Systemic Uptake of Diethyl Phthalate, Dibutyl Phthalate, and Butyl

Jun 26, 2007 - Abstract. In vitro and animal studies have reported endocrine-disrupting activity of chemicals used commonly as additives in cosmetics ...
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Environ. Sci. Technol. 2007, 41, 5564-5570

Systemic Uptake of Diethyl Phthalate, Dibutyl Phthalate, and Butyl Paraben Following Whole-Body Topical Application and Reproductive and Thyroid Hormone Levels in Humans N A D E E M R E Z A Q J A N J U A , * ,† GERDA KROGH MORTENSEN,‡ ANNA-MARIA ANDERSSON,‡ BRIAN KONGSHOJ,† NIELS E. SKAKKEBÆK,‡ AND HANS CHRISTIAN WULF† Department of Dermatology, Bispebjerg Hospital, and Department of Growth and Reproduction, Rigshospitalet, Copenhagen University Hospitals, Denmark

In vitro and animal studies have reported endocrinedisrupting activity of chemicals used commonly as additives in cosmetics and skin care products. We investigated whether diethyl phthalate (DEP), dibutyl phthalate (DBP), and butyl paraben (BP) were systemically absorbed and influenced endogenous reproductive and thyroid hormone levels in humans after topical application. In a twoweek single-blinded study, 26 healthy young male volunteers were assigned to daily whole-body topical application of 2 mg/cm2 basic cream formulation each without (week one) and with (week two) the three 2% (w/ w) compounds. The concentrations of BP and the main phthalate metabolites monoethyl (MEP) and monobutyl phthalate (MBP) were measured in serum together with the following reproductive hormones: follicle stimulating hormone (FSH), lutenising hormone (LH), testosterone, estradiol, and inhibin B and thyroid hormones (thyroid stimulating hormone (TSH), free thyroxine (FT4), total triiodothyroxine (T3), and total thyroxine (T4)). MEP, MBP, and BP peaked in serum a few hours after application, reaching mean ( SEM levels of 1001 ( 81 µg/L, 51 ( 6 µg/ L, and 135 ( 11 µg/L, respectively. Only MEP was detectable in serum before treatment. Minor differences in inhibin B, LH, estradiol, T4, FT4, and TSH were observed between the two weeks, but these were not related to exposure. We demonstrated for the first time that DEP, DBP, and BP could be systemically absorbed in man after topical application. The systemic absorption of these compounds did not seem to have any short-term influence on the levels of reproductive and thyroid hormones in the examined young men.

Introduction Phthalates and parabens have been manufactured in industry since the 1920s. Phthalates are used in a variety of products: * Corresponding author phone: +45 3531 6006; fax +45 3531 6010; e-mail: [email protected]. † Department of Dermatology. ‡ Department of Growth and Reproduction. 5564

