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Urinary metabolites of the UV filter 2-Ethylhexyl salicylate as biomarkers of exposure in humans
Toxicology Letters 309 (2019) 35–41
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Urinary metabolites of the UV filter 2-Ethylhexyl salicylate as biomarkers of exposure in humans
T
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Daniel Burya, , Peter Griemb, Tanja Wildemannb, Thomas Brüninga, Holger M. Kocha a Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-Universität Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany b Symrise AG, Mühlenfeldstrasse 1, 37603, Holzminden, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Ethylhexyl salicylate EHS UV filter Metabolism Oral dosage Human biomonitoring Exposure biomarker Urinary excretion fraction
The UV filter 2-ethylhexyl salicylate (EHS) is used in sunscreens and other personal care products worldwide and has been found in a variety of environmental media. We aimed to provide human toxicokinetic data on EHS as a tool for risk assessment. For that purpose, we investigated metabolism and urinary metabolite excretion after a single oral EHS dose (57.4–75.5 μg/(kg body weight)) in three male volunteers. In a suspect screening, we tentatively identified seven EHS metabolites. Three EHS specific metabolites were quantitatively investigated: 2ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5-oxohexyl 2-hydroxybenzoate (5oxo-EHS), and 5-(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx-EPS). These metabolites were excreted with urinary excretion fractions of 0.28% (range: 0.13−0.54%), 0.11% (0.06−0.20%), and 0.24% (0.14−0.41%), respectively. The elimination was fast: peak urinary concentrations were found 1.6–2.6 h after dose and ≥95% of the total amounts were excreted within 24 h. Elimination kinetics were biphasic, with mean elimination half-lives of 0.8 h (first phase) and 6.6 h (second phase) for 5OH-EHS, 0.8 h and 6.3 h for 5oxo-EHS, and 1.1 h and 5.9 h for 5cx-EPS. After dermal exposure (sunscreen application), we found a considerably delayed EHS elimination. Based on urinary metabolite levels we calculated EHS exposure levels for a small pilot population.
1. Introduction Sunscreens are frequently applied to reduce health risks associated with excessive (solar) UV radiation, both in private life and occupational settings (Greinert et al., 2015; Ruppert et al., 2016). The UV-B filter 2-ethylhexyl salicylate (EHS, ethylhexyl salicylate, octyl salicylate, octisalate; CAS registry no. 118-60-5; EC no. 204-263-4) is used in sunscreen formulations worldwide (Shaath, 2010), as well as in other personal care products (PCP), such as after shave, lipsticks, face creams, make-up foundation, and perfumes (Kerr, 2011; Manová et al., 2013; Uter et al., 2014). The maximum permitted concentration in cosmetic products is 5%, both in the U.S.A. and the European Union (U.S. Food and Drug Administration, 2019; European Parliament and the Council, 2009). in vitro studies on endocrine activity of EHS showed some anti-androgenic activity and weak to no estrogenic, anti-estrogenic, and
androgenic activity (Miller et al., 2001; Morohoshi et al., 2005; Kunz and Fent, 2006; Jiménez-Díaz et al., 2013). Given its potential endocrine activity, EHS has been included in the Community rolling action plan (CoRAP) of the European Union in 2018 (European Chemicals Agency - CoRAP, 2019). Environmental monitoring has provided evidence for the presence of EHS in a large variety of environmental matrices (such as surface freshwater and sea water, beach sand, sediments, wastewater treatment plant effluents, soil, and marine wildlife organisms) and also in indoor dust (examples in Negreira et al. (2009); Albero et al. (2012); Sankoda et al. (2015); Ekpeghere et al. (2016); Apel et al. (2018); Cunha et al. (2018); Vila et al. (2018)). Given the widespread use of EHS in PCPs resulting in direct EHS contact of consumers, and further considering its presence in the environment, the determination of EHS body burdens is of high interest. Human biomonitoring (HBM) provides an integral measure of exposure to chemicals, covering all exposure sources and
Abbreviations: EHS, 2-ethylhexyl salicylate; 5OH-EHS, 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate; 5oxo-EHS, 2-ethyl-5-oxohexyl 2-hydroxybenzoate; 5cx-EPS, 5(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid; 2cx-MHS, 2(1-carboxymethyl)-hexyl salicylate; cx-HeS, carboxyheptyl salicylate; SA, salicylic acid; SUA, salicyluric acid; PCP, personal care product ⁎ Corresponding author. E-mail addresses: [email protected] (D. Bury), [email protected] (P. Griem), [email protected] (T. Wildemann), [email protected] (T. Brüning), [email protected] (H.M. Koch). https://doi.org/10.1016/j.toxlet.2019.04.001 Received 12 February 2019; Received in revised form 20 March 2019; Accepted 1 April 2019 Available online 03 April 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.
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after the dose (oral dosing study) and 96 h after the dose (dermal pilot study), respectively, and stored at −20 °C until analysis. Times of urine collection were recorded by the volunteers. Urine volumes were determined via the weight difference between filled and empty PE containers and urinary creatinine was determined according to Jaffe (1886) by contract analysis (L.u.P. GmbH Labor und Praxisservice; Bochum, Germany).
uptake routes, and is well established for exposure assessment both in large scale population studies and on an individual level (Angerer et al., 2007; Needham et al., 2007; Schindler et al., 2014; Haines et al., 2017; Kolossa-Gehring et al., 2017; Schwedler et al., 2017). Recently, we described an HBM method for the determination of three specific urinary metabolites of EHS (Bury et al., 2019a), which were identified in the current metabolism study: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5-oxohexyl 2-hydroxybenzoate (5oxoEHS), and 5-(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid (5cxEPS). We demonstrated the potential use of these three metabolites as robust and specific biomarkers of EHS exposure after application of a commercial sunscreen containing EHS to one human volunteer. Furthermore, we were able to quantify EHS metabolites in 91% of urine samples from a pilot population from the general population (n = 35). For risk assessment purposes, quantitative data on xenobiotic metabolite formation and elimination (such as urinary excretion fractions) in humans is highly valuable. Urinary excretion fractions can be used to calculate external doses in terms of daily intakes (DI) based on measured urinary biomarker concentrations. These DIs can be related to toxicological threshold values, such as tolerable and daily intakes (TDI/ ADI). With the same data, health-based guidance values for biomarker concentrations (such as human biomonitoring assessment values (HBM values) or biomonitoring equivalents (BE) (Angerer et al., 2011; Hays and Aylward, 2012; Apel et al., 2017)) can be derived, enabling an accurate risk assessment based on urinary metabolite levels (examples in Conrad et al. (2017); Koch et al. (2017); Moos et al. (2017); Ulrich et al. (2018)). Accordingly, this study aimed to investigate EHS metabolism and quantify urinary excretion of specific metabolites.
2.3. Metabolite screening The identification of EHS metabolites was performed using a triplequadrupole-MS suspect screening approach previously applied to the UV filter octocrylene by our group (Bury et al., 2019b). All urine samples from one volunteer were prepared by enzymatic deglucuronidation as described in Bury et al. (2019a), however with omission of internal standards (which were not yet available at that time). These processed urine samples were then analyzed with online-SPE-LC–MS/ MS. For further details see the Supplementary Material. 2.4. Quantification of metabolites 5OH-EHS, 5oxo-EHS, and 5cx-EPS were quantified by stable isotope dilution analysis using an online-SPE-LC–MS/MS method, recently published by our group (Bury et al., 2019a). In short, after addition of a pure β-glucuronidase from E. coli K12, buffer, and stable isotope labeled internal standards, urine samples were incubated at 37 °C for 3 h for the cleavage of glucuronic acid conjugates. Then, samples were frozen overnight, thawed, equilibrated to ambient temperature and centrifuged at 1900 g for 10 min. 100 μL of the supernatant were analyzed. The limits of quantification (LOQ) were 0.05 μg/L (5OH-EHS), 0.15 μg/ L (5oxo-EHS), and 0.01 μg/L (5cx-EPS).
2. Materials and methods 2.1. Chemicals and reagents
2.5. Statistical analysis 2-Ethylhexyl salicylate (‘kosher’, obtained from Sigma-Aldrich, Steinheim, Germany; chemical purity 99.7%) was used in the oral dosing study. For further chemicals and reagents see Bury et al. (2019a). The β-glucuronidase from E. coli K12 applied for metabolite deconjugation (Sections 2.3 and 2.4) was a pure β-glucuronidase preparation without aryl sulfatase activity.
Elimination half-lives were calculated using the equation t1/2 = ln (2)/k, with k being the kinetic constant of the exponential decline in excreted metabolite amounts (Byers and Sarver, 2009). k was obtained by exponential regression of the metabolite excretion rates (ER in μg/h) vs. the midpoint of each time interval (t in h) (described by the mathematical expression ER(t) = ERmax * e−kt, with ERmax the peak metabolite excretion rate) using Microsoft Excel 2010 (Microsoft Corporation, Redmond, U.S.A.). The calculation of urinary excretion fractions (including background correction) and daily intakes is described in the Supplementary Material. Daily intakes were calculated according to Kohn et al. (2000) and Koch et al. (2003) using creatinine excretion rates reported by Harper et al. (1977).
