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Research Article

Biomarkers of Exposure Top

Phthalates are widely used in laboratory equipment, and contamination is possible (Blount et al. 2000a; Kessler et al. 2001). Sample contamination problems are greatly minimized when phthalate metabolites are measured (Blount et al. 2000a). To select the most appropriate biomarkers of exposure, understanding the toxicokinetics of individual phthalates is fundamental. Although differences in absorption of phthalates exist, we address only metabolic differences in this article.

In rats, monoethyl phthalate (MEP) is the principal urinary metabolite of DEP; smaller amounts of phthalic acid and DEP are also found (Albro and Moore 1974). Metabolism in humans is assumed to be similar (ATSDR 1995). Elimination half-lives of DEP and MEP have not been experimentally defined but, like DEHP and its hydrolytic metabolite mono(2-ethylhexyl) phthalate (MEHP), are assumed to be a few hours. These findings suggest that MEP is the most sensitive and specific biomarker of exposure to DEP.

More than 20 urinary metabolites of DEHP have been proposed (Albro 1986). In rodents these consist primarily of terminal oxidation products. In humans the principal DEHP metabolites are side-chain–oxidized metabolites of MEHP (Koch et al. 2004a, 2005b). In two cancer patients receiving an infusion of a platelet concentrate containing DEHP, > 50% of the DEHP disappeared from the blood in about 30 min and appeared as DEHP derivatives in urine within 6 hr (Peck and Albro 1982). In another study of two volunteers who received DEHP orally, the urinary elimination half-life of DEHP was estimated to be 12 hr (Schmid and Schlatter 1985). The urinary excretion of DEHP metabolites in one person after three oral doses of D₄-DEHP followed a multiphase elimination model (Koch et al. 2004a, 2005b). For the first 4–8 hr, excretion half-lives were approximately 2 hr for mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and MEHP. Fourteen to eighteen hours postadministration, half-lives were 5 hr (MEHP) and 10 hr (MEHHP and MEOHP) (Koch et al. 2004a). MEHHP was the major metabolite initially; other metabolites, mono(2-ethyl-5-carboxypentyl) phthalate and mono(2-carboxymethylpentyl) phthalate, were more abundant starting 12 hr after exposure (Koch et al. 2005b). The higher urinary concentrations in humans of MEOHP and MEHHP than of MEHP (Barr et al. 2003; Kato et al. 2004; Koch et al. 2003c, 2004b, 2005b; Silva et al. 2006a, 2006b) suggest that oxidative metabolites may provide greater analytical sensitivity than MEHP. Furthermore, oxidative metabolites cannot be formed as a result of sampling contamination and may be more advantageous as biomarkers of exposure to DEHP than MEHP. DEHP, seldom found in blood or urine except as a consequence of contamination, is not recommended as a biomarker in studies involving these media but may be useful in studies involving other media (e.g., feces).

Highly specific, sensitive, accurate, and precise analytical methods using isotope-dilution–high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry for measuring parts-per-billion levels of selected phthalate metabolites in biologic matrices have been described (Blount et al. 2000a; Calafat et al. 2004b; Kato et al. 2003a, 2003b, 2003c, 2005; Koch et al. 2003b, 2004a; Mortensen et al. 2005; Preuss et al. 2005; Silva et al. 2003, 2004c, 2005a, 2005b; Takatori et al. 2004).

Urine (as matrix) and phthalate metabolite concentrations (as biomarkers) represent the most common approach to investigating phthalate exposure in humans. Phthalate concentrations in blood have been reported, but most assessed concentrations of diesters. Data from such studies are often questionable because of the potential for diester contamination. Consequently, methods were developed to measure concentrations of metabolites in serum (Kato et al. 2003b Kato et al. 2004a; Silva et al. 2005b; Takatori et al. 2004), breast milk (Calafat et al. 2004b; Mortensen et al. 2005), saliva (Silva et al. 2005a), and human amniotic fluid (Silva et al. 2004b). Data from media other than urine could also be used for exposure assessment, but it might be more difficult to collect the samples. Thus, these alternative media may not readily lend themselves to large screening programs but may be useful in specific situations.

Environmental Public Health Uses of Biomonitoring Data Top

Defining human exposure to phthalates requires measuring concentrations of parent compounds or their metabolites in urine and other biomatrices as well as understanding the pharmacokinetics of individual phthalates. The Centers for Disease Control and Prevention (CDC) collects urinary metabolite data for the general population, primarily through the National Health and Nutrition Examination Survey (NHANES), an ongoing national survey designed to evaluate the health and nutritional status of the U.S. population. NHANES is unique in its ability to examine public health issues that can be addressed through physical and laboratory examinations. NHANES 1999–2000 and 2001–2002 (CDC 2005; Silva et al. 2004a) provided nationally representative population-based urinary phthalate metabolite data, based on one specimen per participant, for selected demographic groups in the United States. However, young (i.e., < 6 years of age) and older individuals (i.e., > 60 years of age) were not represented in the population sampled, and no data on prenatal exposures were collected.