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as plasticizers to induce flexibility in the otherwise rigid polyvinyl chloride (PVC) of wall coverings, automotive application, buildings and construction, cables and wires, flooring, food contact materials (food packaging), toys, medical devices, blood bags, and as denaturants or solvents in perfumes, cosmetics, shampoo, clothing, insect repellent, and medication coating (1-4). Because of their almost ubiquitous use, there is a significant exposure to the general population (5) and humans are exposed to phthalates through inhalation, oral, and dermal routes. Diethyl phthalate (DEP), dibutyl phthalate (DBP), and butyl paraben (BP) are commonly used as additives in cosmetics and skin care products. Phthalates are used for their oily texture that makes skin feel soft and their ability to impart flexibility to thin films (i.e., mascara and nail polish). In a study of phthalate levels in 72 different personal care products, DEP was detected in 71% of the products with an average concentration of 0.4% and a maximum concentration of 2.8%. DBP was detected in 8% of the products tested (mainly nail polish) with an average concentration of 5% (6). In Europe, the use of DBP in cosmetics, including nail polish, has been forbidden since April 2005 according to the EU Cosmetics Directive (7). The alkyl esters of p-hydroxybenzoic acid (parabens) are used widely as preservatives to inhibit microbial growth and extend shelf life of products in food, pharmaceuticals, cosmetics, skin care products, shampoos, conditioners, sunscreens, deodorants, and soaps (8). The antimicrobial activity of parabens increases with the length of the alkyl grouping from methyl to n-butyl, but water solubility decreases, while oil solubility increases (8). By far the most prevalent use has been in cosmetics. The European Economic Community (EEC) Directive permits the use of parabens with a maximum concentration for each one of 0.4% (w/w) and total maximum concentration of 0.8% (w/w), expressed as p-hydroxybenzoic acid. A survey of 215 cosmetic products found that parabens were detected in 77% of rinse-off products and in 99% of leave-on products, and the total paraben content in paraben-positive cosmetic products was 0.01-0.87%. The maximum concentration for butylparaben was 0.07% (9). In vitro and animal studies have shown that both parabens and phthalates can penetrate the skin (8, 10). However, there are no studies reporting systemic absorption of these chemicals after dermal application in humans. In the body, DEP and DBP are hydrolyzed to the corresponding monoesters: monoethyl phthalate (MEP) and monobutyl phthalate (MBP). Glucuronidation of the monoester or further degradation of the residual alkyl chain increases water solubility and facilitates excretion (11). However, metabolism of phthalates does not usually detoxify the compounds, and experiments have shown that phthalate monoesters and/or further metabolites of DBP may be responsible for the observed developmental toxic effects (12). DEP, DBP, and BP have all been shown to have a weak estrogenic activity in vitro by promoting estrogen-receptor dependent proliferation of MCF-7 cells. In addition, DBP also has the potential to bind weakly to the estrogen receptor (13-15). Subcutaneous administration of BP has also been shown to increase uterine weight in vivo in both immature rats and mice and in adult ovariectomised mice, hence confirming its estrogenic activity (16-17). In contrast, the estrogenic effects of DEP and DBP observed in vitro have not been confirmed in vivo. Thus, oral treatment with DEP and DBP did not induce mRNA and protein expression of 10.1021/es0628755 CCC: $37.00

 2007 American Chemical Society Published on Web 06/26/2007

TABLE 1. Values of Weight, Height, Age, BMI, Daily Cream Amount, and Body Surface Area for All Volunteersa group

N

weight (kg)

height (cm)

cream age BMI amount (years) (kg/m2) (g)

body surfaceb (m2)

males 26 79 ( 10 182 ( 9 26 ( 4 24 ( 2 40 ( 3 2.00 ( 0.16 a All values are mean ( SD. b Body surface was calculated by the formula of Dubois and Dubois (1916).

calbindin-D9K, a biomarker for estrogen activity, in the uterus of immature rats (18). Animal studies have shown that both DBP and BP exposure are adverse to the male reproduction (19-23). Exposure to BP during fetal development, as well as in young males, results in decreased testoterone concentrations and decreased sperm count (19-23). Fetal and lactational exposures to DBP result in reproductive tract abnormalities including cryptorchidism and hypospadias, decreased testosterone production, and abnormal testis histology with large dysgenetic areas with malformed semeniferous tubules and large Leydig cell clusters (22, 24-25). These effects cannot be explained by the weak estrogenic effect observed for DBP in vitro, but seem to be due to effects on the development and function of the androgen-producing Leydig cells (25). Two recent human studies have found associations between fetal and perinatal exposure to DEP and DBP and effects in the male offspring. Thus, Swan and collaborators found that the maternal urinary concentrations of MEP and MBP were inversely related to the anogenital distance of the boys (26). This study was the first to use anogenital distance, a wellknown biomarker for androgen activity during fetal development used in toxicology, in a human study. A reduced anogenital distance is related to decreased androgen action. In the other human study an association between the concentration of MEP and MBP in breast milk and changes in the reproductive hormone levels in boys at the 3 months of age were found. The observed changes in reproductive hormone levels were compatible with a decreased androgen activity (27). A screening assay by O’Connor et al. reported a dosedependent decrease of T3, T4, after oral administration of DBP for 15 days in male rats (28). DBP has also been shown to have T3 antagonistic activity in an in vitro screening assay but not in vivo in Xenopus laevis (29). So far, no human study has focused on thyroid-disrupting effects of DEP, DBP, or BP. The aim of this study was to investigate whether the three compounds DEP, DBP, and BP applied topically to healthy young men could be found in serum, and whether systemic uptake had any acute effect on the levels of endogenous reproductive and thyroid hormones in humans.