2.2. Experimental design Three healthy male volunteers (age: 28–32 years; body weight: 75–93 kg) each received a single oral dose of approximately 5 mg EHS (weighed exactly), corresponding to 57.4–75.5 μg/(kg body weight). The EHS dose was provided dissolved in 1 mL ethanol diluted with 9 mL water in a chocolate coated waffle cup. The applied doses were more than a factor of 1000 below the no observed adverse effect level (NOAEL) of a short-term (male rats: 28 days, female rats: approx. 7 weeks) repeated dose toxicity study performed in rats (NOAEL: 250 mg/ (kg body weight * d) and 80 mg/(kg body weight * d) for male and female rats, respectively) (ECHA disseminated dossier, 2019). In addition, a dermal pilot study was performed (Bury et al., 2019a): One healthy male volunteer (33 years of age, 90 kg body weight) applied a commercial sunscreen containing 5% EHS to the whole body in a regular use scenario. In total, 10.9 g sunscreen was applied, which corresponds to 543 mg EHS (or 6.03 mg/kg body weight). Any mouth contact with the sunscreen itself or with the exposed hands was avoided to prevent non-dermal EHS uptake. Residual sunscreen was removed from the skin surface 4.75 h after application by taking a shower. No food was touched or eaten between the application and taking the shower. For one week prior to the study, the volunteers abstained from using any products containing EHS to avoid interfering exposures. Urine samples were collected in 250 mL polyethylene (PE) containers immediately before dose (t0) and consecutively and completely for 48 h
3. Results and discussion 3.1. Metabolite screening 3.1.1. Screening approach All urine samples (n = 22) of one volunteer from the oral dosing study were screened for six putative EHS metabolites and EHS itself. Glucuronides were enzymatically hydrolyzed prior to analysis, facilitating the screening procedure (better chromatographic retention; one defined species per phase I metabolite). For details on the choice of SPE column combination and MS conditions see the Supplementary Material. For each putative metabolite, two mass transitions were analyzed in ESI positive ion mode (Table S-3 in the Supplementary Material). Screening for the putative acidic metabolites was additionally performed in negative ion mode. 3.1.2. Sidechain-hydroxylated metabolites In case of the sidechain-hydroxylated EHS metabolite (OH-EHS), two chromatographic peaks were identified, showing a kinetic profile 36
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on both mass transitions (increase in creatinine-adjusted peak area after dose, followed by a slow decline over time; see Figs. S-1 and S-2 in the Supplementary Material). The larger peak at 12.5 min was confirmed as 5OH-EHS, using the custom synthesized analytical standard. The smaller peak at 12.8 min was most likely a second OH-EHS isomer. Metabolites analogous to 5OH-EHS are known for other xenobiotics bearing the 2-ethylhexyl moiety, such as the plasticizers di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) terephthalate (DEHTP), and the UV filter octocrylene (OC) (Koch et al., 2004; Lessmann et al., 2016; Bury et al., 2019b). Sidechain-hydroxy metabolite isomers other than the 5OH-isomer have been reported as minor metabolites (Albro et al., 1982; Peck and Albro, 1982; Schmid and Schlatter, 1985; Preuss et al., 2005). 3.1.3. Sidechain-oxo metabolite The sidechain-oxo metabolite of EHS (oxo-EHS) was found at tR =12.9 min (Fig. S-3 in the Supplementary Material). Oxo metabolites, more specific the 5oxo metabolites, are again known for DEHP and DEHTP (Koch et al., 2004; Lessmann et al., 2016). Thus, we deemed 5oxo-EHS a likely metabolite and obtained it by custom synthesis. This analytical standard was used to confirm this metabolite as 5oxo-EHS. 3.1.4. Sidechain-carboxy metabolites The carboxylic acid metabolite (carboxyheptyl salicylate; ‘cx-HeS’) was found in negative ion mode at tR =12.1 min (Fig. S-4 in the Supplementary Material) and also at tR =12.4 min (data not shown), but with at least 20-fold smaller peak height compared to the peak at tR =12.1 min. Corresponding carboxy metabolites have been described for DEHP and DEHTP. Considering, that in both cases the 2-ethyl-5carboxypentyl metabolite (‘5cx metabolite’) was the major isomer, compared to the 2(1-carboxymethyl)-hexyl metabolite (‘2cx metabolite’) (Koch et al., 2005; Lessmann et al., 2016), we expected the major peak to be 5cx-EPS and the minor peak to be the corresponding 2cx metabolite (2(1-carboxymethyl)-hexyl salicylate; 2cx-MHS). This was further corroborated by the chromatographic elution order, with the 2cx metabolite expected to be less polar and thus eluted later than the 5cx metabolite. Accordingly, 5cx-EPS was obtained by custom synthesis. This standard was used to confirm the major peak at tR =12.1 min as 5cx-EPS.
Fig. 1. Human EHS metabolism as identified in this study. The respective phase II metabolites (glucuronides, sulfates) are not shown. Metabolites investigated quantitatively in this study are underlined. Generic isomers are shown for simplification.
a sensitive HBM method for the determination of real life exposures (Bury et al., 2019a), further enabling us to quantitatively investigate their formation and elimination after defined EHS dosage.
3.2. Quantitative investigation of 5OH-EHS, 5oxo-EHS, and 5cx-EPS elimination 3.2.1. Elimination kinetics after oral dosage Urine samples of all three volunteers (n = 21, 26, and 22; total volumes of 5185, 4826, and 4208 mL) were analyzed for 5OH-EHS, 5oxo-EHS, and 5cx-EPS in duplicate. We were able to quantify 5cx-EPS in each urine sample. 5OH-EHS was found above LOQ in all post-dose urine samples, with exception of one volunteer (above LOQ up to 30 h post dose). 5oxo-EHS was above LOQ up to 5, 23, and 24 h post dose, respectively. Already in the pre-dose samples, low concentrations of 5OH-EHS (< LOQ-0.161 μg/L) and 5cx-EPS (0.024−0.210 μg/L) were found, whereas 5oxo-EHS was not present above LOQ (0.15 μg/L) (see Fig. 2 for exemplary chromatograms for all three metabolites in one volunteer, pre-dose and 12 h post dose). These pre-dose concentrations were in the same range as in the general population without use of PCPs with sun protection factor 5 days prior to sample collection (Bury et al., 2019a). Peak concentrations post dose (cmax; Table 1) were two to three orders of magnitude higher than pre-dose (background) concentrations. The urinary excretion kinetics of 5OH-EHS, 5oxo-EHS, and 5cx-EPS for all three volunteers is depicted in Fig. 3. Creatinine-adjustment was applied to compensate for diuresis-related differences in urinary dilution and clearly resulted in improved smoothness of the curves. The elimination kinetics were quite comparable between the three volunteers with times to reach peak concentrations (tmax; related to creatinine-adjusted cmax) of 1.6–2.6 h for all three metabolites (Table 1). Elimination occurred with biphasic kinetics with the second phase beginning 4.9–6 h (5OH-EHS), around 5–6 h (5oxo-EHS), and 4.0–8.8 h (5cx-EPS) after cmax. Furthermore, elimination kinetics was comparable also between the three metabolites, as is demonstrated by the overall similar elimination half-lives in both phases (Table 1). Based on the total amounts of metabolites excreted within 48 h, urinary excretion fractions (Fue) were calculated for the three EHS
3.1.5. Ester cleavage products Salicylic acid (2-hydroxybenzoic acid, ‘SA’) and its follow-up metabolite salicyluric acid (2-hydroxyhippuric acid, ‘SUA’) were found at tR = 9.6 and 8.4 min, respectively, with rather high peak heights at least for SUA, indicating its presence at rather high concentrations (data not shown). However, apart from EHS, other SA esters used as fragrance ingredients can be expected to be metabolized to SA and SUA to a large extent (Belsito et al., 2007). Furthermore, the frequently applied analgesic acetylsalicylic acid (aspirin) is also known to be metabolized to SA and SUA (Levy, 1980). 3.1.6. Selection of target biomarkers The elution order of the (in part tentatively) identified EHS metabolites was plausible with the most polar metabolite SUA eluting first, followed by SA, 5cx-EPS, tentative 2cx-MHS, 5OH-EHS, the second tentative OH-EHS isomer, and 5oxo-EHS. These urinary metabolites are shown in the metabolism scheme (Fig. 1). The identities of 5OH-EHS, 5oxo-EHS, 5cx-EPS, SA, and SUA were confirmed using analytical standards. Non-metabolized parent EHS eluted very late in the chromatogram (tR =16.6 min) and was only found in traces and in only few samples of the dosing study. This is most likely explained by its low polarity and accordingly low probability of unmodified renal excretion. SA and SUA, although detected in the metabolite screening, are not unique to EHS and thus not suitable as specific biomarkers of EHS exposure. Considering quantitative relevance of the metabolites and their specificity for EHS, 5OH-EHS, 5oxo-EHS, and 5cx-EPS were included in 37
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Fig. 2. Pre and post dose chromatograms. Chromatograms of 5OH-EHS, 5oxo-EHS, and 5cx-EPS in a pre-dose sample (top) and a sample 12 h post dose (bottom). MS/MS transitions for the stable isotope-labeled internal standards are shown in gray and for the native metabolites in black with quantifier transitions as continuous lines and qualifier transitions as dashed lines.
ester moieties of the metabolites. Accordingly, the amount of EHS metabolites conjugated to sulfate could not be quantified.
