Human metabolism and biomonitoring of the UV filter 2-ethylhexyl salicylate after dermal exposure

Exposure to sunlight has various beneficial effects for the human body and is a crucial factor in the endogenous production of vitamin D. However, sunlight, more specifically ultraviolet (UV) radiation can also cause damage to the skin. Such skin damage shows in premature skin aging, discoloration and, in severe cases, can lead to the formation of skin cancers. To counteract skin damage through UV radiation and therefore also partially prevent the resulting conditions and diseases, the use of UV protection is advised. UV protective measures comprise limiting the overall sun exposure, seeking shade, using clothing to cover and using sun protection products such as sunscreens. Sunscreens contain various UV filters to provide a broad coverage of the UV spectrum, one common UV-B filter is 2-ethylhexyl salicylate (EHS). Since sunscreen and other personal care products with UV protection are applied on large body surfaces in various scenarios and often on consecutive days, the dermal absorption and the following metabolism and excretion are of high interest. From a toxicological point of view, EHS is not associated with a high toxicological potential, nevertheless there are indications for an endocrine disrupting potential which is being further investigated. Previous studies on the metabolism of EHS after oral administration revealed relevant oxidative metabolites. Also, the dermal absorption has been shown by the detection of EHS in human plasma after sunscreen use as well as of EHS and an oxidative metabolite in human urine. Thus far, no comprehensive method for the reliable determination of EHS together with several oxidative metabolites known from oral and dermal exposure studies as well as further potentially relevant metabolites is available. Furthermore, there are no existing dermal exposure studies with multiple participants under controlled conditions to assess reliable toxicokinetic data, which is crucial for risk evaluation. Based on the existing studies, a novel methodology was developed for the comprehensive and reliable quantification of EHS together with seven oxidative metabolites in human urine. Within the method development, the stability of EHS in comparison to its glucuronide conjugate was extensively studied, showing that a reliable quantification of EHS requires the use of glucuronidated EHS as analytical and internal standard. Besides the assessment of EHS stability, an analytical method was developed to include seven EHS metabolites, including four isomers, besides EHS itself. This was facilitated using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and a carefully adjusted gradient program as well as evaluation of several analytical columns. Sample preparation was designed efficiently by using an online clean up and enrichment step within the LC system. After chromatography, analytes were detected with negative electrospray ionization followed by tandem mass spectrometry (ESI-MS/MS). The method was extensively validated and exhibited good reliability, sensitivity, and robustness. Therefore, urine samples from an existing sample study could be analyzed. The targeted study was conducted under real-life conditions, including full-body dermal sunscreen exposure, except body parts covered by swimsuits, an outside venue and sunscreen reapplication. With a larger participant number of 18 volunteers, samples could be evaluated regarding the relevance of the metabolites and EHS included in the analytical method. This confirmed oxidation at omega and omega-1 positions of the ethylhexyl side-chain but furthermore revealing relevant oxidation at the omega-2 position in the ethylhexyl sidechain of EHS after dermal exposure. Following the analysis of samples after real-life dermal exposure, an in vivo dermal exposure experiment was conducted under controlled conditions to assess toxicokinetic data of EHS and oxidative metabolites. Three participants were exposed to EHS containing sunscreen on 75% of the body surface area (BSA). The exposure took place indoors to prevent the influence of sun exposure on the dermal absorption. Peak elimination occurred after 10-11 hours after application and elimination half-lives in phase 1 were between 6.6 h and 9.7 h . Additionally, the shares of sulfate and glucuronide conjugation were assessed, revealing up to 40% share of sulfate conjugation for three oxidative metabolites.

Within the framework of this thesis, new information on the metabolism of the UV filter EHS after dermal application was gained. A novel, reliable, and comprehensive analytical method was developed to assess EHS and seven oxidative metabolites in urine. The application of this method to urine samples from a real-life dermal exposure study facilitated an extensive evaluation of relevant parameters for dermal EHS exposure in urine. Furthermore, reliable toxicokinetic data for EHS and oxidative metabolites was obtained from a controlled dermal exposure experiment which was performed within this thesis. The present work therefore contributes to the understanding of the human metabolism of EHS after dermal exposure and provides the tools to comprehensively assess EHS and oxidative metabolites in human urine. This thesis therefore provides an important scientific basis for the exposure and risk assessment of EHS exposure after typical consumer-use of sunscreen products.