Materials and Methods Study Population. Twenty-six healthy and paid Caucasian male volunteers (aged 21-36, mean age 26 years old) were enrolled after informed consent. Male volunteers were chosen to exclude the risk of exposing women during pregnancy as well as to avoid the cyclic variation in reproductive hormone levels in premenupausal women. The study was approved by the ethics committee of Copenhagen and Frederiksberg (No. (KF) 11-039/03). Inclusion criteria for the group were as follows: no sun exposure 3 weeks before enrolment; a body mass index (BMI) < 30 kg/m2; no medication; and no history of allergy. Personal characteristics are shown in Table 1. Study Design. About four weeks prior to the study all the participants were interviewed and informed. Height and weight were measured. Their body surface area (BSA) was

calculated with the DuBois formula: BSA (m2) ) 0.007184 × height (cm)0.725 × weight (kg)0.425 (30). The subjects were only allowed to use a phthalate and butyl paraben free moisturizer and deodorant supplied by us one week before the study and during the study. Physical exercise, sun-bathing, and intake of caffeine, nicotine, or alcohol were not allowed during the study, as these activities may affect the pharmacokinetics of drugs (31-34). Figure 1 shows the study design. The study lasted two consecutive weeks: a control week followed by a treatment week. The study was blinded for the test persons, as they did not know which week was the treatment week. Thus, the subjects served as their own control. In both weeks, subjects visited the Department of Dermatology Monday-Saturday between 10 a.m. and 4 p.m. The exact time for the first cream application and blood sampling was noted, and thereafter performed at the same time each day. Thereby circadian changes in the male hormone levels were avoided. First Week. Immediately after a 0 h blood sample was drawn, basic cream formulation without phthalates and butyl paraben was applied by staff to the whole body (except for the scalp and genital areas). Test persons waited 20 min to let the cream absorb into the skin before dressing. Cream application was repeated every day for 5 days (Figure 1). The same procedure was used in the treatment week except for the fact that a daily prepared test cream formulation was used. Showering, bathing, and swimming were not allowed until 4 h after the daily application. Each subject received 2 mg/cm2 of cream according to his BSA. This is in accordance with the U.S. Food and Drug Administration (FDA) and European Cosmetic Toiletry and Perfumery Association (COLIPA) recommendations. This corresponds to 40 g for an average body area of 2.0 m2. The amount of cream per application varied among the participants from 34 to 48 g with a mean of 40 g. Basic and Active Formulations. The basic formulation was an Essex cream (Schering-Plough, Vnr 462374, Brussels, Belgium). The active formulation consisted of Essex cream with 2% (w/w) of each of the two phthalates, diethyl phthalate (DEP) (Lot A097035701, Acros organic, Bie & Berntsen, Denmark, purity g99%) and dibutyl phthalate (DBP) (Lot 81K0249, Sigma, Steinheim, Germany), and butyl paraben (BP) (Lot 121K11108, Sigma, Steinheim, Germany). Blood Collection. Blood samples were obtained from an antecubital vein via an indwelling catheter. Blood samples were centrifuged (5000 rpm for 10 min). Serum from 10 mL of blood was used for hormone analysis and chemical analysis. The aliquot for chemical analysis was acidified with 0.1 mL phosphoric acid (1.2 M) to inhibit endogenous enzyme activity. Both aliquots were stored at -20 °C until analysis. Chemical Analyses of Blood. Mono ethyl phthalate, mono butyl phthalate, and butyl paraben were analyzed in serum by liquid chromatgraphy quadrupole mass spectrometry (LCMS/MS) following a solid-phase extraction. Details of the method are provided in the Supporting Information (SI). Hormone Analyses. Serum inhibin B was measured in duplicate in a double antibody enzyme immunometric assay (Oxford-bioinnovation, UK) using a monoclonal antibody raised against the inhibin βB-subunit in combination with a labeled antibody raised against the inhibin R-subunit. The detection limit was 20 pg/mL, and the intra- and inter-assay coefficients of variation were 15% and 18%, respectively. Serum FSH and LH were measured by time-resolved immunofluorometric assay (DELFIA, Turku, Finland), with detection limits of 0.06 and 0.05 U/L, respectively. Intra- and inter-assay coefficients of variation were both below 8%. Testosterone and estradiol were measured by radio immunoassay (RIA) (Coat-a-Count, Diagnostic Products, CA and ImmunoDiagnostic Systems, Boldon, UK, respectively). The detection limit for testosterone was 0.23 nmol/L, and the VOL. 41, NO. 15, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 1. Study design: (*) application of basic cream (week one) and test cream (week two); (V) collection of blood samples. Application of cream and blood sampling were done at same time every day at 0, 24, 96 and 120 h. intra- and inter-assay coefficients of variation were both less than 10%. In the estradiol assay, the detection limit was 18 pmol/L, the intra-assay coefficient of variation was less than 8%, and the inter-assay coefficient of variation was less than 13%. All samples from the same subject were analyzed for each hormone in the same assay run in order to exclude interassay variation. A commercially available automated immunoassay system Abbott Architect i 2000 (Abbott Diagnostics Ltd, Illinois) was used to measure serum total thyroxine (T4), total triiodothyroxine (T3), thyrotropin (TSH), and free thyroxine (FT4) by chemiluminescent microparticle immoasssay (CMIA). Assays were performed according to the manufacturer’s specifications. The detection limits for T4, T3, TSH, and FT4 assays were 13 nmol/L, 0.38 nmol/L, 0.004 mU/L, and 5.1 nmol/L, respectively. The intraassay coefficients of variation were 4%, 3.5%, 2.5%, and 5%, respectively, and the interassay coefficients of variation were 7%, 4.5%, 3%, and 6%, respectively. Statistical Analyses. The hormone data were verified for normal distribution with a Kolmogrov-Smirnov normality test. At some time points during the control week and during the subsequent week of treatment, LH and FSH hormone levels did not pass the normality test for all paired samples. Therefore, all hormone data were tested in paired samples using the nonparametric Wilcoxon matched pairs signed rank sum test. The statistical program Prism, version 4.01, was used for all statistical calculations and tests.