Table 1 Excretion kinetics for 5OH-EHS, 5oxo-EHS, and 5cx-EPS in three volunteers after single oral dose (mean values; ranges in parentheses).
cmax [μg/L] cmax [μg/(g creatinine)] tmax [h] t1/2 [h] – phase 1 t1/2 [h] – phase 2
5OH-EHS
5oxo-EHS
5cx-EPS
31.9 (22.1–43.6) 95.2 (41.6–188) 1.8 (1.6–2.3) 0.8 (0.6–1.0) 6.6 (4.4–8.2)
13.1 (9.46–16.5) 37.3 (20.7–68.3) 1.8 (1.6–2.3) 0.8 (0.5–0.9) 6.3 (4.1–8.0)
22.3 (14.9–29.2) 57.5 (30.8–108) 2.1 (1.6–2.6) 1.1 (0.8–1.3) 5.9 (5.5–6.5)
3.2.3. Kinetics and elimination after dermal exposure Compared to the oral application, elimination kinetics observed after the pilot dermal application of a commercial sunscreen, containing 5% EHS (543 mg dermally applied) (Bury et al., 2019a) was rather slow: after dermal application the maximum 5cx-EPS concentration in urine was observed after 9 h and maximum 5OH-EHS and 5oxo-EHS concentrations were observed as a plateau between 3.4 h and 10 h. Elimination half-lives were 4- to 8-fold longer (t1/2 for the 1st and 2nd phase: 5.5 h and 26 h (5OH-EHS), 5.9 h and 29 h (5oxo-EHS), and 5.8 h and 23 h (5cx-EPS)). These differences in elimination kinetics might be caused by a slower uptake and distribution of EHS through the skin and/or the lack of first pass hepatic metabolism, compared to absorption from the gastrointestinal tract. Despite the differences observed in kinetics, the ratios between concentrations of the three metabolites to one another in all samples from the dermal application (5OH-EHS/5cx-EPS: 1.1, 5OH-EHS/5oxo-EHS: 2.4, 5cx-EPS/5oxo-EHS: 2.2; mean values) reflected those after oral dosage (Table 2) quite well. Given the long elimination half-lives, an accumulation of EHS in the skin can be expected after repeated applications. Based on the total amounts of metabolites excreted within 96 h after dermal application (46.3 μg 5OH-EHS, 19.8 μg 5oxo-EHS, and 42.9 μg 5cx-EPS) and using the urinary excretion fractions derived in this study (Table 2), we estimated the amount of EHS that penetrated the skin and became systemically available. We calculated a mean of 16.2 mg EHS (values based on each metabolite: 15.5, 17.0, and 16.0 mg), which corresponds to 3.0% (2.9%, 3.1%, and 2.9%) of the dermally applied EHS dose (543 mg). In a recent in vitro dermal bioavailability study using human excised and dermatomed skin samples from six donors, it was determined that within 24 h 1.82 ± 1.5% of the topically applied dose (10 μL/cm² of 1% EHS in body lotion) became bioavailable (i.e. were found in the receptor fluid and the epidermis and dermis layers of the skin) (Symrise AG, unpublished study). According to the SCCS 10th Notes of Guidance (SCCS, 2018), for risk assessment the mean plus one standard deviation (equaling 3.32%) would be used for dermal bioavailability. Thus, our in vivo approach confirms the in vitro findings, indicating a rather low penetration and low systemic availability of EHS after dermal exposure.
metabolites as well as the sum of these three metabolites; in addition, percentages of metabolite excreted within the first 24 h after dose and in the following 24 h (and Fue) were calculated separately (Table 2). Amounts of 5OH-EHS and 5cx-EPS excreted via urine were comparable, whereas the amounts of 5oxo-EHS were approximately half. After one day, ≥95% of the total metabolite amounts were excreted. In total, the three specific EHS metabolites analyzed in urine account for less than 1% of the orally applied EHS dose. It can be expected, that the major share of the EHS dose was eliminated via urine as the non-specific metabolites SA and SUA. In addition, partial elimination via feces cannot be excluded.
3.2.2. Phase II metabolites We also determined the percentages of 5OH-EHS, 5oxo-EHS, and 5cx-EPS excreted in their free form and as glucuronides after oral application in pooled urine samples from each volunteer (0–6 h, 6–12 h, 12–24 h, and 24–48 h). These samples were analyzed with (free and conjugated metabolites) and without (only free form) the addition of βglucuronidase. Free (non-conjugated) metabolites could only be quantified in the 0–6 h-pooled urine samples, with free 5cx-EPS above LOQ for all three volunteers, and free 5OH-EHS for one volunteer. Free 5oxoEHS was below LOQ in all samples. Therefore, we calculated the percentages of free and glucuronidated metabolites only for the 0–6 h time frame, setting samples with concentrations below the LOQ to the LOQ. Almost all of 5OH-EHS (mean ≥99%) was found in its glucuronidated form with similarly high glucuronide shares for 5oxo-EHS (≥96%) and 5cx-EPS (99%). Some additional share of EHS metabolites might be excreted as sulfate conjugates. However, a pure β-glucuronidase without aryl sulfatase activity had to be used to avoid cleavage of the 38
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Fig. 3. Urinary excretion kinetics of EHS metabolites after oral dose. Elimination kinetics of 5OH-EHS (top), 5oxo-EHS (middle), and 5cx-EPS (bottom) in three male volunteers after oral dose of approximately 5 mg EHS. Absolute concentrations in μg/L (left) and creatinine-adjusted concentrations in μg/g creatinine (middle) of urine samples vs. time of sample collection, and the metabolite excretion rates (ER) in μg/h vs. midpoint of time segment (right) are shown. Table 2 Urinary excretion fractions (Fue, in %) of 5OH-EHS, 5oxo-EHS, 5cx-EPS, and the sum of all three metabolites after a single oral dose in three volunteers and percentage found in the first 24 h after dose and in the following 24 h (assuming excretion within 48 h as 100%). Mean values and ranges (in parentheses) are reported. Fue
0-24 h 24-48 h Total (0-48 h)
Percentage of metabolite found
5OH-EHS
5oxo-EHS
5cx-EPS
Sum
5OH-EHS
5oxo-EHS
5cx-EPS
Sum
0.280% (0.124–0.536%) 0.003% (0.001–0.005%) 0.28% (0.13–0.54%)
0.109% (0.058–0.198%) 0.000% (0.000–0.000%) 0.11% (0.06–0.20%)
0.230% (0.136–0.404%) 0.006% (0.003–0.008%) 0.24% (0.14–0.41%)
0.619% (0.318–1.138%) 0.009% (0.004–0.014%) 0.63% (0.32–1.15%)
99% (97–100%) 1% (0–3%)
100% (100–100%) 0% (0–0%)
97% (95–98%) 3% (2–5%)
98% (97–99%) 2% (1–3%)
3.3. Estimation of daily intakes for a pilot population
concentrations after a single dermal sunscreen application (5OH-EHS, 5oxo-EHS, and 5cx-EPS: 48.7, 20.3, and 41.2 μg/(g creatinine)) and corresponded to concentrations found approximately 1 d after dermal application in our pilot study. We used the 0–48 h urinary excretion fractions derived in this study (Table 2) for the calculation of daily intakes (DI; in μg/(kg body weight * d)) of EHS in that pilot population.
