Five basic types of inhalation toxicology studies have been described: whole body, head only, nose only, lung only, and partial lung (6). Each exposure type has its own unique set of advantages and disadvantages.
The advantages of whole-body inhalation systems include the ability to expose large numbers of animals sinultaneously, accommodate a wide variety of species, employ minimal restraint, and suitability for chronic inhalation exposures. The major disadvantages include the large quantity of test material required for conduct of studies, multiple routes of exposure, distribution of concentration and particle size within the chamber exposure zone, control of chamber environment, and cost. Head only and nose only inhalation exposure systems are similar enough to share advantages and disadvantages. The advantages include relative efficiency in usage of test material, reduction or elimination of multiple exposure routes, and containment of highly toxic materials. The major disadvantages are the labor intensive nature of head/nose only exposure systems, adequate exposure seals about the face or neck of animal models, and stress related to the restraint necessary for head/nose-only exposure studies.
Stress has, until recently, been a major limiting factor in determining the duration of head/nose only exposure studies (4). The need to restrain animals in tubes has been assumed to produce an undesirable level of stress when prolonged exposure is required. Smith et al. (7) have reported that long-term nose only exposures, up to 7 hr per day, were possible with little or no stress. Parameters such as body weight, rectal temperature, clinical pathology, and plasma corticosterone levels were used to indicate the lack of measurable stress in rats and hamsters for prolonged exposure periods. Laper and Burgess (8), however, have reported an increase in the acute inhalation toxicity related to restraint-induced stress.
Lung only and partial lung exposure techniques are also similar enough to share advantages and disadvantages. The major advantages are limited routes of exposure, direct knowledge of the actual quantity of test material delivered to the lung, and uniform delivery of multiple doses. Using other modes of inhalation exposure, i.e., whole body, the dose received during an inhalation exposure is a complex relationship between physiologic (rate, depth, and volume), and physical (particle size, collection efficiency, and retention) parameters. Disadvantages of the limited lung exposure techniques include the need for anesthesia and physiologic support, bypassing the nose, technical difficulty, limited number of animals that can be studied at any one time, and distribution of dose within the lung. Instillation of solutions or suspensions results in heavy, centralized deposits of material. Inhalation results in a lighter, even and widely distributed dose (9). While lung only or partial lung dosing procedures do not lend themselves to large scale screening/testing programs, they do have application in studies of absorption, metabolism, distribution, and excretion.
The design and construction of inhalation chambers have been extensively reviewed (3-5,10,11). Glass and various plastics have been used to construct inhalation chambers. Plastics, in general, tend to age rapidly with use. Both glass and plastic can build areas of high static charge, an undesirable feature in inhalation studies. Stainless steel is a common, and satisfactory material to use in inhalation chamber construction (Fig. 1). Glass windows are provided for observation. The poor thermal insulation properties of steel can be integrated into a chamber environmental control system (discussed later).
Inhalation exposure systems can be either static (no airflow) or dynamic (airflow). Static inhalation exposure systems, where a defined quantity of test material is introduced into a closed system and allowed to mix with the trapped air, are efficient in terms of test chemical usage. Static exposure systems are limited by depletion of oxygen, accumulation of waste, and loss of test agent. They are generally unsuitable for most inhalation toxicology studies under current standards. Most modern inhalation exposure systems are of the dynamic type (Fig. 2). Dynamic exposure systems are characterized by a continuous replacement of chamber air and test material.
The concentration profile in a dynamic inhalation system rises rapidly, then asymptotically approaches a theoretical equilibrium value (Fig. 3). This phenomenon was reported by Silver (12), who also described the time necessary to reach a desired percent of the theoretical equilibrium concentration:
Referring to Figure 3, the exposure duration for a dynamic system is generally considered to be the interval between starting (ta) and stopping (tb) the generation system. The animals, however, remain in the exposure system for a time equivalent to the initial tw (tc - tb). The suggestion by the National Ibxicology Program (NTP) that exposure duration should be defined as the interval tc-ta has not met with universal acceptance (4). The performance of inhalation exposure systems - i.e., leakage, material loss, and uniformity of concentration - has been addressed (13,14) and wrln not be reviewed here.
A requirement for currently performed inhalation studies is the ability to provide a consistently clean air supply, sourced from ambient air, to chambers housing control and treated groups. Environmental control, therefore, is an important aspect of inhalation exposure system design. Unusual environmental conditions may place an additional, and unwanted stress on test animals. The ability to produce, control, and monitor the required environment should be a basic consideration in conducting inhalation studies. Environmental fact ors such as temperature, relative humidity, atmospheric pressure, air flow, air quality, noise, and vibration could affect the evaluation of toxicity (Table 1). Physical activity, respiratory patterns, and respiratory tract mucus may be affected by temperature and humidity. Not only is it important to have a well-controlled environment for a particular inhalation chamber, but all chambers used in a study should be controlled within the same limits. Variations in the inhalation chamber environment may affect experimental animals and exposure atmospheres (15,16).
One environmental parameter that has received particular attention is temperature. Tbmperature could have an important effect on survival of test animals. In a dynamic inhalation system, heat produced by test animals is transferred to chamber walls and then to exposure rooms by radiation, particularly in the case of steel walls. Heat from animals is transferred to the chamber air by convection (16).
Figure 4 presents a schematic of the inhalation chamber control system used at Lilly Research Laboratories. The system was designed to provide very clean air. It also controls, monitors, and reports the chamber's temperature, dew point, air flow and differential pressure, and room temperature. Recognizing that steel chamber walls provide little thermal insulation, the effect on chamber temperature related to the differential between chamber and room temperature was integrated into the control design. The system was designed to provide an initial room temperature 2°C below the chamber temperature set point. The system then adjusts room temperature so that chamber inlet duct heaters operate at 20 to 80% of capacity. Each parameter is controlled within userdefined limits and is accessible through a personal computer.