Results Systemic Uptake. During the control week (week 1) serum levels of MBP and BP were undetectable in most samples with maximum levels not exceeding 4.0 µg/L and 1.0 µg/L, respectively. In contrast, MEP serum levels were measurable in all samples with a mean (SEM) level of 12.0 (1.0) µg/L and before the first application in treatment week (week 2) with a mean (SEM) level of 7.0 (1.0) µg/L (Figure 2a and b). After the first application of cream containing the three compounds, serum levels of the metabolites MEP and MBP increased rapidly. Thus, 2 h after the first application, MEP serum levels had increased to a mean (SEM) peak level of 1001 (81) µg/L (Figure 2a). MBP serum levels increased overall 4 h after the application to reach a mean (SEM) level of 51 (6) µg/L. Also the BP serum level increased rapidly, reaching 5566

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a mean (SEM) peak level of 135 (11) µg/L 3 h after the first application (Figure 2a). At 24 h after the first application, but before the following application, mean (SEM) serum levels were down to 23 (3) µg/L MEP, 8 (1) µg/L MBP, and 18 (3) µg/L BP. However, serum levels did not reach the baseline levels observed during the control week (week 1), and MBP and BP could be detected in most samples throughout week 2 (Figure 2b). Hormone Levels, Hypothalamic-Pituitary-Gonadal Axis. There were no statistically significant differences in the levels of testosterone, FSH, and SHBG in the men (Figure 3) when comparing hormone levels in samples obtained at the same time point in the control week and the treatment week. However, a significant difference in mean inhibin B level was found between the control week and the treatment week at 0, 1, 3, 24, and 120 h, but not at 2, 4, and 96 h. As this difference was evident also in the 0-hour samples when active treatment had not yet started and as it was not consistent, this observed difference was most likely a chance finding not related to the treatment. Likewise, minor but statistically significant differences between the two weeks in estradiol levels in the 3-hour samples and in LH in the 120 h samples most likely reflect chance findings (Figure 3). Hypothalamic-Pituitary-Thyroid Axis. Mean hormone levels obtained in the young men at the same time point during the two weeks gave no statistically significant difference in the levels of T3 (Figure 4). However, a statistical difference (p < 0.05 and p < 0.01) was found in mean TSH and FT4 levels at 96 h between the two weeks. In addition, a statistical difference in mean T4 levels was observed at 2 and 24 h, respectively. Statistical differences in both weeks at different time points could not be explained by the treatment with the three compounds, as T4 and FT4 hormone levels were already higher in the treatment week (Figure 4) before the active cream had been applied. These observed differences were most likely chance findings not related to the treatment.

Discussion Despite the extensive worldwide use of the two phthalates DEP and DBP, and the paraben BP, and in particular their use in personal care products applied to the skin, this is, to our knowledge, the first human study on systemic absorption through the skin after whole-body application of these compounds.

