380 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The protected depilatory was a nfild irritant in 9 of the 25 patients, and the nail polish remover produced a severe reaction in all of the patients. The shampoo, when applied to 25 humans, caused a mild inflammation at the site of application in 16 of the 25 patients, while the cuticle softener resulted in a severe dermatitis in all of the patients treated. It appears from this direct comparison between the responses obtained in the rabbit, and the responses obtained in the human, that the reactions in the rabbit can be used to predict the type of response which will occur in the human. The reaction of the rabbit's skin to a primary irritant appears to be a local one which manifests itself at the site of contact. Systemic changes may also occur following a single application of a cosmetic. The evaluation of a systemic reaction which can be judged by the level of a chemical in the blood stream or in an organ of the body can be quantitatively measured, and lends itself to a greater accuracy of determination than does the local dermal reaction. Most preparations intended for the skin do not show any systemic activity. Exceptions are creams, and lotions, containing estrogenic hormones. These are assayed by virtue of systemic absorption which results in vaginal wall changes, and subsequent estrus in the anestrus ovariectomized rat. In systemic toxicity the entire animal responds to the material rather than just the skin at the point of application, with absorption becoming a necessary adjunct to the final response. To study the acute response of a preparation, rabbits are prepared as previously described but the dose used on the exposed area is greater per kg of animal. The hair is removed from the entire back and sides of rabbits weighing from 2 to 3« kg, and abrasions are made as previously described. The test material is applied to groups of 3 rabbits at dose !evels ranging from 4 to 10 g per kg of body weight. Applications of the test material are introduced under a rubber sleeve which fits over the entire immobilized animal, and contact is for 24 hours. After that time the sleeve is removed, the animals' backs are washed clean, their skin reactions recorded, and they are returned to their individual cages to be observed for at least 14 days after the treatment. If the test material is an ointment or a cream, the use of a rubber dam is not required. Before the test is begun, and 1 week and 2 weeks after the application of the cosmetic, a complete blood count and urinalysis is performed. If the cosmetic contains an ingredient concerning which a chemical method for its ana!ysis in blood or urine is known, this analysis should be performed 24 and 48 hours after the application of the test material. At autopsy, sections of the liver, spleen, bone marrow and kidney of each an.•mal are prepared for histopathological examination. A/though death may not result from this single dermal application, physiological changes may occur, together

RABBIT SKIN STUDIES IN EVALUATING COSMETIC SAFETY 381 with changes in the hematogram and urine constituents. Several examples may be given to indicate the usefulness of this type of experiment. (1) A tin compound which was to be used as a bacteriostatic agent, and which apparently was absorbed by the animal causing neurological changes. The animal was unable to move about due to paralysis of the hindquarters. There was also an increase in sensitivity to pressure, at the site of application. (2) A compound of a heavy metal which was to have a cosmetic effect upon the skin. Enough of the material was, however, absorbed through the skin to accumulate in the kidney, and cause functional damage. Chemical analysis of this organ showed appreciable levels of the metal to be present. Single applications of a new cosmetic in small or large amounts may result in no irritation or systemic toxicity. A repeated exposure to small amounts of the compound may result in a cumulative effect. Since cosmetic preparations are used daily for long periods of time a procedure is followed which involves daily application of the substance under study. 90 daily applications are made to the clipped backs of groups of rabbits, and they are observed for changes in body weight, behaviour pattern, hematological values, and urine constituents. When the animals are sacrificed, a complete histological examination is made of the major organ systems. At least three levels of the material, in terms of body weight, are employed and a blank control group is always included. The levels applied are in direct relation to eventual human use, calculated on a weight basis, and range from three to ten times the amount that would normally be applied. The applications are made daily, and are rubbed into the clipped back of each animal with a glass rod. Approximately 10% of the animals' skin area is covered, and the animals are maintained in multiple animal holders for 4 hours after each treatment. After this period, the remaining material is wiped off, and the animals are returned to their individual cages. This procedure has shown that continuous exposure, with applications always to the same area, can be used to detect substances that are non-irritating on single contact, but do produce a definite change in the skin after multiple applications. If experience indicates that the animals may lick off enough of the material to cause toxicity by ingestion, they are fitted with a harness which does not allow them to lick their backs but does allow them to carry on all their normal functions (1). In a study of the effects of a series of potential lipstick colours upon the skin, it was observed that a particular colour produced death to a number of animals. At first, absorption through the skin was suspected but after the use of the harness none of the animals died. The animals were licking off enough from their backs to cause oral toxicity. An attempt has been made to indicate the usefulness of animal data in evaluating the safety of cosmetic preparations. In 4 examples described,