Data from NHANES and other studies conducted in the United States (Adibi et al. 2003; Blount et al. 2000b; Brock et al. 2002; CDC 2005; Hoppin et al. 2002; Silva et al. 2004a) and abroad (Koch et al. 2003c, 2004b) have confirmed that human exposure to phthalates is widespread (Tables 1–3). Some situations, not specifically addressed by large surveys such as NHANES, may lead to phthalate exposures well above those found in the general population. Examples include the use of certain medications with enteric coatings containing phthalates [e.g., DEP, dibutyl phthalate (DBP)] (Hauser et al. 2004a; Koch et al. 2005d) or related to using DEHP in medical devices (Calafat et al. 2004a; Green et al. 2005; Koch et al. 2005a, 2005c).

Studies of specific health effects with environmental phthalate exposures using urinary metabolite concentrations as exposure surrogates exist (Duty et al. 2003a, 2003b, 2004, 2005; Hoppin et al. 2004; Jonsson et al. 2005; Swan et al. 2005). However, these epidemiologic data are limited and drawing firm conclusions has been difficult (Hauser and Calafat 2005).

Internal Dose and Exposure Assessment Top

Previous exposure assessments for phthalates have been indirect, that is, relying on surveys of product use, measuring phthalates in various media, estimating human contact, and pharmacokinetic assumptions based on animal data. In contrast, direct methods using urinary metabolite concentrations as biomarkers for phthalate exposure may provide the most accurate assessments because these concentrations represent an integrative measure of exposure from multiple sources and routes and can be used to calculate phthalate exposure in the general (Blount et al. 2000b; CDC 2005; Koch et al. 2003c; Silva et al. 2004a) and specific populations (Adibi et al. 2003; Brock et al. 2002; Duty et al. 2003a, 2003b, 2004; Hoppin et al. 2002; Jonsson et al. 2005; Koch et al. 2004b; Swan et al. 2005).

For phthalate metabolite data, two calculation methods produced similar results (David 2000; Kohn et al. 2000). For illustrative purposes, we show the method of David (2000) as expressed by Koch et al. (2003a):

in which DI is the daily intake in milligrams per kilogram per day; UE is the creatinine-corrected urinary metabolite concentration in micrograms per gram; CE is the creatinine clearance rate, normalized for body weight, in milligrams per kilogram per day; Fue is the molar conversion factor that relates urinary excretion of metabolite to diester ingested; and MWd and MWm are the molecular weights of diester and metabolite, respectively. For these calculations, we set CE at 20 mg/kg/day for adults, 11 mg/kg/day for children, and 9.8 mg/kg/day for infants (Jacobs et al. 2001; Tietz 1990). We set Fue at 0.69 mg/kg/day for DEP (as MEP), 0.13 mg/kg/day for DEHP (as MEHP), 0.23 mg/kg/day (as MEHHP), and 0.15 mg/kg/day (as MEOHP).

An Fue value for DEP has not been determined experimentally but is assumed to be similar to the value determined for DBP (Anderson et al. 2001). By contrast, urinary excretion of DEHP metabolites has been studied after oral (Anderson et al. 2001; Koch et al. 2004a; Schmid and Schlatter 1985) and intravenous (Peck and Albro 1982) administration. The earliest reports of Fue for DEHP metabolites came from studies that had either analytical limitations or small sample sizes (Peck and Albro 1982; Schmid and Schlatter 1985). Subsequently, an MEHP Fue value was determined by HPLC–mass spectrometry from a study involving seven individuals dosed orally with both ¹³C-DEHP and ¹³C-diisooctyl phthalate (Anderson et al. 2001). Because the ¹³C-MEHP and ¹³C-monooctyl phthalate signals co-eluted, Fue for these species could not be determined separately, and the MEHP value of 0.13 is the average (Anderson et al. 2001). We used Fue for the oxidative DEHP metabolites (MEHHP, 0.23; MEOHP, 0.15) from a study of one adult man given three single oral doses of D₄-DEHP; the estimated Fue for MEHP was 0.06 (Koch et al. 2005b), about half the value used in the calculations in this case study.

The first data on urinary phthalate metabolite concentrations, including MEP and MEHP, reported in a U.S. population of 289 adults from NHANES III (Blount et al. 2000b), were used to calculate exposures to the corresponding phthalate diesters (David 2000; Kohn et al. 2000). Subsequently, the CDC reported U.S. nationally representative urinary concentrations of seven phthalate metabolites in 2,540 participants of NHANES 1999–2000 (Silva et al. 2004a) and of 10 phthalate metabolites in 2,782 participants of NHANES 2001–2002 (CDC 2005). The frequencies of detection of individual phthalate metabolites were similar. However, the median concentration of MEP was almost 2-fold lower in NHANES 1999–2000 and 2001–2002 than in NHANES III. These differences may have reflected reduced exposures to DEP or have been related to differences in sample sizes. In contrast, the MEHP concentrations remained essentially constant, although they were highest in NHANES 2001–2002 (Table 1). MEHHP and MEOHP were only measured in NHANES 2001–2002. Their median concentrations were 5-fold (MEHHP) and more than 3-fold (MEOHP) higher than the median MEHP concentration. The NHANES 1999–2000 and 2001–2002 data, stratified by age, gender, or ethnicity, indicated some differences in urinary concentrations of phthalate metabolites (CDC 2005; Silva et al. 2004a). For MEHP, MEHHP, and MEOHP, children exhibited higher urinary concentrations than adults, although when accounting for creatinine clearance, the calculated external exposures were similar (Table 2).

