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Environ. Sci. Technol. 2007, 41, 4542-4547

Quantitative Identification of Unknown Exposure Pathways of Phthalates Based on Measuring Their Metabolites in Human Urine H I R O A K I I T O H , * ,†,§ KIKUO YOSHIDA,‡ AND SHIGEKI MASUNAGA† Graduate School of Environment and Information Sciences, Yokohama National University, Tokiwadai 79-7, Hodogaya-ku, Yokohama, 240-8501 Japan, and Research Center for Chemical Risk Management, National Institute of Advanced Industrial Science and Technology, Onogawa 16-1, Tsukuba, 305-8569 Japan

Humans are exposed to ubiquitous phthalates via multiple pathways. Exposures to phthalates have been estimated in some previous risk assessments in Japan based on pointof-contact measurement or scenario evaluation approaches. While the Japanese national government has regulated the use of di(2-ethylhexyl)phthalate (DEHP) and excluded several other phthalates from its regulation based on some of them, it is unclear whether such past exposure assessment studies fully assessed total human exposure to phthalates. In the present study, we measured their urinary metabolites, which show direct evidence of human exposure to phthalates. We recruited voluntary participants (N ) 36) who agreed to donate urine samples, and measured the urinary concentrations of phthalate metabolites using enzymatic deconjugation, solid-phase extraction, and high-performance liquid-chromatography isotope-dilution tandem mass spectrometry. We then derived the daily intakes of their respective phthalates based on steady state assumption and finally compared them with the corresponding estimated daily intakes of each phthalate via diet and air derived from previous exposure or risk assessments in Japan. These comparisons showed that exposures to dimethyl phthalate, diethyl phthalate, and di-n-butyl phthalate via diet and air accounted for less than half of their respective total exposures. On the other hand, it appears that dietary intake was more predictive for the total exposure to n-butyl-benzyl phthalate and DEHP. The probabilities that the lognormal distribution of each phthalate daily intake estimated from the present study exceeds the corresponding tolerable daily intake were estimated to be less than 10-4.

Introduction Humans are exposed to multiple environmental contaminants via various exposure pathways. Phthalic acid diesters, * Corresponding author phone: +81 3 3542 2511 (ext 3391); fax: +81 3 3547 8578; e-mail: [email protected]. † Yokohama National University. ‡ National Institute of Advanced Industrial Science and Technology. § Current address: Epidemiology and Prevention Division, Research Center for Cancer Prevention and Screening, National Cancer Center, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045 Japan. 4542

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commonly known as phthalates, represent one group of such chemicals. Phthalates are well-known for their high production volume, broad range of application, and ubiquity in our immediate environment. The use of certain phthalates, including di(2-ethylhexyl)phthalate (DEHP), has been regulated and controlled in Japan. The Ministry of Health and Welfare (now the Ministry of Health, Labour and Welfare) set a provisional tolerable daily intake (TDI) of DEHP based on the testicular and reproductive toxicities against rodents (1, 2) in 2000. At the same time, the Ministry prohibited the use of gloves made of flexible polyvinylchloride containing DEHP for food preparation (3). The standard was then revised and applied to some kinds of food containers and packages, as well as to certain toys for babies. The altered standard was announced in June 2002, and then enforced in August 2003 (4). These regulations excluded other phthalates, for example, dimethyl phthalate (DMP), diethyl phthalate (DEP), di-nbutyl phthalate (DBP), and n-butyl benzyl phthalate (BBzP), from their target substances because human exposures to these phthalates had been estimated to be sufficiently low. However, those administrative policies were derived from screening level risk assessments based on, for example, the measurement of the concentration of phthalates in diet or air. Exposure to chemicals can be assessed using the following three methods: point-of-contact measurement, scenario evaluation (a mathematical modeling approach), and reconstruction (5). While both of the indirect approaches, namely point-of-contact measurement and scenario evaluation, depend on exposure scenarios described by riskassessment experts and can fail to include all the major human exposure pathways of the chemicals, the reconstruction of exposure based on a direct approach, the measurement of appropriate biomarkers, can faithfully reflect the actual total human exposure to ubiquitous chemicals without omission. When phthalates are experimentally administered to humans, their unique metabolites are quickly excreted in urine (6-9). This relation made it possible to apply a reconstruction approach to evaluate human exposure to phthalates directly. Analytical methods for the measurement of low concentrations of urinary phthalate metabolites have been developed (10-12). Using such methods, a series of population exposure surveys has been performed in the U.S. (13, 14). The measured concentration of phthalate metabolites in human urine can finally be translated into an individual daily intake of the corresponding parent compound (15-17). Now, it is not clear whether exposure to various phthalates via diet and air fully accounts for actual total human exposure to these ubiquitous phthalates. Such indirect estimates need to be validated from the perspective of direct exposure assessment. In this article, we performed direct measurement of urinary biomarkers for phthalates. The mass budgets between direct and indirect assessment were finally quantified. We targeted DMP, DEP, DBP, BBzP, and DEHP for the assessment, and then measured their respective primary metabolites in urine: mono-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-n-butyl phthalate (MBP), monobenzyl phthalate (MBzP), and mono(2-ethylhexyl)phthalate (MEHP). Our results indicate that the past indirect exposureassessment studies of phthalates in Japan have overlooked the critical exposure pathways of at least three of the five phthalates. 10.1021/es062926y CCC: $37.00