In a pilot population from the German general population (n = 35) (Bury et al., 2019a) maximum EHS metabolite concentrations of 8.83, 4.07, and 6.25 μg/(g creatinine) (5OH-EHS, 5oxo-EHS, and 5cx-EPS) were observed. These values were 5-7-fold lower compared to peak 39
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Ethical approval
DIs were separately calculated based on 5OH-EHS, 5oxo-EHS, and 5cxEPS concentrations (in μg/g creatinine), as well as based on the sum of these three metabolites. Based on 5cx-EPS a median DI of 0.584 μg/(kg body weight * d) was calculated. 5OH-EHS and 5oxo-EHS were above LOQ in less than 50% of urine samples, accordingly no median DI could be calculated based on these two metabolites or the sum of metabolites. However, maximum DIs based on 5OH-EHS, 5oxo-EHS, 5cx-EPS, and the sum of metabolites (53.3, 63.1, 41.9, and 50.7 μg/(kg body weight * d)) and 95th percentiles (16.5, 21.0, 17.1, and 17.5 μg/(kg body weight * d)) were in good agreement. The maximum DIs calculated were in the range of the orally applied dose in this study (57.4–75.5 μg/(kg body weight)) and more than a factor of 1000 below the NOAEL from a shortterm repeated dose toxicity study performed in rats (see Section 2.2). These exemplary DIs calculated here have to be regarded as a proof of principle and are probably not representative for certain subpopulations (such as children) or additionally exposed individuals, such as regular users of PCPs containing EHS or occupationally exposed individuals. Furthermore, it has to be mentioned that the inter-individual differences in EHS metabolism observed in this study (with three young, healthy male volunteers) resulted in urinary excretion fractions differing from the mean values up to a factor of 2 in both directions. For risk assessment purposes, possibly higher inter-individual differences have to be expected and considered in larger populations covering all age groups, genders, etc.
The study (oral dosage of 2-ethylhexyl salicylate; pilot dermal application; pilot population) was carried out in accordance with the Code of Ethics of the World Medical Association (1964 Declaration of Helsinki and later amendments) and has been approved by the ethical review board of the medical faculty of the Ruhr-University Bochum (IRB Reg. No.: 3867-10 and 4288-12). The study design was presented to the volunteers in written form and written informed consent was obtained from each participant. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxlet.2019.04.001. References Albero, B., Sánchez-Brunete, C., Miguel, E., Pérez, R.A., Tadeo, J.L., 2012. Determination of selected organic contaminants in soil by pressurized liquid extraction and gas chromatography tandem mass spectrometry with in situ derivatization. J. Chromatogr. A 1248, 9–17. https://doi.org/10.1016/j.chroma.2012.05.078. Albro, P.W., Corbett, J.T., Schroeder, J.L., Jordan, S., Matthews, H.B., 1982. Pharmacokinetics, interactions with macromolecules and species differences in metabolism of DEHP. Environ. Health Perspect. 45, 19–25. https://doi.org/10.1289/ ehp.824519. Angerer, J., Ewers, U., Wilhelm, M., 2007. Human biomonitoring: State of the art. Int. J. Hyg. Environ. Health 210 (3-4), 201–228. https://doi.org/10.1016/j.ijheh.2007.01. 024. Angerer, J., Aylward, L.L., Hays, S.M., Heinzow, B., Wilhelm, M., 2011. Human biomonitoring assessment values: Approaches and data requirements. Int. J. Hyg. Environ. Health 214 (5), 348–360. https://doi.org/10.1016/j.ijheh.2011.06.002. Apel, P., Angerer, J., Wilhelm, M., Kolossa-Gehring, M., 2017. New HBM values for emerging substances, inventory of reference and HBM values in force, and working principles of the German Human Biomonitoring Commission. Int. J. Hyg. Environ. Health 220 (2 Pt A), 152–166. https://doi.org/10.1016/j.ijheh.2016.09.007. Apel, C., Joerss, H., Ebinghaus, R., 2018. Environmental occurrence and hazard of organic UV stabilizers and UV filters in the sediment of European North and Baltic Seas. Chemosphere 212, 254–261. https://doi.org/10.1016/j.chemosphere.2018.08.105. Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2007. A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food Chem. Toxicol. 45 (Suppl 1), S318–61. https://doi.org/10.1016/j.fct.2007.09.066. Bury, D., Brüning, T., Koch, H.M., 2019a. Determination of metabolites of the UV filter 2Ethylhexyl salicylate in human urine by Online-SPE-LC-MS/MS. J. Chromatogr. B 1110-1111, 59–66. Bury, D., Modick-Biermann, H., Leibold, E., Brüning, T., Koch, H.M., 2019b. Urinary Metabolites of the UV Filter Octocrylene in Humans As Biomarkers of Exposure. https://doi.org/10.1007/s00204-019-02408-7. Byers, J.P., Sarver, J.G., 2009. Pharmacokinetic modeling. In: Hacker, M.P., Messer, W.S., Bachmann, K.A. (Eds.), Pharmacology. Principles and Practice. Elsevier/Academic Press, Amsterdam, Boston, pp. 201–277. Conrad, A., Schröter-Kermani, C., Hoppe, H.-W., Rüther, M., Pieper, S., Kolossa-Gehring, M., 2017. Glyphosate in German adults - Time trend (2001 to 2015) of human exposure to a widely used herbicide. Int. J. Hyg. Environ. Health 220 (1), 8–16. https:// doi.org/10.1016/j.ijheh.2016.09.016. Cunha, S.C., Trabalón, L., Jacobs, S., Castro, M., Fernandez-Tejedor, M., Granby, K., Verbeke, W., Kwadijk, C., Ferrari, F., Robbens, J., Sioen, I., Pocurull, E., Marques, A., Fernandes, J.O., Domingo, J.L., 2018. UV-filters and musk fragrances in seafood commercialized in Europe Union: occurrence, risk and exposure assessment. Environ. Res. 161, 399–408. https://doi.org/10.1016/j.envres.2017.11.015. ECHA disseminated dossier, 2019. ECHA Disseminated Dossier on 2-Ethylhexyl Salicylate - Short-term Repeated Dose Toxicity Study in Rats. (Accessed 9 January 2019). https://echa.europa.eu/de/registration-dossier/-/registered-dossier/14203/7/6/2. Ekpeghere, K.I., Kim, U.-J.O., Kim, S.-H., Oh, H.-Y., J.-E, 2016. Distribution and seasonal occurrence of UV filters in rivers and wastewater treatment plants in Korea. Sci. Total Environ. 542 (Pt A), 121–128. https://doi.org/10.1016/j.scitotenv.2015.10.033. European Chemicals Agency - CoRAP, 2019. 2-Ethylhexyl Salicylate - Substance Evaluation - Community Rolling Action Plan (CoRAP). (Accessed 6 December 2018). https://echa.europa.eu/information-on-chemicals/evaluation/community-rollingaction-plan/corap-table/-/dislist/details/0b0236e1820e160c. European Parliament and the Council, 2009. Regulation (EC) No 1223/2009 of the European Parliament and the Council of 30 November 2009 on Cosmetic Products: Reg. (EC) 1223/2009. Greinert, R., Vries de, E., Erdmann, F., Espina, C., Auvinen, A., Kesminiene, A., Schüz, J., 2015. European code against cancer 4th edition: ultraviolet radiation and cancer. Cancer Epidemiol. 39 (Suppl 1), S75–83. https://doi.org/10.1016/j.canep.2014.12. 014. Haines, D.A., Saravanabhavan, G., Werry, K., Khoury, C., 2017. An overview of human biomonitoring of environmental chemicals in the Canadian Health Measures Survey:
4. Conclusions With this study we present the first data on human metabolism and elimination kinetics of the UV filter EHS. We tentatively identified seven metabolites of EHS after oral dosage and quantitatively investigated (using custom synthesized analytical standards and stable isotope labeled internal standards) the urinary excretion of the three most promising metabolites unique to EHS: 5OH-EHS, 5oxo-EHS, and 5cx-EPS. The chemical structure of these three metabolites contains the core structure of EHS, which makes them specific to EHS (unlike e.g. SA) and therefore qualifies them as specific biomarkers of EHS exposure. Urinary excretion fractions (Fue) were rather low for all of these three metabolites (< 1%), indicating that the total breakdown to SA is likely the predominant metabolic pathway. In spite of this apparent and rapid breakdown to unspecific metabolites (such as SA and SUA) we were able to detect and quantify one or more of the above three specific metabolites in the majority of urine samples from the general population, as well as in all urines samples collected after dermal application of an EHS containing sunscreen product (Bury et al., 2019a). The Fue reported in this study enable the reliable conversion of measured biomarker concentrations into daily intakes for robust EHS exposure and risk assessments on individual and population scale.
