used to aerosolize microorganisms if they can be removed from the growth culture and dried. Certain biological particles such as spores are more adaptable to this process than microorganisms requiring moisture to maintain viability. To aid the removal process, methods have been developed so that the microorganisms can be grown on an agar surface and aerosolized by directing a stream of air across the surface. These dispersion methods can generate a more consistent concentration of spores (Reponen et al., 1997).
Bioaerosol Monitoring
A bioaerosol may need to be monitored, not only for mass or number concentration, but also for viability. The amount or proportion of viable microorganisms in the test atmosphere can be the critical factor of interest. Thus the sampling method selected should impose minimum stress on the microorganism and supply nutrients to preserve viability.
Glass Impinger
Bioaerosols in the exposure atmosphere may be drawn through the nozzle of a glass impinger and collected into a liquid. Depending on flow rate and the liquid level in the impinger, a bioaerosol particle can bounce off the bottom surface back into the airstream without being collected into the liquid. Also, collected particles in the liquid can be reaerosolized from bubbles bursting at the liquid surface (Grinshpun et al., 1997). The impingement and impaction process may also stress microorganisms and cause loss of viability (Lin and Li, 1998). The impinger should be carefully evaluated to find the proper operating conditions for maximizing collection and minimizing particle bounce and reaerosolization.
Impactor
A cascade impactor can be used to collect and determine the particle size distribution of a bioaerosol. Impactors have been designed to use petri dishes with agar surfaces as the collection surfaces. The agar surface serves as nutrient media for growing colonies of microorganisms. Some impactors collect onto glass slides for subsequent evaluation by light microscopy (Reponen et al., 2001).
System Operations
An exposure system is an assembly of a generation system, exposure system and monitoring system (Figure 8). The sample line entrance to the monitoring system should be positioned to acquire an atmosphere sample that is representative of what a test subject would inhale, i.e., a breathing zone sample. For aerosol sampling, to avoid losses due to settling, impaction or other mechanisms (Brockman, 2001), a sample line should be short, without sharp bends, and constructed of the appropriate material, as practicable. Some performance tests can be run on the generator-exposure-monitoring system to provide an indication of the stability of concentration, and uniformity of distribution of the test compound throughout the chamber.
Concentration Stability
The exposure atmosphere test material concentration can fluctuate greatly over a length of time such that the average concentration is close to the target concentration, but short-term, real-time concentrations can vary significantly. The temporal stability of test compound concentration inside an exposure chamber is most dependent on the stability of the generation system. One can generally achieve good stability with a gas or vapor generation system since there are effective, reliable ways of metering gases or liquids. Aerosols are notoriously more difficult to maintain a stable concentration because they are more difficult to generate stably and because they are subject to losses in being transported to the chamber. Greater temporal stability in both gas systems and aerosol systems can be achieved by using an active feed back control that monitors the chamber concentration and adjusts the generation system to maintain the target concentration (Wong and Moss, 1996; Wong, 2003).
Uniformity of Distribution
Maintenance of appropriate environmental parameters inside the chamber is insufficient if the concentration of test compound varies from location to location. An exposure system should be checked for uniformity of distribution prior to the start of a study (Moss, 1981; Cheng et al., 1989; Wong, 1999). If the distribution of test compound in a whole-body chamber is uniform, the entire chamber constitutes the breathing zone, and samples may be taken from a single location, usually at the center or near the entrance of the chamber. The breathing zone sample for a nose-only exposure system is usually taken from the approximate location of the test subject’s nose or at the inlet plenum.
Inhaled or Applied Dose
The techniques and methods described above are used to produce a steady exposure atmosphere concentration. However, even though a number of subjects may be exposed to the same exposure concentration, the amount of material that is absorbed by or deposited onto biological tissue and produce pathology can still differ among subjects because of differences in respiratory physiology and anatomy. For example, the tidal volume and breathing frequency may differ between subjects so that the total volume inhaled by one subject is significantly different from another. Some methods have been developed that will deliver a specified dose directly to the respiratory system. Also, exposure methods have been developed that monitor the breathing parameters of the individual subject and expose the subject to a total inhaled volume and thus deliver an inhaled dose (Table 3).
Localized Dose Application
Techniques such as intratracheal instillation, oropharyngeal aspiration, endotracheal inhalation, and tracheostomy, allow the experimenter to isolate certain regions of the respiratory tract and directly deliver materials to the respiratory tract.
Intratracheal Instillation
Instillation may be used to deposit a precisely known amount of compound directly into the lungs. In this technique, a catheter is inserted into the trachea down to the bifurcation of an anesthetized animal. A suspension of the test compound is injected into the lungs. Distribution of the test compound throughout the lungs may not be uniform, and may not replicate the same distribution by inhalation. This method may be used to provide information on the relative toxicity between compounds or over a range of doses. Intratracheal instillation has been used for repeated dosing studies, for example, to test carcinogenicity and establish relative potency of fibrous and nonfibrous particulates (Pott, 1993). Intratracheal instillation is also useful to evaluate particulate material that not readily inhaled by rodents. This method should not be used to determine deposition patterns, or to provide information on the upper respiratory tract toxicity of administered materials (Driscoll et al., 2000).