FIGURE 2. Mean serum concentrations of MBP, MEP, and BP in the treatment week. MBP and BP were undetectable before the first application: (a) from 0 to 4 h, and (b) from 24 to 120 h. All values are means ( SEM. N ) 26. At 0, 24, 96, and 120 h, blood samples were taken at same time every day. Detailed information is provided in Table 3 in the Supporting Information. Animal studies using rodent skin have shown that absorption of phthalates is generally slow, and human skin has in vitro been demonstrated to be less permeable to phthalates than rat skin (35, 36). There are similarly very few studies addressing percutaneous absorption of parabens and most are limited to animal and in vitro studies (17). In these studies, BP usually exhibited low penetration, retention in the epidermis, and/or hydrolysis in the skin. Animal studies have shown that parabens are quickly absorbed from the gastrointestinal tract and from the blood, hydrolyzed to p-hydroxybenzoic acid, conjugated, and then excreted in the urine as glycine, glucuronide, and sulfate conjugates. The unchanged parabens can also be excreted in various forms (17). However, evidence of percutaneous absorption of DBP and BP through human skin in vivo has been presented in a experimental model in which a small skin area of 12 subjects were exposed to a saturated solution. A maximum flux of 10 µg cm -2 h -1 (mean ) 3.8) and 40 µg cm -2 h -1 (mean ) 32) for DBP and BP, respectively, was demonstrated. From these data the authors estimated an absorbed amount of DBP and BP of respectively 66 mg and 576 mg after treating the whole body skin surface of 1.8 m2 with a saturated solution of these compounds (37). Our data support these earlier findings, as we detected higher mean peak concentrations of BP in serum (135 ( 11 µg/L) compared to MBP (51 ( 6 µg/L), showing a higher penetration of BP than DBP. The highest peak concentration was observed for MEP, which also peaked earlier than the two other compounds. Indeed, whereas both MEP and BP seemed to peak within the 4-h window during which we sampled following the first application, the concentration of MBP was still on the rise in the 4-h sample.

Thus, we may have missed the true peak concentration of MBP. These differences in systemic availability of the three compounds correlate with the length of the side chain branching which increases the hydrophobicity, hence showing the highest and fastest absorption of the more hydrophilic compound DEP, and lowest and slowest absorption of the more lipophillic compound DEP. Assuming an average blood volume of 6 L of an adult man, the observed average peak concentrations of MEP, MBP, and BP after application of on average 800 mg of each of the three compounds corresponded to a total systemic amount of 6.0 mg of MEP (corresponding to 6.9 mg of absorbed DEP), 0.30 mg of MBP (corresponding to 0.38 mg absorbed of DBP), and 0.81 mg of BP at the time of the sampling of the blood sample with the peak concentration. The total amount taken up is however larger, but cannot be estimated from serum spot samples. In our study we measured the more bioactive free MEP and MBP metabolites compared to the conjugated monoesters, because we wanted to assess the levels in terms of endocrine bioactivity. We chose also to measure the unchanged parent butyl paraben, because its main metabolite p-hydroxybenzoic acid is not specific for butyl paraben, and different parabens possess different estrogenic bioactivities (17). We observed no biologically significant effects on reproductive and thyroid hormone levels, indicating that the serum levels of the three compounds absorbed did not affect circulating levels of these hormones in adult humans in this short-term study. Minor but statistically significant differences in hormone levels between the control week and the treatment week were observed for estradiol, LH, inhibin B, FT4, and T4 at some time points. However, these differences VOL. 41, NO. 15, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Mean serum concentrations of reproductive hormones during the week of treatment with control cream (O) and during the week of treatment with cream containing DEP, DBP, and BP (b). All values are means ( SD. Significant differences in hormone levels at the same time points between the two weeks are indicated by * P < 0.05, ** P < 0.01, and *** P < 0.001. Differences are compared by a Wilcoxon test. At 0, 24, 96, and 120 h, blood samples were taken at same time every day. Detailed information is provided in Table 4 in the Supporting Information. did not seem to be related to absorption of the compounds, and at least some of these statistically significant differences between the two weeks may be chance findings due to mass significance (when 10 different hormones are tested at 8 different time points, approximately four significant findings might be expected just by chance when using a significance level of 0.05). Normal biological variations in hormone levels may also have contributed to some of the differences. This is supported by the fact that although inhibin B, FT4, and T4 were generally at a lower level during the control week, the higher level of the second week was observed already before the first application of the active formulation. Furthermore, there was no effect on the FSH and TSH levels, which are related to inhibin B levels and FT4 and T4 levels. Estrogens are known to increase serum T4-binding globulin (TBG) concentrations, thereby increasing serum total T4 concentrations. Serum free T4 concentrations, however, remain 5568

