j. Soc. Cosmet. Chem., 46, 117-127 (March/April 1995) In vitro skin absorption and metabolism of Padimate-O and a nitrosamine formed in Padimate-O-containing cosmetic products G. E. KENNEY,• A. SAKR, J. L. LICHTIN, H. CHOU, and R. L. BRONAUGH 2, Hill Top Research, Miamiville, OH 45147 (G. E. K, ), College of Pharmacy, University of Cincinnati, Cincinnati, OH 45221 (A.S., J.L.L.), and Food and Drug Administration, Laurel, MD 20708 (H.C., R.L.B.). Received August 5, 1994, Synopsis 2-Ethylhexyl p-(N,N-dimethylamino) benzoic acid (Padimate-O) has been a popular sunscreen agent. In 1988 it was reported by the U.S. Food and Drug Administration to sometimes contain a nitrosamine contaminant, 2oethylhexyl p-(N-methyl-N-nitrosamino) benzoic acid (NMPABAO). The nitrosamine was initially reported to be mutagenic in the Ames test, but this finding was not confirmed by others. The in vitro percutaneous absorption and metabolism of NMPABAO and Padimate-O were determined in hairless guinea pig and human skin. The viability of excised skin was maintained for 24 h in flow-through diffusion cells. The percutaneous absorption of Padimate-O was almost fourfold greater from a volatile vehicle (ethanol) than from a lotion vehicle. Substantial amounts of the absorbed compound were hydrolyzed by esterase enzymes in skin. The percentages of the applied dose of NMPABAO absorbed (lotion vehicle) were 5.2 (hairless guinea pig) and 2.3 (human skin). About 26% (hairless guinea pig) and 85% (human) of the NMPABAO absorbed into the receptor fluid were hydrolyzed to p-(N-methyl-N-nitrosamino) benzoic acid (NMPABA). In some experiments, NMPABAO applied to hairless guinea pig skin was immediately irradiated with UVA and UVB light from a solar simulator. The amount of NMPABAO absorbed was reduced 17-fold as a result of the instability of the nitrosamine in UV light. However, UV instability generally cannot be considered a safety factor because many sunscreen users apply these products on a regular basis regardless of whether exposure to the sun is expected. INTRODUCTION 2-Ethylhexyl p-(N,N-dimethylamino) benzoic acid (Padimate-O), a tertiary amine de- rivative ofp-aminobenzoic acid (PABA), is a UV-absorbing chemical used in cosmetics • Results submitted as partial fulfillment of requirements for the M.S. degree in Cosmetic Science, College of Pharmacy, University of Cincinnati, Cincinnati, OH 45221. 2 Reprint requests should be addressed to R. L. Bronaugh, Food and Drug Administration, HFS-128, Office of Cosmetics and Colors, 8301 Muirkirk Road, Laurel, MD 20708. 117

118 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS and sunscreen formulations. Its efficiency as a UV absorber is due in part to its high molar absorptivity in various solvents and its ability to absorb UV radiation in the spectral range of 300 nm (in nonpolar solvents) to 316 nm (in polar solvents) without the extensive oxidation and intermolecular hydrogen bonding that PABA and some of its derivatives undergo. Recently, however, concern has been growing about the possible nitrosation of Padimate-O to a nitrosamine, 2-ethylhexyl p-(N-methyl-N-nitrosamino) benzoic acid (NMPABAO), in cosmetic products. In May 1988, the U.S. Food and Drug Administration (FDA) reported that a new nitrosamine was found in certain sunscreen products. Of the 17 sunscreen products tested, 14 were found to contain NMPABAO at levels varying from 60 to 1960 ppb (1). Animal studies have shown that nitrosamines are carcinogenic and may be mutagenic and teratogenic as well (2). More than 120 N-nitroso compounds have been examined for carcinogenic activity in animals, and approximately 80% of these have been found to be carcinogenic to some degree (2,3). NMPABAO was reported to be mutagenic in the Ames test using two strains of Salmonella typhimurium (4). However, in a more recent FDA study, NMPABAO was not found to be mutagenic in a battery of S. typhimurium studies (including the same assay in reference 4) or in the mouse lymphoma assay (5). The possible carcinogenicity of NMPABAO has not been evaluated in an animal bio- assay. We investigated the penetration and metabolism of NMPABAO in hairless guinea pig and human skin to evaluate the compound's safety. The viability of the skin was maintained in flow-through diffusion cells. The results of some of the experiments in which the viability of the skin was maintained were compared with those from exper- iments that used nonviable skin. The effects of two cosmetic vehicles (ethanol and a lotion) on the penetration and cutaneous metabolism of Padimate-O were also deter- mined. The nitrogen-nitrogen bond of nitrosamines is weak and can be broken by relatively low energy inputs, including energy from UV radiation (6). Therefore, the photodecompo- sition of NMPABAO in a cosmetic vehicle was investigated in preliminary studies (without skin) and in diffusion-cell skin absorption studies. MATERIALS AND METHODS [•4C]NMPABAO (specific activity 20.85 mCi/mmol, purity 98%) and [•4C]Padi- mate-O (specific activity 20.0 mCi/mmol, purity 86%) were synthesized by Research Triangle Institute, Research Triangle Park, NC. Padimate-O was further purified on Sep-Pak © silica cartridges (Waters Associates, Milford, MA) immediately before each experiment, giving a final purity of 96%. Absorption and metabolism experiments were conducted in vitro, using flow-through diffusion cells (7). The system was cleaned with a 70% ethanol solution to prevent interference due to bacterial metabolism. Most studies used skin from the hairless guinea pig (female, 3-6-months old) that was prepared with a Padgett dermatome (Padgett Instruments, Kansas City, MO) at a thickness of approximately 200 •m. Several studies were performed using surgical specimens of viable human skin obtained from abdominoplasty and dermatomed to 200 •m. Barrier integrity of the human specimens was verified by using [3H]water absorp- tion as a standard (8).