 2007 American Chemical Society Published on Web 06/01/2007

Materials and Methods Urine Sample Collection. We collected urine samples for the present study between late May and early June 2004 (18). The protocol was approved by the ethics committee of the Japan Epidemiological Association, and all participants or parents provided their written informed consent. Thirty-six volunteers, who ranged from 4 to 70 years of age, participated. There were 35 adults and 1 child. Twenty-six of the participants were from 20 to 29 years of age. Twenty-five of the participants were males. Most of the participants lived in the Tokyo-Yokohama area. A single spot urine sample was collected in a polypropylene centrifuge tube, and then stored at -20 °C until the analysis was performed. Information including height, body weight, age, and gender was acquired through a brief, self-administered questionnaire completed by the participants (18). These sample containers and questionnaires were anonymous but numbered. The informed consent forms were not numbered, and were automatically anonymized at the point of submission. Additionally, all samples were newly renumbered before analysis. Urine Sample Analysis. We measured five urinary phthalate metabolites using enzymatic deconjugation, solid-phase extraction, and high-performance liquid-chromatography isotope-dilution tandem mass spectrometry following the procedures by Itoh et al. (18) and Silva et al. (12) (see the Supporting Information). Along with this series of analyses, we analyzed one pooled urine sample spiked with phthalate monoesters and one method blank in each analytical batch to check the between-run reproducibility of the measurement values and background contamination. Analytical methods and conditions are detailed in the Supporting Information section. Urinary creatinine concentration was also measured to correct urine dilution. An aliquot of collected urine sample was shipped to Mitsubishi Kagaku Bio-Clinical Laboratories, Inc. (Tokyo, Japan) for the measurement of urinary creatinine concentration using an enzymatic method. Translation into Daily Intake. The concentration of phthalate metabolite measured in individual urine samples from adults (20 years old and above, n ) 35) was then converted into the individual daily intake of the corresponding phthalate diester. We assumed steady-state exposure and then used eq 1 as in the previously published studies (15, 17, 18). Here, ME is the creatinine-adjusted concentration of each phthalate monoester [µg/g creatinine], CE is the personal daily urinary creatinine-excretion rate normalized by individual body weight [g/kg/day], f is the molar fraction of the urinary excreted monoester related to the ingested diester, and MWd and MWm are the molecular weights of the phthalate diesters and monoesters, respectively.

