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-

TESTING FOIl INHALATION TOXICITY 287 tions at autopsy, and to the blood data, to get a picture of the effect of the test material. Special Measurements Besides histological examination of tissue sections, another parameter which may be of interest to the inhalation toxicologist is the observation of the muco- ciliary activity .of the respiratory system. This activity is one of the ways that the lungs are protected from the deposition of foreign material. Basi- cally, the cells lining the trachea, larynx, and upper airways have special modifications called cilia which are microfine, whisker-like projections from the free surfaces of these cells. These cilia have an inherent, rhythmic beat, whose total activity produces a wave-like motion directed towards the pharynx. In addition to the ciliated cells, interspersed among the latter are the so-called goblet cells which secrete a mucous substance. This mucous forms a blanket overlying the cilia so that fine inhaled particles may become entrapped in the mucous and removed from the respiratory system by move- ment of the blanket layer via the underlying cilia. It is known that certain inhaled materials impede this mucociliary stream- ing, and some actually paralyze it. To determine whether it is still operating effectively, a fresh tracheal preparation taken from an animal that has been exposed to the suspect material can be examined. Usually, pollen grains or synthetic microspheres are placed on the tracheal membrane, and their pro- gress between two fixed points is observed under a microscope. Another meth- o,d for monitoring mucociliary function involves administration of an aerosol containing radiolabelled particles to an animal (usually of large size). The tnovement of these particles back up the airways is then monitored by scintil- lation detectors and their rate of clearance is deternfined. If the inhaled particle escapes the mueoeiliary system and actually gains access to the alveoli of the lungs, it is confronted with a second system which is concerned with entrapment and removal of foreign matter. There is pres- ent in the interalveolar walls a specialized cell called the septal cell whose job it is to act like the policeman for the alveolus. In performing this func- tion, termed phagoeytosis, projections of the septal eytoplasm sun'ound the particle, and then join, enclosing it within a vacuole. Soon after this, such a cell becomes detached from the alveolar wall. In this free state it is commonly referred to as an alveolar maerophage it still participates in phagoeytie activ- ity, and it also appears to. be capable of ameboid movement. It is by way of this movement that the maerophage reaches the ciliated lining of the air- ways through which it is carried toward the nasopharynx, where it enters and is swallowed. The possibility exists that the septaI cells too may be adversely affected by aerosol inhalation. The most common derangement might be a simple over-