Urinary concentrations of DEP and DEHP metabolites in other smaller groups (Adibi et al. 2003; Brock et al. 2002; Duty et al. 2004; Hoppin et al. 2002; Koch et al. 2003c, 2004b) were largely consistent with the NHANES 1999–2002 data (Table 3). In general, differences between various segments of the population were smaller than the differences across the population, that is, from lowest to the most highly exposed individuals. The underlying explanation for the range of exposures is unknown but may be related to individual lifestyle choices. However, selection of study subjects (at least for NHANES) did not exclude those occupationally exposed, and specific situations may contribute to higher exposures for some individuals (Calafat et al. 2004a; Green et al. 2005; Hauser et al. 2004a; Koch et al. 2005a, 2005d, 2005c). Median urinary MEP concentrations in 85 German children and adults were approximately half those in NHANES 1999–2002 (Koch et al. 2003c, 2004b). By contrast, median urinary MEHP, MEHHP, and MEOHP concentrations were approximately twice those in NHANES 1999–2002, but 95th percentile values were similar (Becker et al. 2004; Koch et al. 2003c, 2004b) (Table 1). Whether these findings reflect differences in sampling (e.g., first morning vs. non-first morning voids, nonrepresentative nature of the population examined in Germany) or in exposure patterns between the United States and Germany is unknown.

Estimates of DEP exposure resulting from its use in personal care products, based on conservative assumptions, were not realistic (730 μg/kg/day from fragrances and 100 μg/kg/day from personal care products) (Api 2001). With food as the largest identified contributor to exposure for most individuals, calculated median DEP exposure ranges were 2–6 μg/kg/day for most of the population, with somewhat higher estimates for toddlers and lower estimates for infants (Clark et al. 2003). For DEHP, relying heavily on a previous study (Huber et al. 1996), estimated DEHP exposure ranges within the general population were 3–30 μg/kg/day, with higher exposures likely in occupational settings and the highest associated with certain medical procedures (Doull et al. 1999). Estimates from other researchers (Clark et al. 2003; Meek and Chan 1994) also fall in this range.

For DEP a comparison of the biomarker-based and indirect approaches indicates that, in adults, mean estimates derived by indirect methods (Clark et al. 2003) were about half the mean exposures calculated from bio-marker-based data (Table 4). Because this indirect approach did not consider DEP exposure from cosmetics use, these differences are expected. The 95th percentile exposures calculated from biomonitoring data were above these indirect estimates but far below unrealistic estimates of exposures from cosmetic and personal care products (Api 2001). One might hypothesize that exposure from sources other than personal care products accounts for approximately half the mean total DEP exposure, with exposure from personal care products comprising the remainder. In agreement with this hypothesis, children have lower exposures to DEP than adults (Tables 1, 2). Three overall conclusions emerge from this example: a) indirect methods can provide realistic estimates of exposure only if reasonable assumptions are used; b) use of biomonitoring data can yield precise exposure estimates because it does not require overly conservative assumptions; and c) it may identify situations in which not all potential sources of exposure were considered.

For DEHP, urinary MEHP data produced estimates of mean exposure that were lower than those using the indirect methods, although the 95th percentile values were similar (Table 4). Using DEHP oxidative metabolite data, the estimated DEHP exposures are about twice those calculated from MEHP data (Tables 1, 2, 4). That mean DEHP exposures within the general population, calculated from urinary metabolite data, are approximately 4-fold lower than the indirect estimates may be due, in part, to reliance on older measurements of phthalates in various media, particularly food, as the basis for indirect estimates (Clark et al. 2003). Conservatism may also be introduced by assumptions about absorption based on results of animal studies. Nevertheless, this comparison suggests that, for DEHP, all relevant sources of exposure were taken into consideration when using the indirect approach.

Risk Assessment Top

Biomonitoring data can also be used to address the exposure component of risk assessment. In risk assessment, exposure estimates are compared with NOAELs that for phthalates were from studies in rats. Important and controversial issues relating to these hazard data include choice of species, identification of critical end points, and relevance to humans (Bosgra et al. 2005; Foster 2005). Discussing those issues in detail is beyond the scope of this article. Rather, this section relates results of risk assessments based on phthalate exposures calculated from urinary metabolite data to conclusions of previous risk assessments.

The most reasonable indirect estimates of mean exposure to DEP were 2–6 μg/kg/day, depending on the ages of the groups considered and neglecting consideration of cosmetics and personal care products (Clark et al. 2003). Estimates of DEP exposure in the general population, based on biomonitoring data, are 5.4 μg/kg/day, with a 95th percentile of 64.7 μg/kg/day (Table 4). Thus, indirect and biomarker-based methods produced comparable estimates and indicated that within the United States most individuals are exposed to DEP levels well below the RfD (800 μg/kg/day).