Intake )

ME × CE MWd × f MWm

(1)

The individual creatinine-adjusted concentration of phthalate monoesters was substituted for ME in eq 1. For f for DBP, BBzP, and DEHP, human experimental data were available. For DEHP, a value of f ) 0.062, which was obtained from a low-dose study (9), was newly employed because the low dose (4.7 µg/kg) is appropriate for the actual range of DEHP intake. According to Anderson et al. (8), it was found that 0.69 and 0.73 molar fractions of single dietary-administered doses of DBP-13C and BBzP-d4, which are isotopically labeled phthalate diesters, were excreted in human urine as MBP13C and MBzP-d , respectively. Certainly, a part of the 4 observed MBP will also be derived from BBzP exposure; however, we disregarded its contribution because only a 6%

FIGURE 1. Population risk is defined as the probability that a lognormal distribution of estimated daily intake exceeds the corresponding TDI. molar fraction of dosed BBzP is recovered as MBP in urine (8). The contribution is negligible because of the low exposure to BBzP. For the other hydrophilic phthalate esters, DMP and DEP, no human data were available. However, their hydrophilic property indicates that their f should be reasonably inferable to be higher than 0.69; hence, as the value of f for DMP and DEP, both ends of the possible range, 0.69 and 1.0, were substituted. CE [g/kg/day] was calculated for each individual (18); while previous studies (16, 17, 19) treated CE as a constant. Individual daily urinary creatinine excretion rate (PRCr [mg/day]) was predicted based on the individual gender, age, body weight, and height of participants, along with a special regression equations (20): PRCr (male) ) -12.63 × age (years) + 15.12 × bodyweight (kg) + 7.39 × height (cm) - 79.90; PRCr (female) ) -4.72 × age (years) + 8.58 × bodyweight (kg) +5.09 × height (cm) -74.95. Thus, the concentration of their urinary metabolites determined the daily intake of phthalate diesters. Incidentally, probabilistic risk of phthalates, which is a probability that a lognormal distribution fitted into estimated daily intake exceeds the corresponding TDI, was also calculated as shown in Figure 1. Mass Budget. We explored whether the past indirect exposure assessments reported in Japan fully accounted for total human exposure to each phthalate. We constructed graphs to compare the phthalate daily intake levels that we estimated from the measurement of urinary metabolites to the levels of exposure to phthalates estimated from the other studies, which measured dietary intake or air inhalation. Several past exposure assessment studies of phthalates in Japan were found in the literature (21-25). These studies at least covered or targeted general population or hospitals in Japan in recent years. They estimated daily intake of phthalates based on eq 2 or 3 assuming steady-state exposure. Here, Intakediet [µg/kg/day] is the daily intake of each phthalate via diet, Intakeair [µg/kg/day] is the daily intake of each phthalate via air, Cdiet and Cair,i are the concentrations of each phthalate in the diet [µg/g] and in air (i ) indoors or outdoors) [µg/m3], IRdiet is the diet-consumption rate [g/day], IRair is the air-inhalation rate [m3/day], ACTi is the time spent daily (i ) indoors or outdoors) [hours], 24 is a normalization factor [hours], and BW is human body weight [kg].

Intakediet )

∑ Intakeair )

i

Cdiet × IRdiet BW

Cair,i ×

ACTi 24

(2)

× IRair (3)

BW

We finally calculated Intaketotal, which is the theoretical aggregate exposure to phthalates based on the indirect VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Mass budgets of human exposures to five phthalates in Japan. The combinations of paired indirect exposure-assessment data from other studies, exposure via diet (Intakediet) and exposure via indoor air (Intakeair), and relevant to the symbols in the brackets are cited from literature as follows. Theoretical aggregate exposure (a) is the combination of data for Intakediet from Ministry of the Environment (26) and data for Intakeair from Ministry of Environment (26) respectively. (b) Similarly, data from Tsumura et al. (21) and Hasegawa et al. (24). (c) Data from Takahashi et al. (23) and Hasegawa et al. (24). (d) Data from Tsumura et al. (22) and Hasegawa et al. (24). (e) Data from Tsumura et al. (21) and Hasegawa et al. (24). (f) Data from Takahashi et al. (23) and Hasegawa et al. (24). (g) Data from Tsumura et al. (22) and Hasegawa et al. (24). (h) Data from Tsumura et al. (21) and Hasegawa et al. (24). (i) Data from Yoshida and Naito (25) using scenario evaluation and Hasegawa et al. (24). (j) Data from Tsumura et al. (22) and Hasegawa et al. (24). (k) Data for Intaketotal from Yoshida and Naito (25) using point-of-contact measurement, respectively. The theoretical aggregate exposures to DMP, DEP, and DBP via diet and air account for only limited fractions of their respective actual total exposures. On the other hand, exposures to BBzP and DEHP via diet were the main contributors to their respective actual total exposures.