Oropharyngeal Aspiration
Aspiration may be used to deliver a specific dose into the lungs. The tongue of an anesthetized animal is extended, and a small volume containing the test material is placed at the base of the tongue. During inhalation, material is aspirated into the lungs. In this technique, intubation is not required. Dye studies and radiolabeled colloid tracer studies showed that the material is distributed through the lungs. However, the clearance of the material showed a different pattern between intratracheal instillation or oropharyngeal aspiration, and nose-only inhalation (Foster et al., 2001). Another study with fluorescent polystyrene latex beads and beryllium oxide particles also showed that the material was distributed throughout the lungs (Rao et al., 2003). This method may be used under similar circumstances and conditions as intratracheal instillation (Table 3).
Endotracheal Inhalation
This technique uses a catheter that is inserted into an anesthetized animal’s trachea. The catheter is sized such that it forms a tight fit inside the trachea. A system of valves and air under slightly positive pressure inflates the lung. When the pressure is released, the lungs deflate. The test compound is administered in the inhaled air. This technique has been used to deliver ultrafines and noninhalable aerosols to rats (Oberdorster et al., 1997; Owen Moss, August 1997, personal communication).
Tracheostomy
An opening in the trachea may be used to deliver a test compound to the lungs. The trachea is surgically exposed, and a cannula is inserted towards the lung. The test compound may be injected into the lungs, or fluids and cells may be obtained from the lungs (Phalen, 1997). A variation of this method has also been used to study nasal uptake or deposition. In this system, 2 cannuli are used. One is inserted toward the lung, allowing the animal to breathe. The other cannula, inserted toward the larynx, is used to draw an atmosphere through the nasal passages (Gerde et al., 1991; Kelly et al., 2001; Schroeter et al., 2006).
The advantage of these specialized techniques is that a very precise dose can be delivered to the lungs or respiratory system. However, the test subjects must be anesthetized and may require surgical procedures. These techniques are not suited for long-term studies. These techniques are useful to provide a ranking of respiratory toxicity of different materials, particularly if only small quantities of materials are available. Other advantages and disadvantages are listed in Table 3.
Exposure by Inhaled Dose
An alternative to direct delivery of a dose to the lungs is to monitor the breathing of an animal and estimate the exposure dose based on the volume of atmosphere that an animal has inhaled. The breathing parameters of an animal may be monitored using a specially designed plethysmography chamber. The chamber is equipped with sensitive pressure transducers to detect pressure changes resulting from an animal’s breathing activity. Two general types are available, a whole-body and a dual-chamber plethysmograph. In the whole-body plethysmograph, the animal is unrestrained, and the pressure signal occurs from the expansion of air due to warming and humidification by the animal.
In the dual chamber system, the animal is restrained by a flexible neck collar between 2 compartments, one compartment enclosing the head, and the other the remainder of the body. Pressure transducers in the compartments sense the movement of air and the expansion of the thoracic cavity. Both of these types of chambers can be used to measure the breathing frequency and tidal volume of an individual animal leading to a determination of the volume of air inhaled (DeLorme and Moss, 2002). This type of feed back system has been used to expose primates to a predetermined bioaerosol dose (Hartings and Roy, 2004).
If the animal is inhaling a known concentration of test atmosphere during this period, the amount of inhaled test material can be calculated. If the uptake or deposited fraction is known, the delivered dose of the inhaled material can be calculated:
where D = Dose, C = concentration of test material, f = breathing frequency, V T = tidal volume, t = exposure duration, and Fr = fraction of material that is deposited or absorbed (determined from dosimetry experiments (Miller, 1999). Also, minute ventilation can be determined from:
Average values for the minute ventilation, V m can be estimated from body mass using empirical allometric scaling formulae (Guyton, 1947; Bide et al., 2000). However, actual breathing parameters under specific conditions can often be quite different. For example, in an inhalation study, young male Wistar rats were exposed in a nose-only system (Cassee et al, 2002). During the exposure, breathing parameters were measured, resulting in an average minute ventilation of 314 cm3/min. In contrast, the empirical formulas for a 200 g rat give an average minute ventilation of 116 cm3/min (Guyton) or 136 cm3/min (Bide). The use of the calculated average minute ventilation versus the experimentally measured values would give a very different estimate for the delivered dose.
As a part of that study, Cassee et al. (2002) exposed the rats to aerosols with particle sizes ranging from 33 to 1495 nm (count median diameter) at approximately the same mass concentration. The amount of test compound deposited in the lung was dependent on particle size, as predicted by particle dosimetry and modeling (Cassee, 2002). Thus, calculation of the delivered dose depends on a knowlege of the minute ventilation and the effect of particle size on the deposition of the inhaled material.
The fourth factor that affects observed variability is biological sensitivity, or the way that individual animals handle the inhaled materials on a regional, or even cellular level. The responses of tissues, cells, and intracellular components to the presence of inhaled exogeneous materials are the observed biological results of primary interest of the toxicologic pathologist, and the reason for conducting inhalation studies.
Summary
The overall goal in an inhalation study is to control the environmental surroundings of the subject, provide a well-characterized exposure concentration that can be related to inhaled dose, and be able to associate biologic responses to the dose of administered compound. A facility conducting inhalation studies strives to provide a uniform environment with relatively consistent temperature, humidity, air flow, oxygen content, and other major environmental factors for all groups of experimental animals. The system used to provide the exposure atmosphere, either a whole-body or nose/head only exposure system is selected to provide a controllable and consistent exposure to the test material. The appropriate generation and monitoring system is selected for the physical and chemical properties of the test material. If necessary, a direct route of administration such as intratracheal instillation or oropharyngeal aspiration may be used to directly deposit the test material in the lungs. If these factors are carefully considered and controlled, then the toxicologic pathologist can determine the biologic responses and make a link to the dose.