For DEHP, indirect estimates of mean exposure were 5.8–8.2 μg/kg/day (Clark et al. 2003) and a range of 3–30 μg/kg/day (Doull et al. 1999). From urinary metabolite data, estimated mean exposures are in the range of 1–2 μg/kg/day, with a 95th percentile of 7–17 μg/kg/day depending on the metabolite used (Table 4). This comparison suggests that both indirect and biomarker-based methods produced mean estimates below the RfD (20 μg/kg/day) and TDI (37 μg/kg/day), although the upper ranges of exposure approximated the RfD. As another example, the National Toxicology Program (NTP) CERHR determined that the NOAEL for reproductive effects in rats was 5–8 mg/kg/day (CERHR 2005) and expressed concern over the potential for reproductive risk among infants younger than 1 year, if their exposures were significantly higher than those of the general population (1–30 μg/kg/day). Biomonitoring data are unavailable for healthy infants younger than 1 year, so this specific question cannot be addressed from the available data. However, for those 6 or more years of age, biomonitoring data indicate that ambient exposures to DEHP within the United States are lower than estimates used by the NTP-CERHR and that children’s and adults’ exposures are comparable. Some medical interventions may result in higher exposures to DEHP (Calafat et al. 2004a; Green et al. 2005; Koch et al. 2005a, 2005c). These medical treatments entail risk–benefit calculations that make risk assessments substantially different from those relating to ambient exposures (U.S. Food and Drug Administration 2001) and are beyond the scope of this exercise. Note that for children and adults, exposure estimates calculated from the oxidized DEHP metabolites were approximately twice those calculated from MEHP (Tables 1, 2). However, for premature neonates the differences were in the range of an order of magnitude (Table 2), presumably from differences in metabolism and/or excretion in these preterm infants.

Recommendations for Future Research Top

We make the following recommendations for future research:

• Improve the understanding of human metabolism and pharmacokinetics. The most relevant urinary metabolites and appropriate metabolites for other matrices that provide the greatest analytical sensitivity must be measured. Differences in metabolic patterns among phthalates are important both toxicologically and in exposure assessment, especially when comparing relative exposures to different phthalates because the complex metabolism of high-molecular-weight phthalates leads to additional metabolic products (e.g., oxidative metabolites).

• Refine molar conversion factors to relate external phthalate exposure to urinary metabolite concentrations. Based on available data, the largest uncertainties appear in premature neonates.

• Determine the biologic media best suited for biomarker studies. If media other than urine are evaluated, methodologic issues must be considered.

• Improve the understanding of the mechanisms of action of phthalates in humans.

• Determine whether more highly exposed groups can be identified and, if so, identify the sources of exposure. Potentially vulnerable segments of the population (e.g., children, women of reproductive age, minorities) should be evaluated.

• Determine whether use of urinary metabolite data as an adjunct to epidemiology studies is possible. One specific issue relates to categorizing exposure from a limited number of urine samples (Hauser et al. 2004b; Hoppin et al. 2002).

Figure and Tables Top

Figure 1.

Generic chemical structure of phthalates. R and R′ are ethyl groups for DEP and 2-ethylhexyl groups for DEHP.

Table 1.

Urinary concentrations (micrograms per gram creatinine) of MEP, MEHP, MEHHP, and MEOHP and estimated exposures (in parentheses, micrograms per kilogram per day) to DEP and DEHP calculated using urinary concentrations from several studies of adults or the general population.

Table 2.

Urinary concentrations (micrograms per gram creatinine) of MEP, MEHP, MEHHP, and MEOHP and estimated exposures (in parentheses, micrograms per kilogram per day) to DEP and DEHP calculated using urinary concentrations from several studies of children.

Table 3.

Urinary concentrations (micrograms per gram creatinine) of MEP, MEHP, MEHHP, and MEOHP and estimated exposures (in parentheses, micrograms per kilogram per day) to DEP and DEHP calculated using urinary concentrations from specific populations.

Table 4.

Estimates of the geometric mean (95th percentiles in parentheses) exposures (in micrograms per kilogram per day) to DEP and DEHP using the geometric mean (95th percentile) urinary phthalate metabolite concentrations compared with indirect estimates based on phthalate diester levels in various media (e.g., food, air, water, soil, and dust).^a