TABLE 1. Urinary Concentration of Phthalate Metabolites (n ) 36) and Estimated Daily Intake of Their Parent Compounds (n ) 35) urinary primary metabolitea

MMP MEP MBP MBzP MEHP

parent compounda

raw concentration median [µg/L]

geometric standard deviation

33 18 36 2.4 5.0

3.89 3.97 3.35 3.18 2.52

DMP DEP DBP BBzP DEHP

intake [µg/kg/day]

f

mean (95% CI)

0.69-1.0 0.69-1.0 0.69 0.73 0.062

1.4 (0.64-2.1)-2.0 (0.93-3.1) 0.77 (0.39-1.2)-1.2 (0.56-1.7) 1.7 (1.2-2.2) 0.093 (0.074-0.11) 2.7 (2.0-3.3)

a Dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), n-butyl benzyl phthalate (BBzP), and di(2-ethylhexyl)phthalate (DEHP) and their corresponding urinary primary metablites, mono-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), and mono(2-ethylhexyl)phthalate (MEHP).

assessments from other studies (after-mentioned), using eq 4.

Intaketotal ) Intakediet + Intakeair

(4)

For example, Intakediet of DEP provided by Tsumura et al. (22) and Intakeair of DEP provided by Hasegawa et al. (24) were combined to be compared to the Intake of DEP obtained from the present study. We employed the dietary DEHP studies performed only after 2001 because dietary DEHP intake in Japan might have dropped over the period from 1998 to 2001 (18, 22). We recalculated some of other study results for standardization to be comparable to the daily intakes [µg/kg/ day] we estimated. Hasegawa et al. (24) and the Ministry of Environment (26) assumed BW to be 50 kg. We replaced the 50 kg with 60 kg for the sake of standardization because the mean body weight of the adult participants (n ) 35) in the present study was 60 kg. On the other hand, Tsumura et al. (21, 22) and Takahashi et al. (23) did not assume BW. For the BW in these studies, we substituted 60 kg as well. Additionally, 4544

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we disregarded exposure to phthalates via drinking water because the levels (0.002-0.004 µg/kg/day) were less than the limits of detection or much lower than dietary exposure (26-30). Thus, these Intaketotal obtained from the paired indirect studies were comparable to the Intake in the present study.

Results and Discussion We measured urinary phthalate metabolite concentrations and then translated them into the individual daily intake levels of the corresponding parent compounds as shown in Table 1. These estimated daily intakes were finally compared to the theoretically aggregated exposures based on the past indirect exposure-assessment data in Japan as shown in Figure 2. These results elucidated the sufficiency or insufficiency of the understanding of human exposure to each phthalate in the previous exposure-assessment studies. Exposure via diet and air accounted for only half or less of the total exposure to DMP, DEP, and DBP. These results suggest the possible contribution of unknown exposure pathways of such phthalates. A certain percentage of the