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normal (38). We did not observe any of these effects on the thyroid hormone levels. A positive association between adult male urinary levels of MBP and inhibin B, but not with any of the other reproductive hormones, has previously been found in a population-based study (39). However, there is no clear biological explanation for this observed association and it may be a chance finding. In another study by Jonsson et al. no clear pattern of associations between urinary MBP levels in young men and none of the reproductive biomarkers were found. However, subjects within the highest quartile for MEP had fewer motile sperm, more immotile sperms, and lower LH values (40). In both studies the authors concluded that there was a weak association between biomarkers of phthalate exposure and hormone alterations. However, if the weak associations found in these two population-based studies are genuine, they are likely to reflect long-term differences

FIGURE 4. Mean serum concentrations of thyroid hormones during the week of treatment with control cream (0) and during the week of treatment with cream containing DEP, DBP, and BP (9). All values are means ( SD. Significant differences in hormone levels at the same time points between the two weeks are indicated by * P < 0.05 and ** P < 0.01. Differences are compared by a Wilcoxon test. At 0, 24, 96, and 120 h, blood samples were taken at same time every day. Detailed information is provided in Table 5 in the Supporting Information. in exposure levels, whereas the exposure in our study was only for one week. To our knowledge there are no data available on the background serum levels of MEP, MBP, and BP in young men. In our control week, MBP and BP serum levels were below the limit of detection in most of the samples and the average concentration of MEP was 12 µg/L, however, these levels do not represent the normal background levels in the population, as our volunteers were asked to restrain from using personal care products containing the three compounds during the week before the study started. In our exposure week we used a cream containing 2% (w/w) of all three compounds, which with a mean cream amount of 40 g gives a total daily mean exposure of each compound of 800 mg in the treatment week. Thus, the mean exposure in µg/kg bodyweight (bw)/day(d) can be estimated with a mean bodyweight of 79 kg (Table 1) to be 10 mg/kg bodyweight/d. In order to make sure that we would attain measurable levels, we chose to expose the men to this amount although it was higher than the expected exposure from normal use of cosmetics and personal care products. However, at the same time the concentration of 2% (w/w) was not unreasonably high compared to the levels measured in commercial cosmetics and personal care products. Based on the review of the toxicological literature for butylparaben, the National Toxicology Program (NTP) and National Institute of Environmental Health Sciences (NEIHS) have estimated that adult humans are exposed to butylparaben in personal care products with a dose of 14.6 mg/day (8). Based on

measured levels of DEP and DBP in different personal care product brands combined with questionnaire data on the use of cosmetics and personal care products, the median exposure levels to phthalates in cosmetics by dermal absorption was estimated to be 0.6 µg/kg bw/d for DEP, and 0.103 µg/kg bw/d for DBP (41). However, this estimate is presumably too low considering that concentrations of 180 µg/L MEP and 23 µg/L DEP have been measured in adult spot urine (42). In conclusion, this study showed that with a whole-body dermal application of 2 mg cream/cm2 containing 2% (w/w) of DEP, DBP, and BP each, we were able to detect their main metabolites of DEP and DBP, MEP and MBP, and the parent compound BP in serum within 1 h, showing a rapid skin penetration and systemic uptake of all three compounds in humans. Furthermore, our results confirm that there was a background exposure of DEP detectable in serum before treatment. The systemic concentrations achieved from the three compounds tested in this short term study did not seem to interfere with the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-thyroid axes, as the endogenous levels of reproductive and thyroid hormones were unaffected.

Acknowledgments We thank Mette Tage and Anne-Marie Bangsgaard for assistance and Kirsten Helbo at the Department of Clinical Biochemistry, Bispebjerg Hospital, for technical assistance with the thyroid analyses. Kirsten Jørgensen is thanked for VOL. 41, NO. 15, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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technical assistance with the assays at the Department of Growth and Reproduction, Rigshospitalet. This work was supported by Aage Bangs Foundation, The Velux Foundation, Lundbeck Foundation, and the Danish Medical Research Council (grant no. 2107-04-0006).

Supporting Information Available Description and validation of the LC-MS/MS method used to measure the phthalate metabolites and butyl paraben in serum; mean serum levels of MEP, MBP, and BP, and mean reproductive and thyroid hormone levels during both weeks. This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review December 4, 2006. Revised manuscript received May 15, 2007. Accepted May 16, 2007. ES0628755