References Top

1. Adibi JJ, Perera FP, Jedrychowski W, Camann DE, Barr D, Jacek R, et al. 2003. Prenatal exposures to phthalates among women in New York City and Krakow, Poland Environ Health Perspect 111:1719–1722. Find this article online
2. Albro PW 1986. Absorption, metabolism, and excretion of di(2-ethylhexyl) phthalate by rats and mice Environ Health Perspect 65:293–298. Find this article online
3. Albro PW, Moore B 1974. Identification of the metabolites of simple phthalate diesters in rat urine J Chromatogr 94:209–218. Find this article online
4. Anderson WAC, Castle L, Scotter MJ, Massey RC, Springall C 2001. A biomarker approach to measuring human dietary exposure to certain phthalate diesters Food Addit Contam 18:1068–1074. Find this article online
5. Api AM 2001. Toxicological profile of diethyl phthalate: a vehicle for fragrance and cosmetic ingredients Food Chem Toxicol 39:97–108. Find this article online
6. ATSDR 1995. Toxicological Profile for Diethyl Phthalate (DEP). Atlanta:Agency for Toxic Substances and Disease Registry. Available: http://www.atsdr.cdc.gov/toxprofiles/tp7​3.html [accessed 26 July 2004]
7. ATSDR 2002. Toxicological Profile for Di(2-ethylhexyl)phthalate (DEHP). Atlanta:Agency for Toxic Substances and Disease Registry. Available: http://www.atsdr.cdc.gov/toxprofiles/tp9​.html [accessed 26 July 2004]
8. Awal MA, Kurohmaru M, Ishii M, Andriana BB, Kanai Y, Hayashi Y 2004. Mono-(2-ethyl hexyl) phthalate (MEHP) induces spermatogenic cell apoptosis in guinea pig testes at prepubertal stage in vitro Int J Toxicol 23:349–355. Find this article online
9. Barber ED, Cifone M, Rundell J, Przygoda R, Astill BD, Moran E, et al. 2000. Results of the L5178Y mouse lymphoma assay and the Balb/3T3 cell in vitro transformation assay for eight phthalate esters J Appl Toxicol 20:69–80. Find this article online
10. Barlow NJ, Foster PMD 2003. Pathogenesis of male reproductive tract lesions from gestation through adulthood following in utero exposure to di(n-butyl) phthalate Toxicol Pathol 31:397–410. Find this article online
11. Barlow NJ, McIntyre BS, Foster PMD 2004. Male reproductive tract lesions at 6, 12, and 18 months of age following in utero exposure to di(n-butyl) phthalate Toxicol Pathol 32:79–90. Find this article online
12. Barr DB, Silva MJ, Kato K, Reidy JA, Malek NA, Hurtz D, et al. 2003. Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers Environ Health Perspect 111:1148–1151. Find this article online
13. Becker K, Seiwert M, Angerer J, Heger W, Koch HM, Nagorka R, et al. 2004. DEHP metabolites in urine of children and DEHP in house dust Int J Hyg Environ Health 207:409–417. Find this article online
14. Bility MT, Thompson JT, McKee RH, David RM, Butala JH, Vanden Heuvel JP, et al. 2004. Activation of mouse and human peroxisome proliferator-activated receptors (PPARs) by phthalate monoesters Toxicol Sci 82:170–182. Find this article online
15. Blount BC, Milgram KE, Silva MJ, Malek NA, Reidy JA, Needham LL, et al. 2000a. Quantitative detection of eight phthalate metabolites in human urine using HPLC-APCI-MS/MS Anal Chem 72:4127–4134. Find this article online
16. Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson EJ, et al. 2000b. Levels of seven urinary phthalate metabolites in a human reference population Environ Health Perspect 108:979–982. Find this article online
17. Bosgra S, Bos PMJ, Vermeire TG, Luit RJ, Slob W 2005. Probabilistic risk characterization: an example with di(2-ethylhexyl) phthalate Regul Toxicol Pharmacol 43:104–113. Find this article online
18. Brock JW, Caudill SP, Silva MJ, Needham LL, Hilborn ED 2002. Phthalate monoesters levels in the urine of young children Bull Environ Contam Toxicol 68:309–314. Find this article online
19. Calafat AM, Needham LL, Silva MJ, Lambert G 2004a. Exposure to di-(2-ethylhexyl) phthalate among premature neonates in a neonatal intensive care unit Pediatrics 113:e429–e434. Find this article online
20. Calafat AM, Slakman AR, Silva MJ, Herbert AR, Needham LL 2004b. Automated solid phase extraction and quantitative analysis of human milk for 13 phthalate metabolites J Chromatogr B 805:49–56. Find this article online
21. Carruthers CM, Foster PMD 2005. Critical window of male reproductive tract development in rats following gestational exposure to di-n-butyl phthalate Birth Defects Res B Dev Reprod Toxicol 74:277–285. Find this article online
22. CDC 2005. Third National Report on Human Exposure to Environmental Chemicals. Atlanta:Centers for Disease Control and Prevention. Available: http://www.cdc.gov/exposurereport/3rd/pd​f/thirdreport.pdf [accessed 11 August 2005]
23. CERHR 2005. NTP-CERHR Expert Panel Update Report on the Reproductive and Developmental Toxicity of Di(2-ethyl-hexyl)phthalate. Research Triangle Park, NC:National Toxicology Program Center for the Evaluation of Risks to Human Reproduction. Available: http://cerhr.niehs.nih.gov/chemicals/deh​p/DEHP__Report_final.pdf [accessed 20 March 2006]
24. Clark K, Cousins I, Mac Kay D 2003. Assessment of critical exposure pathways. In: The Handbook of Environmental Chemistry, 3Q. Phthalate Esters (Staples C, ed). New York: Springer, 227–262