unassigned exposure pathways may be attributable to the use of personal care products to some extent because DMP, DEP, and DBP are known to be ingredients in personal care products (31) such as cosmetics, nail polishes, hair sprays, deodorants, and perfumes (31, 32). It has not been confirmed whether phthalates in such products are actually transferred into the human blood stream; however, the following suggestive evidence has been reported. Koch et al. (33) previously reported that the urinary MBP concentration of cosmetic-products users was significantly higher than that of nonusers in their study. Duty et al. (34) also pointed out that the use of certain personal care products was predictive of the urinary concentration of MEP and MBP. These exposure pathways should also contribute to the human exposure to these phthalates to some degree. Therefore, understanding the characteristics of the use of these chemicals is also crucial to evaluating human exposure and effective countermeasures. Although dermal intake or inhalation of volatile portion or aerosol of phthalates like DEP may be considerably high, to our regret, there has been no available data, which assessed human exposure to phthalates in Japan based on point-of-contact measurement or scenario evaluation approach. On the other hand, considering Figure 2 and the mean and 95% confidence intervals of estimated intake shown in Table 1, the level of total exposure to DEHP and BBzP estimated in the present study was similar to the theoretical aggregate exposures estimated by the past indirect studies in Japan. The mean intakes of BBzP and DEHP from other studies fell at least within the 95% confidence intervals of the corresponding mean intakes we estimated. It appears that dietary intake is responsible for most human exposure to DEHP and BBzP in Japan. Here, we particularize the previous exposure-assessment studies for phthalates in Japan and the respective indirect methods they employed. Tsumura et al. (21, 22) and Takahashi et al. (23) measured Cdiet and IRdiet, and then calculated the mean of Cdiet × IRdiet for each phthalate. Tsumura et al. (21) surveyed the concentrations of 11 phthalates including DEP, DBP, BBzP, and DEHP in one-week duplicate diet samples (n ) 63) obtained from three hospitals in Osaka, Aichi, and Niigata Prefecture in October or December 1999. Tsumura et al. (22) surveyed the concentrations of four phthalates including DBP, BBzP, and DEHP in one-week duplicate diet samples (n ) 63) obtained from the same three hospitals between July 2001 and September 2001. Tsumura et al. (22) have stated that the intake estimated from hospital diets reflects typical intake from Japanese foods. Takahashi et al. (23) also surveyed the concentrations of four phthalates including DEP, DBP, and DEHP in the 3 day duplicate diet samples (n ) 18) obtained from three households in Sendai in 1998 and 1999. They also measured each IRdiet at the same time. Hasegawa et al. (24) surveyed the concentrations of eight phthalates including DEP, DBP, BBzP, and DEHP in indoor (living room and bedroom) and outdoor air samples obtained from 95 households in seven blocks all over the country in 2001. Hasegawa et al. (24) then estimated their Intakeair based on the assumption that IRair, BW, and ACTi were 15 m3/day, 50 kg, and the average time spent daily for a Japanese homemaker (ACTi ) 14 h in living room; 7 h in bedroom; 3 h outdoor), respectively. Yoshida and Naito (25) performed two DEHP exposure assessments. One is based on point-of-contact measurement; the other is based on scenario evaluation. First, they estimated Intaketotal of DEHP using eqs 5 and 6 with point-of-contact measurement data. Here, Cair,i,j is each phthalate concentration in air (i ) indoors or outdoors; j ) in summer or in winter) [µg/m3]. They randomly and iteratively sampled 10 000 virtual data sets from each probabilistic distribution

of DEHP concentration in diet (undermentioned), in indoor air, and in outdoor air (35, 36), IRdiet (37), IRair (eq 6), and BW (37) simultaneously, and finally integrated them into a probabilistic distribution of Intaketotal of DEHP using eq 5 and Monte Carlo simulation. Japan Food Research Laboratories surveyed DEHP concentrations in duplicate diet samples (81 day meals including drinking water) obtained from 27 households in Tokyo Metropolitan Prefecture three households in every nine blocks all over the country) 3 days in a row in 2001 (25). The Tokyo Metropolitan Government (35) surveyed DEHP concentrations in indoor air (n ) 68 in summer and n ) 68 in winter, in fiscal year 2000) and outdoor air (n ) 17 in summer and n ) 17 in winter, in fiscal year 2001) in Tokyo Metropolitan Prefecture. The general population was broken down by age groups. For each of these age groups, exposures were quantified using eq 5. The IRair was corrected using BW by age bracket based on eq 5.

Cdiet × IRdiet + Intaketotal )

∑C i,j

air,i,j

×

ACTi × IRair 24

BW IRair ) 20 ×

(5)

(BW 70 )

2/3

(6)

