386 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS The hair is first removed over an area of the back, using electric clippers. 'This may be followed by chemical depilation with barium sulphide paste, but there is a danger of a damaging keratolytic effect. Acute effects are determined from a single application, chronic effects from repeated application of the compound. Acute effects The compound is applied to the skin and covered with a piece of gauze held in place with adhesive piaster. Solids may be applied direct or in solution. Formulated preparations, such as ointments, creams and lotions, may be lightly rubbed in. The trunk of the animal is then wrapped in a plaster of Paris bandage to keep the test patch in position. The skin is examined after 24 and 72 hours, and the reactions scored according to an arbitrary scale which records the degree of damage. Mildly irritant substances produce no more than a slight erythema, moderate irritants a severe erythema, and severe irritants a marked necrotic effect with scab formation. In addition to tests on intact skin, the substance may be applied to abraded skin. Chronic effects These are determined following repeated application of the compound to the skin over a period of 10 to 14 days. Again the damage may be scored at the completion of the test. Some observers keep the skin covered throughout the test, others do not. Covering does prevent the animal from removing the compound, and it is not difficult to apply the substance under the piaster of Paris bandage. Covering does not in itself produce damage, but there is some evidence that covered skin is more sensitive than uncovered skin. A more sensitive method for assessing primary skin irritation is to inject a single dose of the compound intradermally into the skin of the guineapig or the rabbit. Again the hair is removed from an area on the back or flanks. A solution of the compound is injected intradermaliy using a volume of 0.05 to 0.25 m!. Several injection sites can be included in the same area, so that direct comparisons can be made in the same animal of different compounds. The injection sites are observed at intervals, over 7 days, and scored accord- ing to an arbitrary scale for the amount of damage produced. For inter- pretation of the results it is desirable to make comparisons with substances of known irritancy. Mucous membranes It is necessary to determine the effects of compounds on mucous mere-

TESTING DRUGS FOR DERMAL TOXICITY 387 branes when there is a danger of contamination or when it is intended to apply a medicament for a therapeutic purpose. Tests should be carried out on the specific mucosa to which the compound is to be applied, but this may not always be the best for quantitative comparisons. The eye of the rabbit is most commonly used. 0.1 ml of a solution of the compound is instilled into the cupped lower lid of one eye and held for 30 seconds. The other eye is left untreated as the control. The solution may be washed out after a certain period of time, but it is more usual to leave it in contact unless it is causing severe damage. The effects on the cornea and conjunctiva are read at intervals over 7 days and scored according to an arbitrary scale, such as the one described by Draize (1). Substances which do not irritate the eye are unlikely to be irritant to the skin, but eye irritation does not preclude the use of a chemical on the skin. In addition to the eye, tests may also be carried out on the penile mucosa of the rabbit, and in the vagina of the rabbit, guineapig, rat, monkey or dog. SENSITIZATION STUDIES Sensitization reactions in the skin represent an allergic reaction to the chemical, and require prior exposure for development. They are prone to occur in humans, but are difficult to produce in animals. Little reliance can be placed on animal tests, and trials need to be made in human volunteers. DraJze (1) does describe a test in guineapigs which will detect severe sensitizers. A solution, or suspension, of the compound in normal saline is injected intradermally into the skin of guineapigs every other day up to a total of 10 injections over an area of 3 to 4 cm •. The first injection is 0.05 ml, and the remaining nine, 0.1 mi. Two weeks after the last injection a retest injection of 0.05 ml is made in an area just below the region of the sensitizing injections. 24 hours later readings are made of the diameter, height and colour of the reaction, and compared with the average of those observed for the original injections. Sensitization studies may include photosensitization, and this may be determined in rabbits submitted to ultraviolet light. SYSTEMIC I)ERMAL TOXICITY When drugs are applied to the skin for a local therapeutic effect it is possible for systemic absorption to occur, leading to distant toxic effects. The dermis forms a barrier to absorption, and if it is damaged considerable absorption may occur. Absorption is much greater from mucous membranes. For systemic effects, the drug must be absorbed into the tissues and general circulation, and the damage may occur in distant organs such as