Funding The presented study and development of the analytical method applied herein are part of a large-scale 10-year project on the advancement of human biomonitoring in Germany. This project is a cooperation agreed in 2010 between the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety (BMU) and the German Chemical Industry Association (VCI -Verband der Chemischen Industrie e.V.) and is managed by the German Environment Agency (UBA). Experts from governmental scientific authorities, industry and science provide advice in the selection of substances to be investigated and during method development. The analytical method development and the human metabolism study are financed by the Chemie Wirtschaftsförderungsgesellschaft mbH while the first application of the novel methodology in a larger population study will be financed by the German Environment Agency. 40
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Urinary metabolites of the UV filter 2-Ethylhexyl salicylate as biomarkers of exposure in humans
T
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Daniel Burya, , Peter Griemb, Tanja Wildemannb, Thomas Brüninga, Holger M. Kocha a Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-Universität Bochum (IPA), Bürkle-de-la-Camp-Platz 1, 44789, Bochum, Germany b Symrise AG, Mühlenfeldstrasse 1, 37603, Holzminden, Germany
A R T I C LE I N FO
A B S T R A C T
Keywords: Ethylhexyl salicylate EHS UV filter Metabolism Oral dosage Human biomonitoring Exposure biomarker Urinary excretion fraction
The UV filter 2-ethylhexyl salicylate (EHS) is used in sunscreens and other personal care products worldwide and has been found in a variety of environmental media. We aimed to provide human toxicokinetic data on EHS as a tool for risk assessment. For that purpose, we investigated metabolism and urinary metabolite excretion after a single oral EHS dose (57.4–75.5 μg/(kg body weight)) in three male volunteers. In a suspect screening, we tentatively identified seven EHS metabolites. Three EHS specific metabolites were quantitatively investigated: 2ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5-oxohexyl 2-hydroxybenzoate (5oxo-EHS), and 5-(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid (5cx-EPS). These metabolites were excreted with urinary excretion fractions of 0.28% (range: 0.13−0.54%), 0.11% (0.06−0.20%), and 0.24% (0.14−0.41%), respectively. The elimination was fast: peak urinary concentrations were found 1.6–2.6 h after dose and ≥95% of the total amounts were excreted within 24 h. Elimination kinetics were biphasic, with mean elimination half-lives of 0.8 h (first phase) and 6.6 h (second phase) for 5OH-EHS, 0.8 h and 6.3 h for 5oxo-EHS, and 1.1 h and 5.9 h for 5cx-EPS. After dermal exposure (sunscreen application), we found a considerably delayed EHS elimination. Based on urinary metabolite levels we calculated EHS exposure levels for a small pilot population.
1. Introduction Sunscreens are frequently applied to reduce health risks associated with excessive (solar) UV radiation, both in private life and occupational settings (Greinert et al., 2015; Ruppert et al., 2016). The UV-B filter 2-ethylhexyl salicylate (EHS, ethylhexyl salicylate, octyl salicylate, octisalate; CAS registry no. 118-60-5; EC no. 204-263-4) is used in sunscreen formulations worldwide (Shaath, 2010), as well as in other personal care products (PCP), such as after shave, lipsticks, face creams, make-up foundation, and perfumes (Kerr, 2011; Manová et al., 2013; Uter et al., 2014). The maximum permitted concentration in cosmetic products is 5%, both in the U.S.A. and the European Union (U.S. Food and Drug Administration, 2019; European Parliament and the Council, 2009). in vitro studies on endocrine activity of EHS showed some anti-androgenic activity and weak to no estrogenic, anti-estrogenic, and
androgenic activity (Miller et al., 2001; Morohoshi et al., 2005; Kunz and Fent, 2006; Jiménez-Díaz et al., 2013). Given its potential endocrine activity, EHS has been included in the Community rolling action plan (CoRAP) of the European Union in 2018 (European Chemicals Agency - CoRAP, 2019). Environmental monitoring has provided evidence for the presence of EHS in a large variety of environmental matrices (such as surface freshwater and sea water, beach sand, sediments, wastewater treatment plant effluents, soil, and marine wildlife organisms) and also in indoor dust (examples in Negreira et al. (2009); Albero et al. (2012); Sankoda et al. (2015); Ekpeghere et al. (2016); Apel et al. (2018); Cunha et al. (2018); Vila et al. (2018)). Given the widespread use of EHS in PCPs resulting in direct EHS contact of consumers, and further considering its presence in the environment, the determination of EHS body burdens is of high interest. Human biomonitoring (HBM) provides an integral measure of exposure to chemicals, covering all exposure sources and
Abbreviations: EHS, 2-ethylhexyl salicylate; 5OH-EHS, 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate; 5oxo-EHS, 2-ethyl-5-oxohexyl 2-hydroxybenzoate; 5cx-EPS, 5(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid; 2cx-MHS, 2(1-carboxymethyl)-hexyl salicylate; cx-HeS, carboxyheptyl salicylate; SA, salicylic acid; SUA, salicyluric acid; PCP, personal care product ⁎ Corresponding author. E-mail addresses: [email protected] (D. Bury), [email protected] (P. Griem), [email protected] (T. Wildemann), [email protected] (T. Brüning), [email protected] (H.M. Koch). https://doi.org/10.1016/j.toxlet.2019.04.001 Received 12 February 2019; Received in revised form 20 March 2019; Accepted 1 April 2019 Available online 03 April 2019 0378-4274/ © 2019 Elsevier B.V. All rights reserved.
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after the dose (oral dosing study) and 96 h after the dose (dermal pilot study), respectively, and stored at −20 °C until analysis. Times of urine collection were recorded by the volunteers. Urine volumes were determined via the weight difference between filled and empty PE containers and urinary creatinine was determined according to Jaffe (1886) by contract analysis (L.u.P. GmbH Labor und Praxisservice; Bochum, Germany).
uptake routes, and is well established for exposure assessment both in large scale population studies and on an individual level (Angerer et al., 2007; Needham et al., 2007; Schindler et al., 2014; Haines et al., 2017; Kolossa-Gehring et al., 2017; Schwedler et al., 2017). Recently, we described an HBM method for the determination of three specific urinary metabolites of EHS (Bury et al., 2019a), which were identified in the current metabolism study: 2-ethyl-5-hydroxyhexyl 2-hydroxybenzoate (5OH-EHS), 2-ethyl-5-oxohexyl 2-hydroxybenzoate (5oxoEHS), and 5-(((2-hydroxybenzoyl)oxy)methyl)heptanoic acid (5cxEPS). We demonstrated the potential use of these three metabolites as robust and specific biomarkers of EHS exposure after application of a commercial sunscreen containing EHS to one human volunteer. Furthermore, we were able to quantify EHS metabolites in 91% of urine samples from a pilot population from the general population (n = 35). For risk assessment purposes, quantitative data on xenobiotic metabolite formation and elimination (such as urinary excretion fractions) in humans is highly valuable. Urinary excretion fractions can be used to calculate external doses in terms of daily intakes (DI) based on measured urinary biomarker concentrations. These DIs can be related to toxicological threshold values, such as tolerable and daily intakes (TDI/ ADI). With the same data, health-based guidance values for biomarker concentrations (such as human biomonitoring assessment values (HBM values) or biomonitoring equivalents (BE) (Angerer et al., 2011; Hays and Aylward, 2012; Apel et al., 2017)) can be derived, enabling an accurate risk assessment based on urinary metabolite levels (examples in Conrad et al. (2017); Koch et al. (2017); Moos et al. (2017); Ulrich et al. (2018)). Accordingly, this study aimed to investigate EHS metabolism and quantify urinary excretion of specific metabolites.
2.3. Metabolite screening The identification of EHS metabolites was performed using a triplequadrupole-MS suspect screening approach previously applied to the UV filter octocrylene by our group (Bury et al., 2019b). All urine samples from one volunteer were prepared by enzymatic deglucuronidation as described in Bury et al. (2019a), however with omission of internal standards (which were not yet available at that time). These processed urine samples were then analyzed with online-SPE-LC–MS/ MS. For further details see the Supplementary Material. 2.4. Quantification of metabolites 5OH-EHS, 5oxo-EHS, and 5cx-EPS were quantified by stable isotope dilution analysis using an online-SPE-LC–MS/MS method, recently published by our group (Bury et al., 2019a). In short, after addition of a pure β-glucuronidase from E. coli K12, buffer, and stable isotope labeled internal standards, urine samples were incubated at 37 °C for 3 h for the cleavage of glucuronic acid conjugates. Then, samples were frozen overnight, thawed, equilibrated to ambient temperature and centrifuged at 1900 g for 10 min. 100 μL of the supernatant were analyzed. The limits of quantification (LOQ) were 0.05 μg/L (5OH-EHS), 0.15 μg/ L (5oxo-EHS), and 0.01 μg/L (5cx-EPS).
2. Materials and methods 2.1. Chemicals and reagents
2.5. Statistical analysis 2-Ethylhexyl salicylate (‘kosher’, obtained from Sigma-Aldrich, Steinheim, Germany; chemical purity 99.7%) was used in the oral dosing study. For further chemicals and reagents see Bury et al. (2019a). The β-glucuronidase from E. coli K12 applied for metabolite deconjugation (Sections 2.3 and 2.4) was a pure β-glucuronidase preparation without aryl sulfatase activity.