Second, Yoshida and Naito (25) performed another indirect DEHP exposure-assessment study based on scenario evaluation (mathematical modeling approach). The study also targeted the Tokyo-Yokohama area in 2001. They estimated the DEHP concentration in diet using eq 2 and several mathematical models, for example, an atmospheric dispersion model (38) and plant uptake models (39). The estimated DEHP concentrations in various environmental media were checked using point-by-point comparisons with the corresponding existing measured concentrations in air, apple, milk, and other stock farm products. No assessment data for mean dietary DMP exposure are available in Japan, but a worst-case assessment has been performed based on a survey performed in 1999 (26). In that assessment, because the DMP concentration in diet was too low to be detectable, a value equal to the analytical limit of detection in the dietary DMP analysis (0.01 µg/kg) was substituted as a high-end estimate of DMP concentration in diet. This DMP assessment result was also provisionally employed in the present study. The IRdiet was assumed to be 2 × 103 g/day (26). This assumption will, at least, not underestimate dietary DMP exposure. Incidentally, some uncertainties and limitations exist in the present study. The following points need to be considered in future studies. The source or sources that account for the unknown shortfall of exposure to DMP, DEP, and DBP remains to be identified. Further study is recommended to determine the unknown exposure pathways of DMP, DEP, and DBP quantitatively. We have to point out that the present study does not necessarily represent the general population in Japan because of the limited sample size and geographic location. In particular, children were not covered in the present study. The results presented here may only be applicable to adults. It is still not clear whether babies and neonates are exposed to phthalates including BBzP and DEHP via unknown exposure pathways. To be precise, the difference in the bioavailability of phthalates between exposure via inhalation and exposure via ingestion and the interindividual variation of the metabolism of phthalates should also be considered in the future. Temporal variability of urinary concentration derived from the short biological half-lives of phthalate metabolites as shown in two previous studies (40, 41) would VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 2. Probabilistic Risk of Phthalates Estimated from Measured Concentration of Their Metabolites in Human Urine intake compound

geometric mean [µg/kg/day]

DMP DEP DBP BBzP DEHP

0.60-0.87 0.39-0.56 1.19 0.077 1.75

geometric standard deviation 3.39 3.09 2.30 1.98 2.18

TDI [µg/kg/day]

riske

(not available) 800a 100b 200b 40-140c (48d)

(not calculated) 7 × 10-12 - 6 × 10-11 6 × 10-8 < 10-12 3 × 10-8 - 7 × 10-5 (3 × 10-5)

a Reference dose (42). b EU tolerable daily intake (TDI) (43). c Japanese provisional TDI (3). d EU TDI (44). e Risk shows a probability that a lognormal distribution of estimated daily intake exceeds the corresponding TDI (see Figure 1).

have increased the confidence intervals of the mean of estimated intake. The direct and indirect assessments, which were compared with each other, were conducted in different years. In particular, exposure to DEHP may have historically changed during 2001-2004 (18). Such a time lag could affect the conclusions of our analysis. The number of past indirect studies cited in the present study was also limited. Further studies in the future will ensure our conclusion. Although currently researchers are using the secondary metabolites of DEHP to assess DEHP exposure, we did not measure them because they had not been commercially available analytical standard substances when we analyzed urine sample. They can be more suitable to assess exposure to DEHP than MEHP because they show longer half-lives of urinary excretion. The question arises as to whether the health risks of phthalates were really lower than the safe levels. We stochastically estimated their population risk based on the respective TDIs and the assumption of a lognormal distribution of exposure (see Figure 2) using each geometric mean and geometric standard deviation (see Table 2). The results are summarized in Table 2. Every noncancer risk estimated here was lower than 10-4. Exposures to every phthalate were lower than the safe levels as long as these TDIs were employed as the benchmarks of the health risk of these phthalates. Although the government omitted the use of DEP, DBP, and BBzP from the regulation, this policy decision will eventually prove not to be in a serious mistake. In conclusion, the study presented here clearly demonstrated that the past indirect studies had overlooked the critical exposure pathways of at least three of five phthalates. Surprisingly, the theoretically aggregated exposures via diet and air accounted for less than half of the total exposures to DMP, DEP, and DBP. On the other hand, it appears that dietary intake is a main contributor to the total human exposure to BBzP and DEHP. The conclusions are based on a limited number of subjects and may not apply to the general Japanese population.

Acknowledgments We thank the anonymous reviewers for the critical reading of the manuscript and valuable suggestions. We are grateful to the participants in our study, as well as to the others who gave their assistance. We also thank the experts at JASCO International (Tokyo, Japan) and Waters Japan (Tokyo, Japan) for providing us with valuable technical support and for maintaining our instruments. An HPLC column was obtained from YMC (Kyoto, Japan) during a column-monitor campaign. This study was supported by a part of the university research expenditure of Yokohama National University. In addition, H.I. is an awardee of two travel fellowships: the SRA 2005 International Travel Award and the Foundation for Environmental Research of Showa Shell Sekiyu K.K., for his attending the SRA 2005 Annual Meeting (Society for Risk Analysis). 4546

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Supporting Information Available Analytical methods and analytical conditions of urinary phthalate monoesters. This material is available free of charge via the Internet at http://pubs.acs.org.

