284 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS INHALATION METHODOLOGY Systems The first factor to be considered with regard to the design of an inhalation experiment is the design of the exposure system. Many considerations, includ- ing the number and species of animals to be used, space available, nature of the sample, etc., are involved in the selection of equipment for this type of toxicity testing. There are two basic types of inhalation exposure (and sev- eral variables of these): head-only and whole-body exposure. In the former type of experiment, the animal may be in an enclosure which is external to the actual chamber only its head protrudes into the chamber and is exposed to the test material. This approach finds frequent use in noncosmetic areas where the possibility of oral ingestion of the test material should be separated from the inhalation route of entry oral intake of the material can occur quite easily if the whole animal is exposed and goes through the normal routine of preening its fur which has been saturated with an aerosolized prod- uct. A modification of the head-only type of exposure is one in xvhich the aerosol is delivered through a face-mask which has been fitted to the animal an antiasthma preparation was recently reported as having been tested in this manner ( 1 ). The whole-body method of exposure probably best approximates the types of contact with which we must deal in the use of cosmetic aerosols. In expos- ing the entire animal to an aerosolized material, of course, it must be under- stood that in addition to conducting an experiment in inhalation toxicology, we are also performing qualitative tests in eye irritation, oral toxicity, and derreal toxicity since all of these organ systems are being exposed to the aerosol in addition to the lungs. It makes good sense, then, to fully investigate the potential for toxicity on these other organ systems before investing time and effort in an inhalation study. Many differcnt chamber designs have been suggested since the original plexiglass box used by Draize (2). In one type, the axis of orientation is hori- zontal and the test material is introduced on one side, while the chamber is evacuated on the other side. In a second type, the axis is vertical and sample movement is from top to bottom. The latter seems to be more popular and is generally used in conjunction with a chamber whose body is basically cubical and tapers towards the top and bottom. Allusion was made to the introduction and distribution of sample. Again, there are two basic approaches available. In the so-called "static chamber" system, the dose of material is introduced into the chamber atmosphere, the system is sealed, and the animals breathe only thb air which is present in the chamber. In the "dynamic" system, the gas or suspended liquid is introduced into a stream of air which is continuously sweeping through the chamber at a constant rate. With proper chamber design, good distribution of the aerosol is almost assured if the airflow is rapid enough.

TESTING FOR INHALATION TOXICITY 285 No matter what type of exposure chamber is used, size is very important. This does not mean that a big chamber is better than a small chamber, or vice versa. The essential point is that the volume which the animals occupy within the chamber should be small in relation to total chamber volume a generally accepted figure for animal volume is around 5% (3). In such a situation, especially in a long-term study, the inhaled doses are more easily standardized than if a large portion of the chamber volume is removed with each inspiration. This chamber-to-animal volume ratio is even more critical if a static exposure system is being used, because here the available oxygen is being used up and not replaced. Animal Selection It cannot be stated that, for example, "the rat is the animal of choice for inhalation studies." All species are obviously quite different and may react differently to the material being tested. Whatever animals are chosen, the species best suited to the particular experiment should be used. This means that the way in which the animal metabolizes the test material, for example, should be taken into account. The vital capacity of each species should also be a part of the calculation of dose. Chamber Environment As in the animal colony, the conditions of temperature and humidity within the inhalation chamber should be standardized-usually at 25øC and 50% relative humidity. Needless to say, there must be a sufficient level of oxygen for the animals to breathe, and there must be an efficient method of removal of expired CO2 from the chamber. In a static system, these factors limit the length of time that the animals can be exposed. The homogeneous distribution of gases or suspended liquids within the inhalation chamber is also of critical impm'tance. This is affected by several factors among them, method of generation of the aerosol, rate of airflow, and chamber geometry. Also to be taken into consideration is the equilibra- tion time, that is, how long it will take the test material to attain a constant level within the chamber. If this is significant, then the overall duration of the experiment must be modified accordingly. The duration of the experiment is variable and depends on the type of material to be tested and the intended dose level. A commonly used exposure time in a dynamic system is 4 hours per day. As mentioned above, this would be practically impossible in a static chamber, unless the chamber-to-animal volume ratio• were extremely large. While the animals are in the chamber, there should be facilities for monitoring the concentration and, if desirable, the particle size of the test material in the experimental atmosphere gas- liquid chromatography, infrared, and standard wet chemistry can be per- formed for analytical purposes. The techniques for particle sizing are many