25. Corton JC, Lapinskas PJ 2005. Peroxisome proliferator-activated receptors: mediators of phthalate ester-induced effects in the male reproductive tract? Toxicol Sci 83:4–17. Find this article online
26. CSTEE 1998. Opinion on Phthalate Migration from Soft PVC Toys and Child-Care Articles—Opinion Expressed at the 6th CSTEE Plenary Meeting, 26/27 November 1998, Brussels, Belgium. Brussels:Scientific Committee for Toxicity, Ecotoxicity and the Environment
27. David RM 2000. Exposure to phthalate esters Environ Health Perspect 108:A440. Find this article online
28. David RM, McKee RH, Butala JH, Barter RA, Kayser M 2001. Esters of aromatic mono-, di-, and tricarboxylic acids, aromatic diacids, and di-, tri-, or polyalcohols. In: Patty’s Toxicology, Vol 6, 5th ed. (Bingham E, Cohrssen B, Powell CH, eds). New York:John Wiley and Sons, 635–932
29. Doull J, Cattley R, Elcombe C, Lake BG, Swenberg J, Wilkinson C, et al. 1999. A cancer risk assessment of di(2-ethylhexyl)-phthalate: application of the new US EPA risk assessment guidelines Regul Toxicol Pharmacol 29:327–357. Find this article online
30. Duty SM, Calafat AM, Silva MJ, Brock JW, Ryan L, Chen ZY, et al. 2004. The relationship between environmental exposure to phthalates and computer-aided sperm analysis motion parameters J Androl 25:293–302. Find this article online
31. Duty SM, Calafat AM, Silva MJ, Ryan L, Hauser R 2005. Phthalate exposure and reproductive hormones in adult men Human Reprod 20:604–610. Find this article online
32. Duty SM, Silva MJ, Barr DB, Brock JW, Ryan L, Chen ZY, et al. 2003a. Phthalate exposure and human semen parameters Epidemiology 14:269–277. Find this article online
33. Duty SM, Singh NP, Silva MJ, Barr DB, Brock JW, Ryan L, et al. 2003b. The relationship between environmental exposures to phthalates and DNA damage in human sperm using the neutral comet assay Environ Health Perspect 111:1164–1169. Find this article online
34. Ema M, Miyawaki E 2001. Effects of monobutyl phthalate on reproductive function in pregnant and pseudopregnant rats Reprod Toxicol 15:261–267. Find this article online
35. Ema M, Miyawaki E, Hirose A, Kamata E 2003. Decreased ano-genital distance and increased incidence of undescended testes in fetuses of rats given monobenzyl phthalate, a major metabolite of butyl benzyl phthalate Reprod Toxicol 17:407–412. Find this article online
36. Fisher JS 2004. Environmental anti-androgens and male reproductive health: focus on phthalates and testicular dysgenesis syndrome Reproduction 127:305–315. Find this article online
37. Foster PMD 2005. Mode of action: impaired fetal Leydig cell function—effects on male reproductive development produced by certain phthalate esters Crit Rev Toxicol 35:713–719. Find this article online
38. Foster PMD, Cattley RC, Mylchreest E 2000. Effects of di-n-butyl phthalate (DBP) on male reproductive development in the rat: implications for human risk assessment Food Chem Toxicol 38:S97–S99. Find this article online
39. Gray LE, Ostby J, Furr J, Price M, Veeramachaneni DNR, Parks L 2000. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentiation of the male rat Toxicol Sci 58:350–365. Find this article online
40. Gray TJB, Beamand JA 1984. Effect of some phthalate esters and other testicular toxins on primary cultures of testicular cells Food Chem Toxicol 22:123–131. Find this article online
41. Gray TJB, Gangolli SD 1986. Aspects of the testicular toxicity of phthalate-esters Environ Health Perspect 65:229–235. Find this article online
42. Green R, Hauser R, Calafat AM, Weuve J, Schettler T, Ringer S, et al. 2005. Use of di(2-ethylhexyl) phthalate-containing medical products and urinary levels of mono(2-ethylhexyl) phthalate in neonatal intensive care unit infants Environ Health Perspect 113:1222–1225. Find this article online
43. Hauser R, Calafat AM 2005. Phthalates and human health Occup Environ Med 62:806–818. Find this article online
44. Hauser R, Duty S, Godfrey-Bailey L, Calafat AM 2004a. Medications as a source of human exposure to phthalates Environ Health Perspect 112:751–753. Find this article online
45. Hauser R, Meeker JD, Park S, Silva MJ, Calafat AM 2004b. Temporal variability of urinary phthalate metabolite levels in men of reproductive age Environ Health Perspect 112:1734–1740. Find this article online
46. Heindel JJ, Powell CJ 1992. Phthalate ester effects on rat Sertoli-cell function in vitro—effects of phthalate side-chain and age of animal Toxicol Appl Pharmacol 115:116–123. Find this article online
47. Hoppin JA, Brock JW, Davis BJ, Baird DD 2002. Reproducibility of urinary phthalate metabolites in first morning urine samples Environ Health Perspect 110:515–518. Find this article online
48. Hoppin JA, Ulmer R, London SJ 2004. Phthalate exposure and pulmonary function Environ Health Perspect 112:571–574. Find this article online
49. Huber WW, GraslKraupp B, SchulteHermann R 1996. Hepato-carcinogenic potential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk Crit Rev Toxicol 26:365–481. Find this article online
50. IARC 2000. Some industrial chemicals IARC Monogr Eval Carcinog Risks Hum 77:41–148. Find this article online
