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

286 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS and varied. They range from simple collection on a glass slide, followed by visual classification, to the sophisticated technique of laser holography. Again, the method of choice is the one that gives the investigator the informa- tion he wants within the realistic limitations of his experiment. Length o[ Study The overall duration of the experiment can be acute, subacute, or chronic. As in other types of toxicological investigation, the acute study is conducted to enable us to get an idea of the to•xic potential of the material in question in relation to other materials. This is normally referred to as the LD5o-the amount of test material which will kill 50% of the population to which it is administered in one do'se. In inhalation toxicology this value is called the LCa0, C representing the concentration of the material in air, and it is us- ually expressed with relation to time in terms of ppm, mg/l., or mg/m s. It should now be clear that it is even more important to know chamber and animal volume so that the nominal dose of inhaled material can be calculated. In most cases, when dealing with cosmetic aerosols, the LC•o is difficult if not impossible to calculate because the airborne concentration of material which might produce death in the animals is impractically high. For this rea- son, the procedure of selecting some fractional multiple of the LC50 to use for the subacute study is inappropriate. Instead, the test material may be aerosolized at a nominal chamber concentration of 20-200 mg/m 3, the pre- cise dose being left to the investigator's iudgment and experience, and his understanding of how the product is intended to be used. Chronic studies, which may run for oe years, have rarely been used in the area of cosmetic inhalation toxicology. In the future, however, some empha- sis may shift toward this variety of test, particularly with reference to. the in- vestigation of raw materials and aerosol propellants. Larger species of ani- mals such as dogs or monkeys are many times preferred for such testing. Observations Throughout any inhalation experiment, the animals should be observed carefully and regularly for any visible signs of toxicity such as changes in behavior, physical appearance, locomotor activity, etc. In addition, before a subacute or chronic study is initiated, while it is in progress, and after its completion, the animals should be weighed and a record kept of their food consumption. At the termination of the experiment blood samples are taken from the ani- mals and the standard hematological parameters are examined serum en- zyme levels and other clinical tests are also performed. After sacrifice, tissues from the various organ systems are removed and fixed for histological section- ing. The histopathological findings can then be added to the gross observa-