Elimination half-lives were calculated using the equation t1/2 = ln (2)/k, with k being the kinetic constant of the exponential decline in excreted metabolite amounts (Byers and Sarver, 2009). k was obtained by exponential regression of the metabolite excretion rates (ER in μg/h) vs. the midpoint of each time interval (t in h) (described by the mathematical expression ER(t) = ERmax * e−kt, with ERmax the peak metabolite excretion rate) using Microsoft Excel 2010 (Microsoft Corporation, Redmond, U.S.A.). The calculation of urinary excretion fractions (including background correction) and daily intakes is described in the Supplementary Material. Daily intakes were calculated according to Kohn et al. (2000) and Koch et al. (2003) using creatinine excretion rates reported by Harper et al. (1977).
2.2. Experimental design Three healthy male volunteers (age: 28–32 years; body weight: 75–93 kg) each received a single oral dose of approximately 5 mg EHS (weighed exactly), corresponding to 57.4–75.5 μg/(kg body weight). The EHS dose was provided dissolved in 1 mL ethanol diluted with 9 mL water in a chocolate coated waffle cup. The applied doses were more than a factor of 1000 below the no observed adverse effect level (NOAEL) of a short-term (male rats: 28 days, female rats: approx. 7 weeks) repeated dose toxicity study performed in rats (NOAEL: 250 mg/ (kg body weight * d) and 80 mg/(kg body weight * d) for male and female rats, respectively) (ECHA disseminated dossier, 2019). In addition, a dermal pilot study was performed (Bury et al., 2019a): One healthy male volunteer (33 years of age, 90 kg body weight) applied a commercial sunscreen containing 5% EHS to the whole body in a regular use scenario. In total, 10.9 g sunscreen was applied, which corresponds to 543 mg EHS (or 6.03 mg/kg body weight). Any mouth contact with the sunscreen itself or with the exposed hands was avoided to prevent non-dermal EHS uptake. Residual sunscreen was removed from the skin surface 4.75 h after application by taking a shower. No food was touched or eaten between the application and taking the shower. For one week prior to the study, the volunteers abstained from using any products containing EHS to avoid interfering exposures. Urine samples were collected in 250 mL polyethylene (PE) containers immediately before dose (t0) and consecutively and completely for 48 h
3. Results and discussion 3.1. Metabolite screening 3.1.1. Screening approach All urine samples (n = 22) of one volunteer from the oral dosing study were screened for six putative EHS metabolites and EHS itself. Glucuronides were enzymatically hydrolyzed prior to analysis, facilitating the screening procedure (better chromatographic retention; one defined species per phase I metabolite). For details on the choice of SPE column combination and MS conditions see the Supplementary Material. For each putative metabolite, two mass transitions were analyzed in ESI positive ion mode (Table S-3 in the Supplementary Material). Screening for the putative acidic metabolites was additionally performed in negative ion mode. 3.1.2. Sidechain-hydroxylated metabolites In case of the sidechain-hydroxylated EHS metabolite (OH-EHS), two chromatographic peaks were identified, showing a kinetic profile 36
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on both mass transitions (increase in creatinine-adjusted peak area after dose, followed by a slow decline over time; see Figs. S-1 and S-2 in the Supplementary Material). The larger peak at 12.5 min was confirmed as 5OH-EHS, using the custom synthesized analytical standard. The smaller peak at 12.8 min was most likely a second OH-EHS isomer. Metabolites analogous to 5OH-EHS are known for other xenobiotics bearing the 2-ethylhexyl moiety, such as the plasticizers di(2-ethylhexyl) phthalate (DEHP) and di(2-ethylhexyl) terephthalate (DEHTP), and the UV filter octocrylene (OC) (Koch et al., 2004; Lessmann et al., 2016; Bury et al., 2019b). Sidechain-hydroxy metabolite isomers other than the 5OH-isomer have been reported as minor metabolites (Albro et al., 1982; Peck and Albro, 1982; Schmid and Schlatter, 1985; Preuss et al., 2005). 3.1.3. Sidechain-oxo metabolite The sidechain-oxo metabolite of EHS (oxo-EHS) was found at tR =12.9 min (Fig. S-3 in the Supplementary Material). Oxo metabolites, more specific the 5oxo metabolites, are again known for DEHP and DEHTP (Koch et al., 2004; Lessmann et al., 2016). Thus, we deemed 5oxo-EHS a likely metabolite and obtained it by custom synthesis. This analytical standard was used to confirm this metabolite as 5oxo-EHS. 3.1.4. Sidechain-carboxy metabolites The carboxylic acid metabolite (carboxyheptyl salicylate; ‘cx-HeS’) was found in negative ion mode at tR =12.1 min (Fig. S-4 in the Supplementary Material) and also at tR =12.4 min (data not shown), but with at least 20-fold smaller peak height compared to the peak at tR =12.1 min. Corresponding carboxy metabolites have been described for DEHP and DEHTP. Considering, that in both cases the 2-ethyl-5carboxypentyl metabolite (‘5cx metabolite’) was the major isomer, compared to the 2(1-carboxymethyl)-hexyl metabolite (‘2cx metabolite’) (Koch et al., 2005; Lessmann et al., 2016), we expected the major peak to be 5cx-EPS and the minor peak to be the corresponding 2cx metabolite (2(1-carboxymethyl)-hexyl salicylate; 2cx-MHS). This was further corroborated by the chromatographic elution order, with the 2cx metabolite expected to be less polar and thus eluted later than the 5cx metabolite. Accordingly, 5cx-EPS was obtained by custom synthesis. This standard was used to confirm the major peak at tR =12.1 min as 5cx-EPS.
Fig. 1. Human EHS metabolism as identified in this study. The respective phase II metabolites (glucuronides, sulfates) are not shown. Metabolites investigated quantitatively in this study are underlined. Generic isomers are shown for simplification.
a sensitive HBM method for the determination of real life exposures (Bury et al., 2019a), further enabling us to quantitatively investigate their formation and elimination after defined EHS dosage.
3.2. Quantitative investigation of 5OH-EHS, 5oxo-EHS, and 5cx-EPS elimination 3.2.1. Elimination kinetics after oral dosage Urine samples of all three volunteers (n = 21, 26, and 22; total volumes of 5185, 4826, and 4208 mL) were analyzed for 5OH-EHS, 5oxo-EHS, and 5cx-EPS in duplicate. We were able to quantify 5cx-EPS in each urine sample. 5OH-EHS was found above LOQ in all post-dose urine samples, with exception of one volunteer (above LOQ up to 30 h post dose). 5oxo-EHS was above LOQ up to 5, 23, and 24 h post dose, respectively. Already in the pre-dose samples, low concentrations of 5OH-EHS (< LOQ-0.161 μg/L) and 5cx-EPS (0.024−0.210 μg/L) were found, whereas 5oxo-EHS was not present above LOQ (0.15 μg/L) (see Fig. 2 for exemplary chromatograms for all three metabolites in one volunteer, pre-dose and 12 h post dose). These pre-dose concentrations were in the same range as in the general population without use of PCPs with sun protection factor 5 days prior to sample collection (Bury et al., 2019a). Peak concentrations post dose (cmax; Table 1) were two to three orders of magnitude higher than pre-dose (background) concentrations. The urinary excretion kinetics of 5OH-EHS, 5oxo-EHS, and 5cx-EPS for all three volunteers is depicted in Fig. 3. Creatinine-adjustment was applied to compensate for diuresis-related differences in urinary dilution and clearly resulted in improved smoothness of the curves. The elimination kinetics were quite comparable between the three volunteers with times to reach peak concentrations (tmax; related to creatinine-adjusted cmax) of 1.6–2.6 h for all three metabolites (Table 1). Elimination occurred with biphasic kinetics with the second phase beginning 4.9–6 h (5OH-EHS), around 5–6 h (5oxo-EHS), and 4.0–8.8 h (5cx-EPS) after cmax. Furthermore, elimination kinetics was comparable also between the three metabolites, as is demonstrated by the overall similar elimination half-lives in both phases (Table 1). Based on the total amounts of metabolites excreted within 48 h, urinary excretion fractions (Fue) were calculated for the three EHS
3.1.5. Ester cleavage products Salicylic acid (2-hydroxybenzoic acid, ‘SA’) and its follow-up metabolite salicyluric acid (2-hydroxyhippuric acid, ‘SUA’) were found at tR = 9.6 and 8.4 min, respectively, with rather high peak heights at least for SUA, indicating its presence at rather high concentrations (data not shown). However, apart from EHS, other SA esters used as fragrance ingredients can be expected to be metabolized to SA and SUA to a large extent (Belsito et al., 2007). Furthermore, the frequently applied analgesic acetylsalicylic acid (aspirin) is also known to be metabolized to SA and SUA (Levy, 1980). 3.1.6. Selection of target biomarkers The elution order of the (in part tentatively) identified EHS metabolites was plausible with the most polar metabolite SUA eluting first, followed by SA, 5cx-EPS, tentative 2cx-MHS, 5OH-EHS, the second tentative OH-EHS isomer, and 5oxo-EHS. These urinary metabolites are shown in the metabolism scheme (Fig. 1). The identities of 5OH-EHS, 5oxo-EHS, 5cx-EPS, SA, and SUA were confirmed using analytical standards. Non-metabolized parent EHS eluted very late in the chromatogram (tR =16.6 min) and was only found in traces and in only few samples of the dosing study. This is most likely explained by its low polarity and accordingly low probability of unmodified renal excretion. SA and SUA, although detected in the metabolite screening, are not unique to EHS and thus not suitable as specific biomarkers of EHS exposure. Considering quantitative relevance of the metabolites and their specificity for EHS, 5OH-EHS, 5oxo-EHS, and 5cx-EPS were included in 37
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Fig. 2. Pre and post dose chromatograms. Chromatograms of 5OH-EHS, 5oxo-EHS, and 5cx-EPS in a pre-dose sample (top) and a sample 12 h post dose (bottom). MS/MS transitions for the stable isotope-labeled internal standards are shown in gray and for the native metabolites in black with quantifier transitions as continuous lines and qualifier transitions as dashed lines.
ester moieties of the metabolites. Accordingly, the amount of EHS metabolites conjugated to sulfate could not be quantified.