51. Jacobs DS, Oxley DK, DeMott WR 2001. Laboratory Test Handbook. Hudson, OH:Lexi-Comp
52. Jonsson BAG, Richthoff J, Rylander L, Giwercman A, Hagmar L 2005. Urinary phthalate metabolites and biomarkers of reproductive function in young men Epidemiology 16:487–493. Find this article online
53. Kato K, Shoda S, Takahashi M, Doi N, Yoshimura Y, Nakazawa H 2003a. Determination of three phthalate metabolites in human urine using on-line solid-phase extraction-liquid chromatography-tandem mass spectrometry J Chromatogr B 788:407–411. Find this article online
54. Kato K, Silva MJ, Brock JW, Reidy JA, Malek NA, Hodge CC, et al. 2003b. Quantitative detection of nine phthalate metabolites in human serum using reversed-phase high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry J Anal Toxicol 27:284–289. Find this article online
55. Kato K, Silva MJ, Needham LL, Calafat AM 2005. Determination of 16 phthalate metabolites in urine using automated sample preparation and on-line preconcentration/high-performance liquid chromatography/tandem mass spectrometry Anal Chem 77:2985–2991. Find this article online
56. Kato K, Silva MJ, Reidy JA, Hurtz D, Malek NA, Needham LL, et al. 2004. Mono(2-ethyl-5-hydroxyhexyl) phthalate and mono-(2-ethyl-5-oxohexyl) phthalate as biomarkers for human exposure assessment to di-(2-ethylhexyl) phthalate Environ Health Perspect 112:327–330. Find this article online
57. Kato K, Yamauchi T, Higashiyama K, Nakazawa H 2003c. High throughput analysis of di-(2-ethylhexyl)phthalate metabolites in urine for exposure assessment J Liq Chromatogr Relat Technol 26:2167–2176. Find this article online
58. Kavlock R, Boekelheide K, Chapin R, Cunningham M, Faustman E, Foster P, et al. 2002. NTP Center for the Evaluation of Risks to Human Reproduction: phthalates expert panel report on the reproductive and developmental toxicity of di(2-ethylhexyl) phthalate Reprod Toxicol 16:529–653. Find this article online
59. Kessler W, Phokha W, Csanady GA, Filser JG 2001. No background concentrations of di(2-ethylhexyl) phthalate and mono(2-ethylhexyl) phthalate in blood of rats Arch Toxicol 75:62–64. Find this article online
60. Klaunig JE, Babich MA, Baetcke KP, Cook JC, Corton JC, David RM, et al. 2003. PPAR alpha agonist-induced rodent tumors: modes of action and human relevance Crit Rev Toxicol 33:655–780. Find this article online
61. Koch HM, Angerer J, Drexler H, Eckstein R, Weisbach V 2005a. Di(2-ethylhexyl)phthalate (DEHP) exposure of voluntary plasma and platelet donors Int J Hyg Environ Health 208:489–498. Find this article online
62. Koch HM, Bolt HM, Angerer J 2004a. Di(2-ethylhexyl)phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuterium-labelled DEHP Arch Toxicol 78:123–130. Find this article online
63. Koch HM, Bolt HM, Preuss R, Angerer J 2005b. New metabolites of di(2-ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP Arch Toxicol 79:367–376. Find this article online
64. Koch HM, Bolt HM, Preuss R, Eckstein R, Weisbach V, Angerer J 2005c. Intravenous exposure to di(2-ethylhexyl)phthalate (DEHP): metabolites of DEHP in urine after a voluntary platelet donation Arch Toxicol 79:689–693. Find this article online
65. Koch HM, Drexler H, Angerer J 2003a. An estimation of the daily intake of di(2-ethylhexyl)phthalate (DEHP) and other phthalates in the general population Int J Hyg Environ Health 206:1–7. Find this article online
66. Koch HM, Drexler H, Angerer J 2004b. Internal exposure of nursery-school children and their parents and teachers to di(2-ethylhexyl)phthalate (DEHP) Int J Hyg Environ Health 207:15–22. Find this article online
67. Koch HM, Gonzalez-Reche LM, Angerer J 2003b. On-line cleanup by multidimensional liquid chromatography-electrospray ionization tandem mass spectrometry for high throughput quantification of primary and secondary phthalate metabolites in human urine J Chromatogr B 784:169–182. Find this article online
68. Koch HM, Muller J, Drexler H, Angerer J 2005d. Dibutyl-phthalate (DBP) in medications: are pregnant women and infants at risk? Umweltmed Forsch Prax 10:144–146. Find this article online
69. Koch HM, Rossbach B, Drexler H, Angerer J 2003c. Internal exposure of the general population to DEHP and other phthalates—determination of secondary and primary phthalate monoester metabolites in urine Environ Res 93:177–185. Find this article online
70. Kohn MC, Parham F, Masten SA, Portier CJ, Shelby MD, Brock JW, et al. 2000. Human exposure estimates for phthalates Environ Health Perspect 108:A440–A442. Find this article online
71. Li H, Kim KH 2003. Effects of mono-(2-ethylhexyl) phthalate on fetal and neonatal rat testis organ cultures Biol Reprod 69:1964–1972. Find this article online
72. Medeiros AM, Devlin DJ, Keller LH 1999. Evaluation of skin sensitization response of dialkyl (C6-C13) phthalate esters Contact Derm 41:287–289. Find this article online
73. Meek ME, Chan PKL 1994. Bis(2-ethylhexyl)phthalate—evaluation of risks to health from environmental exposure in Canada J Environ Sci Health Part C 12:179–194. Find this article online