Literature Cited (1) Poon, R.; Lecavalier, P.; Mueller, R.; Valli, V. E.; Procter, B. G.; Chu, I. Subchronic oral toxicity of di-n-octyl phthalate and di(2-Ethylhexyl) phthalate in the rat. Food Chem. Toxicol. 1997, 35 (2), 225-239. (2) Lamb 4th, J. C.; Chapin, R. E.; Teague, J.; Lawton, A. D.; Reel, J. R. Reproductive effects of four phthalic acid esters in the mouse. Toxicol. Appl. Pharmacol. 1987, 88 (2), 255-269. (3) Ministry of Health and Welfare: Report on the conclusion at the joint meeting of toxicity group and tools (in Japanese). Vessels and Wraps Group in Committee for Food Hygiene, Tokyo 14 June 2000. http://www1.mhlw.go.jp/houdou/1206/h06141_13.html. (4) Ministry of Health Labour and Welfare: Report on partial revision of the standard for food, additive, etc. (in Japanese). No. 0802005, Tokyo 2 August 2002. http://www.mhlw.go.jp/ topics/bukyoku/iyaku/kigu/dl/1.pdf. (5) U. S. Environmental Protection Agency. Guidelines for exposure assessment. Fed. Regist. 1992, 57 (104), 22888-22938. (6) Peck, C. C.; Albro, P. W. Toxic potential of the plasticizer Di(2-ethylhexyl) phthalate in the context of its disposition and metabolism in primates and man. Environ. Health Perspect. 1982, 45, 11-17. (7) Schmid, P.; Schlatter, C. Excretion and metabolism of di(2ethylhexyl)phthalate in man. Xenobiotica 1985, 15 (3), 251256. (8) Anderson, W. A.; Castle, L.; Scotter, M. J.; Massey, R. C.; Springall, C. A biomarker approach to measuring human dietary exposure to certain phthalate diesters. Food Addit. Contam. 2001, 18 (12), 1068-1074. (9) Koch, H. M.; Bolt, H. M.; Preuss, R.; Angerer, J. New metabolites of di(2-ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP. Arch. Toxicol. 2005, 79 (7), 367-376. (10) Blount, B. C.; Milgram, K. E.; Silva, M. J.; Malek, N. A.; Reidy, J. A.; Needham, L. L.; Brock, J. W. Quantitative detection of eight phthalate metabolites in human urine using HPLC-APCIMS/MS. Anal. Chem. 2000, 72 (17), 4127-4134. (11) Silva, M. J.; Malek, N. A.; Hodge, C. C.; Reidy, J. A.; Kato, K.; Barr, D. B.; Needham, L. L.; Brock, J. W. Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry. J. Chromatogr., B: Analyt. Technol. Biomed. Life Sci. 2003, 789 (2), 393-404. (12) Silva, M. J.; Slakman, A. R.; Reidy, J. A.; Preau, J. L.; Jr.; Herbert, A. R.; Samandar, E.; Needham, L. L.; Calafat, A. M. Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2004, 805 (1), 161-167. (13) Blount, B. C.; Silva, M. J.; Caudill, S. P.; Needham, L. L.; Pirkle, J. L.; Sampson, E. J.; Lucier, G. W.; Jackson, R. J.; Brock, J. W. Levels of seven urinary phthalate metabolites in a human reference population. Environ. Health Perspect. 2000, 108 (10), 979-982. (14) Silva, M. J.; Barr, D. B.; Reidy, J. A.; Malek, N. A.; Hodge, C. C.; Caudill, S. P.; Brock, J. W.; Needham, L. L.; Calafat, A. M. Urinary levels of seven phthalate metabolites in the U.S. population

(15) (16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

(25) (26)

(27)

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Received for review December 10, 2006. Revised manuscript received April 4, 2007. Accepted April 4, 2007. ES062926Y

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