Table 1 Excretion kinetics for 5OH-EHS, 5oxo-EHS, and 5cx-EPS in three volunteers after single oral dose (mean values; ranges in parentheses).
cmax [μg/L] cmax [μg/(g creatinine)] tmax [h] t1/2 [h] – phase 1 t1/2 [h] – phase 2
5OH-EHS
5oxo-EHS
5cx-EPS
31.9 (22.1–43.6) 95.2 (41.6–188) 1.8 (1.6–2.3) 0.8 (0.6–1.0) 6.6 (4.4–8.2)
13.1 (9.46–16.5) 37.3 (20.7–68.3) 1.8 (1.6–2.3) 0.8 (0.5–0.9) 6.3 (4.1–8.0)
22.3 (14.9–29.2) 57.5 (30.8–108) 2.1 (1.6–2.6) 1.1 (0.8–1.3) 5.9 (5.5–6.5)
3.2.3. Kinetics and elimination after dermal exposure Compared to the oral application, elimination kinetics observed after the pilot dermal application of a commercial sunscreen, containing 5% EHS (543 mg dermally applied) (Bury et al., 2019a) was rather slow: after dermal application the maximum 5cx-EPS concentration in urine was observed after 9 h and maximum 5OH-EHS and 5oxo-EHS concentrations were observed as a plateau between 3.4 h and 10 h. Elimination half-lives were 4- to 8-fold longer (t1/2 for the 1st and 2nd phase: 5.5 h and 26 h (5OH-EHS), 5.9 h and 29 h (5oxo-EHS), and 5.8 h and 23 h (5cx-EPS)). These differences in elimination kinetics might be caused by a slower uptake and distribution of EHS through the skin and/or the lack of first pass hepatic metabolism, compared to absorption from the gastrointestinal tract. Despite the differences observed in kinetics, the ratios between concentrations of the three metabolites to one another in all samples from the dermal application (5OH-EHS/5cx-EPS: 1.1, 5OH-EHS/5oxo-EHS: 2.4, 5cx-EPS/5oxo-EHS: 2.2; mean values) reflected those after oral dosage (Table 2) quite well. Given the long elimination half-lives, an accumulation of EHS in the skin can be expected after repeated applications. Based on the total amounts of metabolites excreted within 96 h after dermal application (46.3 μg 5OH-EHS, 19.8 μg 5oxo-EHS, and 42.9 μg 5cx-EPS) and using the urinary excretion fractions derived in this study (Table 2), we estimated the amount of EHS that penetrated the skin and became systemically available. We calculated a mean of 16.2 mg EHS (values based on each metabolite: 15.5, 17.0, and 16.0 mg), which corresponds to 3.0% (2.9%, 3.1%, and 2.9%) of the dermally applied EHS dose (543 mg). In a recent in vitro dermal bioavailability study using human excised and dermatomed skin samples from six donors, it was determined that within 24 h 1.82 ± 1.5% of the topically applied dose (10 μL/cm² of 1% EHS in body lotion) became bioavailable (i.e. were found in the receptor fluid and the epidermis and dermis layers of the skin) (Symrise AG, unpublished study). According to the SCCS 10th Notes of Guidance (SCCS, 2018), for risk assessment the mean plus one standard deviation (equaling 3.32%) would be used for dermal bioavailability. Thus, our in vivo approach confirms the in vitro findings, indicating a rather low penetration and low systemic availability of EHS after dermal exposure.
metabolites as well as the sum of these three metabolites; in addition, percentages of metabolite excreted within the first 24 h after dose and in the following 24 h (and Fue) were calculated separately (Table 2). Amounts of 5OH-EHS and 5cx-EPS excreted via urine were comparable, whereas the amounts of 5oxo-EHS were approximately half. After one day, ≥95% of the total metabolite amounts were excreted. In total, the three specific EHS metabolites analyzed in urine account for less than 1% of the orally applied EHS dose. It can be expected, that the major share of the EHS dose was eliminated via urine as the non-specific metabolites SA and SUA. In addition, partial elimination via feces cannot be excluded.
3.2.2. Phase II metabolites We also determined the percentages of 5OH-EHS, 5oxo-EHS, and 5cx-EPS excreted in their free form and as glucuronides after oral application in pooled urine samples from each volunteer (0–6 h, 6–12 h, 12–24 h, and 24–48 h). These samples were analyzed with (free and conjugated metabolites) and without (only free form) the addition of βglucuronidase. Free (non-conjugated) metabolites could only be quantified in the 0–6 h-pooled urine samples, with free 5cx-EPS above LOQ for all three volunteers, and free 5OH-EHS for one volunteer. Free 5oxoEHS was below LOQ in all samples. Therefore, we calculated the percentages of free and glucuronidated metabolites only for the 0–6 h time frame, setting samples with concentrations below the LOQ to the LOQ. Almost all of 5OH-EHS (mean ≥99%) was found in its glucuronidated form with similarly high glucuronide shares for 5oxo-EHS (≥96%) and 5cx-EPS (99%). Some additional share of EHS metabolites might be excreted as sulfate conjugates. However, a pure β-glucuronidase without aryl sulfatase activity had to be used to avoid cleavage of the 38
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Fig. 3. Urinary excretion kinetics of EHS metabolites after oral dose. Elimination kinetics of 5OH-EHS (top), 5oxo-EHS (middle), and 5cx-EPS (bottom) in three male volunteers after oral dose of approximately 5 mg EHS. Absolute concentrations in μg/L (left) and creatinine-adjusted concentrations in μg/g creatinine (middle) of urine samples vs. time of sample collection, and the metabolite excretion rates (ER) in μg/h vs. midpoint of time segment (right) are shown. Table 2 Urinary excretion fractions (Fue, in %) of 5OH-EHS, 5oxo-EHS, 5cx-EPS, and the sum of all three metabolites after a single oral dose in three volunteers and percentage found in the first 24 h after dose and in the following 24 h (assuming excretion within 48 h as 100%). Mean values and ranges (in parentheses) are reported. Fue
0-24 h 24-48 h Total (0-48 h)
Percentage of metabolite found
5OH-EHS
5oxo-EHS
5cx-EPS
Sum
5OH-EHS
5oxo-EHS
5cx-EPS
Sum
0.280% (0.124–0.536%) 0.003% (0.001–0.005%) 0.28% (0.13–0.54%)
0.109% (0.058–0.198%) 0.000% (0.000–0.000%) 0.11% (0.06–0.20%)
0.230% (0.136–0.404%) 0.006% (0.003–0.008%) 0.24% (0.14–0.41%)
0.619% (0.318–1.138%) 0.009% (0.004–0.014%) 0.63% (0.32–1.15%)
99% (97–100%) 1% (0–3%)
100% (100–100%) 0% (0–0%)
97% (95–98%) 3% (2–5%)
98% (97–99%) 2% (1–3%)
3.3. Estimation of daily intakes for a pilot population
concentrations after a single dermal sunscreen application (5OH-EHS, 5oxo-EHS, and 5cx-EPS: 48.7, 20.3, and 41.2 μg/(g creatinine)) and corresponded to concentrations found approximately 1 d after dermal application in our pilot study. We used the 0–48 h urinary excretion fractions derived in this study (Table 2) for the calculation of daily intakes (DI; in μg/(kg body weight * d)) of EHS in that pilot population.