74. Mortensen GK, Main KM, Andersson AM, Leffers H, Skakkebaek NE 2005. Determination of phthalate monoesters in human milk, consumer milk, and infant formula by tandem mass spectrometry (LC-MS-MS) Anal Bioanal Chem 382:1084–1092. Find this article online
75. Mylchreest E, Cattley RC, Foster PMD 1998. Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci 43:47–60. Find this article online
76. Parks LG, Ostby JS, Lambright CR, Abbott BD, Klinefelter GR, Barlow NJ, et al. 2000. The plasticizer diethylhexyl phthalate induces malformations by decreasing fetal testosterone synthesis during sexual differentiation in the male rat Toxicol Sci 58:339–349. Find this article online
77. Peck CC, Albro PW 1982. Toxic potential of the plasticizer di(2-ethylhexyl) phthalate in the context of its disposition and metabolism in primates and man Environ Health Perspect 45:11–17. Find this article online
78. Preuss R, Koch HM, Angerer J 2005. Biological monitoring of the five major metabolites of di-(2-ethylhexyl)phthalate (DEHP) in human urine using column-switching liquid chromatography-tandem mass spectrometry J Chromatogr B 816:269–280. Find this article online
79. Saillenfait AM, Langonne I, Leheup B 2001. Effects of mono-n-butyl phthalate on the development of rat embryos: in vivo and in vitro observations Pharmacol Toxicol 89:104–112. Find this article online
80. Schmid P, Schlatter C 1985. Excretion and metabolism of di(2-ethylhexyl)-phthalate in man Xenobiotica 15:251–256. Find this article online
81. Silva MJ, Barr DB, Reidy JA, Malek NA, Hodge CC, Caudill SP, et al. 2004a. Urinary levels of seven phthalate metabolites in the US population from the National Health and Nutrition Examination Survey (NHANES) 1999–2000 Environ Health Perspect 112:331–338. Find this article online
82. Silva MJ, Malek NA, Hodge CC, Reidy JA, Kato K, Barr DB, et al. 2003. Improved quantitative detection of 11 urinary phthalate metabolites in humans using liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry J Chromatogr B 789:393–404. Find this article online
83. Silva MJ, Reidy JA, Herbert AR, Preau JL, Needham LL, Calafat AM 2004b. Detection of phthalate metabolites in human amniotic fluid Bull Environ Contam Toxicol 72:1226–1231. Find this article online
84. Silva MJ, Reidy JA, Samandar E, Herbert AR, Needham LL, Calafat AM 2005a. Detection of phthalate metabolites in human saliva Arch Toxicol 79:647–652. Find this article online
85. Silva MJ, Reidy JA, Samandar E, Preau JLJ, Needham LL, Calafat AM 2006a. Measurement of eight urinary metabolites of di(2-ethylhexyl) phthalate as biomarkers for human exposure assessment Biomarkers 11:1–13. Find this article online
86. Silva MJ, Samandar E, Preau JL, Reidy JA, Needham LL, Calafat AM 2005b. Automated solid-phase extraction and quantitative analysis of 14 phthalate metabolites in human serum using isotope dilution-high-performance liquid chromatography-tandem mass spectrometry J Anal Toxicol 29:819–824. Find this article online
87. Silva MJ, Samandar E, Preau JLJ, Needham LL, Calafat AM 2006b. Urinary oxidative metabolites of di(2-ethylhexyl) phthalate in humans Toxicology 219:22–32. Find this article online
88. Silva MJ, Slakman AR, Reidy JA, Preau JL, Herbert AR, Samandar E, et al. 2004c. Analysis of human urine for fifteen phthalate metabolites using automated solid-phase extraction J Chromatogr B 805:161–167. Find this article online
89. Stroheker T, Cabaton N, Nourdin G, Regnier JF, Lhuguenot JC, Chagnon MC 2005. Evaluation of anti-androgenic activity of di-(2-ethylhexyl)phthalate Toxicology 208:115–121. Find this article online
90. Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, et al. 2005. Decrease in anogenital distance among male infants with prenatal phthalate exposure Environ Health Perspect 113:1056–1061. Find this article online
91. Takatori S, Kitagawa Y, Kitagawa M, Nakazawa H, Hori S 2004. Determination of di(2-ethylhexyl)phthalate and mono(2-ethylhexyl)phthalate in human serum using liquid chromatography-tandem mass spectrometry J Chromatogr B 804:397–401. Find this article online
92. Tietz NW 1990. Clinical Guide to Laboratory Tests. Philadelphia:W.B. Saunders
93. U.S. EPA 1993a. Di(2-ethylhexyl)phthalate (DEHP) (CASRN 117-81-7). U.S. Environmental Protection Agency. Available: http://www.epa.gov/IRIS/subst/0014.htm [accessed 11 August 2003]
94. U.S. EPA 1993b. Diethyl Phthalate (CASRN 84-66-2). U.S. Environmental Protection Agency. Available: http://www.epa.gov/iris/subst/0226.htm [accessed 11 August 2003]
95. U.S. EPA 2002. Bis(2-ethylhexyl) Phthalate (DEHP). U.S. Environmental Protection Agency. Available: http://www.epa.gov/ttn/atw/hlthef/eth-ph​th.html [accessed 11 August 2003]
96. U.S. Food and Drug Adminstration 2001. Safety Assessment of Di(2-ethylhexyl)phthalate (DEHP) Released from PVC Medical Devices. Rockville, MD:Center for Devices and Radiological Health, U.S. Food and Drug Administration. Available: http://www.fda.gov/cdrh/ost/dehp-pvc.pdf [accessed 11 August 2003]
97. Ward JM, Peters JM, Perella CM, Gonzalez FJ 1998. Receptor and nonreceptor-mediated organ-specific toxicity of di(2-ethylhexyl)phthalate (DEHP) in peroxisome proliferator-activated receptor alpha-null mice Toxicol Pathol 26:240–246. Find this article online