SUNSCREEN EFFICACY 277 Hill Top Research Inc. for confirmatory clinical data, according to the FDA monograph (14). ANIMAL PREPARATION Two young adult male Hartley guinea pigs from Murphy Breeding Laboratories Inc. (Plainfield, IN), weighing approximately 750 grams each, were used for each trial. The large size of this animal provided a broad dorsal surface area for exposure and irradia- tion, although this size of animal is not critical to the observation. On the day prior to treatment, the entire dorsal surface of each animal was clipped using a no. 40 clipper blade. This area was then depilated using a commercial depila- tory that was applied and left for no more than 15 minutes. The depilatory was washed off thoroughly with warm running water. The animals were towel dried and returned to their cages. TEST MATERIAL APPLICATION On the day following animal preparation, a rectangular area (approximately 3 X 10 cm), parallel to the dorsal midline on the right side of each animal, was delineated with a waterproof marker. Using a microliter syringe, the test material was deposited evenly across the rectangle, resulting in a total application of 2 I•l/cm 2. This material was spread using a gloved fingertip. For the evaluation of UVA screening potential, approx- imately two hours prior to test material treatment, 8MOP was administered orally at a dose of 20 mg/kg. This rendered the animals photosensitive to the UVA spectrum. IRRADIATION The animals were restrained on an irradiation table in a circular arrangement (15). After the animals had been properly positioned, opaque templates were applied, isolating three test sites, arranged linearly, on each side of the dorsal midline. A fourth skin site was isolated on one side of each animal as a nonirradiated primary irritation control site. A light wheel (15), holding either twelve UVA black light bulbs (20 watt F20T12/BL) or twenty four UVB sunlamp bulbs (FS20 USA, 20 watt) in a spoke-like fashion, was started and revolved over the restrained animals. Approximately two minutes after the wheel had been started, the lamps were turned on. The three untreated test sites on the left side of the animal were sequentially irradiated from head to tail for three, six, and nine minutes, respectively, when testing with UVB radiation and for six, eight, and ten minutes, respectively, when testing with UVA radiation. The exposed skin sites were occluded, as appropriate, with plastic tape to discontinue exposure. The middle time value had been predetermined to approximate the respective minimal erythemic dose (MED) for the experimental conditions. The opposite three treated test sites were each irradiated for time periods that corresponded to the product of the target value to be investigated and the irradiated time period of the paired untreated site. WASH-OFF When wash-off data were to be obtained, the appropriate animals were individually

278 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS restrained in a specially designed wash-off tank. The tank was filled and the animals were exposed to mildly turbulent tap water, held at an approximate temperature of 38øC. The total duration of wash-off was forty minutes, consisting of two 20-minute periods. Each 20-minute wash period was followed by a 20-minute rest period. The tank was refilled with fresh water just prior to each period. One should not expect to fully discriminate between water-resistant and waterproof claims in the guinea pig, even though it is intended that the wash-off procedure will estimate substantivity. The main concern is to establish a degree of substantivity under mild water activity. The finer analysis should be left for the clinical trial. SKIN SITE EVALUATION The skin sire evaluation system used for each animal is based on the system established for evaluating human skin responses as described in the OTC monograph (14). In this instance, relative increases or decreases in responsiveness are recorded for the treated sires, as compared to nontreated, in order to describe the test material as effective or ineffective at the expected target value. It is the trend observed in each animal which is combined and subjectively evaluated to give the final results for a given test material. The day following UVB irradiation or three days following UVA irradiation, the entire dorsal surface of each animal was depilated using a commercial depilatory. The depila- tory was applied for no more than fifteen minutes and was thoroughly washed off with warm running water. The animals were rowel dried and returned to their cages. A minimum of two hours following depilation the irradiated sires of each animal were evaluated and scored. The six irradiated skin sires of each animal were progressively read from left to right, head to rail, using the following symbols: -, no appreciable response +, response =, equal to •, less than •, greater than The symbols •, (less than or equal to), and •, (greater than or equal to), although not used for formal scoring, are used for the presentation of data. Each treated/nontreated pair was evaluated separately. There was no marching of com- parable degrees of erythema on the opposite sides of the animal in order to determine a specific protection factor. We feel this approach provides a superior evaluation of the sunscreen's actual potential. In addition to this evaluation, the quality of response at the treated and untreated skin sires of test animals was evaluated, as necessary, to fully define test material activity. The primary irritation control sire was also evaluated and taken into consideration during skin sire evaluation, in order to determine the primary irritative effects of the rest material. Since some test materials may be inherently irritating and produce an erythematous background, this effect must be subjectively evaluated. We have found that this may complicate the evaluation of sunscreen effectiveness, but it does not neces- sarily obviate the interpretation. The raw data, thus obtained, indicates when a response was noted on an untreated sire, whether or not the treated sire was comparable, and the differential quality of the reactions. The values determined for the guinea pig are presented as sun protection estimates (SPE) to differentiate them from human values (10).