In a pilot population from the German general population (n = 35) (Bury et al., 2019a) maximum EHS metabolite concentrations of 8.83, 4.07, and 6.25 μg/(g creatinine) (5OH-EHS, 5oxo-EHS, and 5cx-EPS) were observed. These values were 5-7-fold lower compared to peak 39
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Ethical approval
DIs were separately calculated based on 5OH-EHS, 5oxo-EHS, and 5cxEPS concentrations (in μg/g creatinine), as well as based on the sum of these three metabolites. Based on 5cx-EPS a median DI of 0.584 μg/(kg body weight * d) was calculated. 5OH-EHS and 5oxo-EHS were above LOQ in less than 50% of urine samples, accordingly no median DI could be calculated based on these two metabolites or the sum of metabolites. However, maximum DIs based on 5OH-EHS, 5oxo-EHS, 5cx-EPS, and the sum of metabolites (53.3, 63.1, 41.9, and 50.7 μg/(kg body weight * d)) and 95th percentiles (16.5, 21.0, 17.1, and 17.5 μg/(kg body weight * d)) were in good agreement. The maximum DIs calculated were in the range of the orally applied dose in this study (57.4–75.5 μg/(kg body weight)) and more than a factor of 1000 below the NOAEL from a shortterm repeated dose toxicity study performed in rats (see Section 2.2). These exemplary DIs calculated here have to be regarded as a proof of principle and are probably not representative for certain subpopulations (such as children) or additionally exposed individuals, such as regular users of PCPs containing EHS or occupationally exposed individuals. Furthermore, it has to be mentioned that the inter-individual differences in EHS metabolism observed in this study (with three young, healthy male volunteers) resulted in urinary excretion fractions differing from the mean values up to a factor of 2 in both directions. For risk assessment purposes, possibly higher inter-individual differences have to be expected and considered in larger populations covering all age groups, genders, etc.
The study (oral dosage of 2-ethylhexyl salicylate; pilot dermal application; pilot population) was carried out in accordance with the Code of Ethics of the World Medical Association (1964 Declaration of Helsinki and later amendments) and has been approved by the ethical review board of the medical faculty of the Ruhr-University Bochum (IRB Reg. No.: 3867-10 and 4288-12). The study design was presented to the volunteers in written form and written informed consent was obtained from each participant. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.toxlet.2019.04.001. References Albero, B., Sánchez-Brunete, C., Miguel, E., Pérez, R.A., Tadeo, J.L., 2012. Determination of selected organic contaminants in soil by pressurized liquid extraction and gas chromatography tandem mass spectrometry with in situ derivatization. J. Chromatogr. A 1248, 9–17. https://doi.org/10.1016/j.chroma.2012.05.078. Albro, P.W., Corbett, J.T., Schroeder, J.L., Jordan, S., Matthews, H.B., 1982. Pharmacokinetics, interactions with macromolecules and species differences in metabolism of DEHP. Environ. Health Perspect. 45, 19–25. https://doi.org/10.1289/ ehp.824519. Angerer, J., Ewers, U., Wilhelm, M., 2007. Human biomonitoring: State of the art. Int. J. Hyg. Environ. Health 210 (3-4), 201–228. https://doi.org/10.1016/j.ijheh.2007.01. 024. Angerer, J., Aylward, L.L., Hays, S.M., Heinzow, B., Wilhelm, M., 2011. Human biomonitoring assessment values: Approaches and data requirements. Int. J. Hyg. Environ. Health 214 (5), 348–360. https://doi.org/10.1016/j.ijheh.2011.06.002. Apel, P., Angerer, J., Wilhelm, M., Kolossa-Gehring, M., 2017. New HBM values for emerging substances, inventory of reference and HBM values in force, and working principles of the German Human Biomonitoring Commission. Int. J. Hyg. Environ. Health 220 (2 Pt A), 152–166. https://doi.org/10.1016/j.ijheh.2016.09.007. Apel, C., Joerss, H., Ebinghaus, R., 2018. Environmental occurrence and hazard of organic UV stabilizers and UV filters in the sediment of European North and Baltic Seas. Chemosphere 212, 254–261. https://doi.org/10.1016/j.chemosphere.2018.08.105. Belsito, D., Bickers, D., Bruze, M., Calow, P., Greim, H., Hanifin, J.M., Rogers, A.E., Saurat, J.H., Sipes, I.G., Tagami, H., 2007. A toxicologic and dermatologic assessment of salicylates when used as fragrance ingredients. Food Chem. Toxicol. 45 (Suppl 1), S318–61. https://doi.org/10.1016/j.fct.2007.09.066. Bury, D., Brüning, T., Koch, H.M., 2019a. Determination of metabolites of the UV filter 2Ethylhexyl salicylate in human urine by Online-SPE-LC-MS/MS. J. Chromatogr. B 1110-1111, 59–66. Bury, D., Modick-Biermann, H., Leibold, E., Brüning, T., Koch, H.M., 2019b. Urinary Metabolites of the UV Filter Octocrylene in Humans As Biomarkers of Exposure. https://doi.org/10.1007/s00204-019-02408-7. Byers, J.P., Sarver, J.G., 2009. Pharmacokinetic modeling. In: Hacker, M.P., Messer, W.S., Bachmann, K.A. (Eds.), Pharmacology. Principles and Practice. Elsevier/Academic Press, Amsterdam, Boston, pp. 201–277. Conrad, A., Schröter-Kermani, C., Hoppe, H.-W., Rüther, M., Pieper, S., Kolossa-Gehring, M., 2017. Glyphosate in German adults - Time trend (2001 to 2015) of human exposure to a widely used herbicide. Int. J. Hyg. Environ. Health 220 (1), 8–16. https:// doi.org/10.1016/j.ijheh.2016.09.016. Cunha, S.C., Trabalón, L., Jacobs, S., Castro, M., Fernandez-Tejedor, M., Granby, K., Verbeke, W., Kwadijk, C., Ferrari, F., Robbens, J., Sioen, I., Pocurull, E., Marques, A., Fernandes, J.O., Domingo, J.L., 2018. UV-filters and musk fragrances in seafood commercialized in Europe Union: occurrence, risk and exposure assessment. Environ. Res. 161, 399–408. https://doi.org/10.1016/j.envres.2017.11.015. ECHA disseminated dossier, 2019. ECHA Disseminated Dossier on 2-Ethylhexyl Salicylate - Short-term Repeated Dose Toxicity Study in Rats. (Accessed 9 January 2019). https://echa.europa.eu/de/registration-dossier/-/registered-dossier/14203/7/6/2. Ekpeghere, K.I., Kim, U.-J.O., Kim, S.-H., Oh, H.-Y., J.-E, 2016. Distribution and seasonal occurrence of UV filters in rivers and wastewater treatment plants in Korea. Sci. Total Environ. 542 (Pt A), 121–128. https://doi.org/10.1016/j.scitotenv.2015.10.033. European Chemicals Agency - CoRAP, 2019. 2-Ethylhexyl Salicylate - Substance Evaluation - Community Rolling Action Plan (CoRAP). (Accessed 6 December 2018). https://echa.europa.eu/information-on-chemicals/evaluation/community-rollingaction-plan/corap-table/-/dislist/details/0b0236e1820e160c. European Parliament and the Council, 2009. Regulation (EC) No 1223/2009 of the European Parliament and the Council of 30 November 2009 on Cosmetic Products: Reg. (EC) 1223/2009. 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4. Conclusions With this study we present the first data on human metabolism and elimination kinetics of the UV filter EHS. We tentatively identified seven metabolites of EHS after oral dosage and quantitatively investigated (using custom synthesized analytical standards and stable isotope labeled internal standards) the urinary excretion of the three most promising metabolites unique to EHS: 5OH-EHS, 5oxo-EHS, and 5cx-EPS. The chemical structure of these three metabolites contains the core structure of EHS, which makes them specific to EHS (unlike e.g. SA) and therefore qualifies them as specific biomarkers of EHS exposure. Urinary excretion fractions (Fue) were rather low for all of these three metabolites (< 1%), indicating that the total breakdown to SA is likely the predominant metabolic pathway. In spite of this apparent and rapid breakdown to unspecific metabolites (such as SA and SUA) we were able to detect and quantify one or more of the above three specific metabolites in the majority of urine samples from the general population, as well as in all urines samples collected after dermal application of an EHS containing sunscreen product (Bury et al., 2019a). The Fue reported in this study enable the reliable conversion of measured biomarker concentrations into daily intakes for robust EHS exposure and risk assessments on individual and population scale.
Funding The presented study and development of the analytical method applied herein are part of a large-scale 10-year project on the advancement of human biomonitoring in Germany. This project is a cooperation agreed in 2010 between the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety (BMU) and the German Chemical Industry Association (VCI -Verband der Chemischen Industrie e.V.) and is managed by the German Environment Agency (UBA). Experts from governmental scientific authorities, industry and science provide advice in the selection of substances to be investigated and during method development. The analytical method development and the human metabolism study are financed by the Chemie Wirtschaftsförderungsgesellschaft mbH while the first application of the novel methodology in a larger population study will be financed by the German Environment Agency. 40
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