Urinary Concentrations of Diisoheptyl Phthalate Biomarkers in Convenience Samples of U.S. Adults in 2000 and 2018-2019.

We know little about the potential health risks from exposure to diisoheptyl phthalate (DiHpP), a plasticizer used in commercial applications. The production of DiHpP ended in the United States in 2010, but DiHpP may still be present in phthalate diester mixtures. To investigate human exposure to DiHpP, we used three oxidative metabolites of DiHpP: Monohydroxyheptyl phthalate (MHHpP), mono-oxoheptylphthalate (MOHpP), and monocarboxyhexyl phthalate (MCHxP) as exposure biomarkers. We analyzed urine collected anonymously in 2000 (N = 144) and 2018-2019 (N = 205) from convenience groups of U.S. adults using high-performance liquid chromatography coupled with isotope-dilution high-resolution mass spectrometry. We detected MCHxP in all the samples tested in 2000 (GM = 2.01 ng/mL) and 2018-2019 (GM = 1.31 ng/mL). MHHpP was also detected in 100% of the 2018-2019 samples (GM = 0.59 ng/mL) and 96% of the 2000 urine samples analyzed (GM = 0.38 ng/mL). MOHpP was detected in 57% (2018-2019, GM = 0.03 ng/mL) and 92% (2000, GM = 0.19 ng/mL) of samples. The presence of MHHpP, MOHpP, and MCHxP in the 2018-2019 samples suggests recent exposure to DiHpP. Intercorrelations between metabolite concentrations were more significant in samples collected in 2000 than in samples collected in 2018-2019. The differences in urinary metabolite profiles and intercorrelations from samples collected during 2000 and 2018-2019 likely reflects changes in the composition of commercial DiHpP formulations before and after 2010.

Exposure to di-2-ethylhexyl terephthalate in the U.S. general population from the 2015-2016 National Health and Nutrition Examination Survey.

BACKGROUND: Di-2-ethylhexyl terephthalate (DEHTP) is used as a replacement plasticizer for other phthalates, including di-2-ethylhexyl phthalate (DEHP). Use of consumer products containing DEHTP may result in human exposure to DEHTP. OBJECTIVE: To assess exposure to DEHTP in a nationally representative sample of the U.S. general population 3 years and older from the 2015-2016 National Health and Nutrition Examination Survey (NHANES). METHOD: We quantified two DEHTP metabolites, mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP) and mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) in 2970 urine samples by using online solid-phase extraction coupled with isotope dilution-high-performance liquid chromatography-tandem mass spectrometry. We used linear regression to examine associations between MEHHTP and MECTPP and several parameters including age, sex, race/ethnicity, and household income. We also compared the MEHHTP and MECPTP results to those of their corresponding DEHP metabolite analogs, namely mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP) and mono-2-ethyl-5-carboxypentyl phthalate (MECPP). RESULTS: The weighted detection frequencies were 96% (MEHHTP) and 99.9% (MECPTP); urinary concentrations of the two metabolites correlated significantly (Pearson correlation coefficient = 0.89, p < 0.0001). MECPTP concentrations were higher than MEHHTP in all age, sex, race/ethnicity groups examined. Furthermore, MECPTP adjusted geometric mean (GM) concentrations were significantly higher in samples collected in the evening than in the morning or afternoon. Females had significantly higher adjusted GM concentrations of MEHHTP and MECPTP than males. We observed no significant associations between the adjusted GM concentrations of the metabolites and race/ethnicity. Both metabolite adjusted GM concentrations increased significantly with household income, and decreased significantly with age. Only household income was significantly associated with the concentrations of MECPP, but not of MEHHP, the two DEHP metabolites. The adjusted GM of the [MEHHTP]:[MECPTP] molar concentrations ratio increased with age, and was significantly higher in samples collected in the morning than in those collected in the afternoon or evening. CONCLUSIONS: Exposure to DEHTP is widespread in the U.S. general population 3 years and older. These data represent the first U.S. population-based representative background exposure to DEHTP.

Prenatal phthalate exposures and child temperament at 12 and 24 months.

INTRODUCTION: Gestational phthalate exposures have been adversely associated with attention, externalizing, and internalizing behaviors in childhood. Early childhood temperament may be a marker of later behavioral patterns. We therefore sought to determine whether gestational phthalate exposures were associated with infant and toddler temperament. METHODS: The Mount Sinai Children's Environmental Health Study is a prospective cohort study of children born between May 1998 and July 2001 in New York City (N=404). Phthalate metabolites were measured in spot urine samples collected from pregnant women in their third trimester. Child temperament was assessed by parental report at 12-months using the Infant Behavior Questionnaire (IBQ) (N=204) and at 24-months using the Toddler Behavior Assessment Questionnaire (TBAQ) (N=279). We used multiple linear regression to evaluate associations between urinary phthalate metabolites and eleven temperament domains. RESULTS: Phthalate biomarker concentrations were weakly associated with lower gross motor activity levels as well as higher duration of orienting at the 12-month assessment. Mono(3-carboxypropyl) phthalate (MCPP), monobenzyl phthalate (MBzP) and the sum of metabolites of di(2-ethylhexyl) phthalate (∑DEHP) were associated with lower levels of smiling and laughing at 12 months. At 24-months, social fear and lower pleasure was linked to higher concentrations of MCPP and MBzP, and higher ∑DEHP was weakly associated with increased anger levels at 24-months. CONCLUSIONS: Though we observed some weak associations between biomarkers of prenatal exposure to phthalates and temperament at 12- and 24-months, overall phthalates biomarkers were not strongly associated with alterations in temperament.

Exposure to di-2-ethylhexyl terephthalate in a convenience sample of U.S. adults from 2000 to 2016.

Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of di-2-ethylhexyl phthalate (DEHP), is a plasticizer used in a variety of commercial applications, but data on Americans' exposure to DEHTP do not exist. We investigated the exposure to DEHTP in a convenience group of U.S. adults by analyzing urine collected anonymously in 2000 (N = 44), 2009 (N = 61), 2011 (N = 81), 2013 (N = 92), and 2016 (N = 149) for two major DEHTP oxidative metabolites: mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) and mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP). For comparison, we also quantified the analogous DEHP metabolites mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP) and mono-2-ethyl-5-carboxypentyl phthalate (MECPP). We detected MECPTP, MEHHP, and MECPP in all samples collected in 2016 with geometric means of 13.1, 4.1, and 6.7 ng/mL, respectively; we detected MEHHTP in 91% of the samples (geometric mean = 3.1 ng/mL). Concentrations of MECPTP correlated well with those of MEHHTP (R 2 = 0.8, p < 0.001), but did not significantly correlate with those of MEHHP (p > 0.05) suggesting different sources of exposure to DEHP and DEHTP. We also evaluated the fraction of the metabolites eliminated in their free (i.e., unconjugated) form. The median percent of unconjugated species was lower for the DEHP metabolites (MECPP [45.5%], MEHHP [1.9%]) compared to the DEHTP metabolites (MECPTP [98.8%], MEHHTP [21.2%]). Contrary to the downward trend from 2000 to 2016 in urinary concentrations of MEHHP and MECPP, we observed an upward trend for MEHHTP and MECPTP. These preliminary data suggest that exposure to DEHTP may be on the rise. Nevertheless, general population exposure data using MEHHTP and MECPTP as exposure biomarkers would increase our understanding of exposure to DEHTP, one of the known DEHP alternatives.

Associations of urinary phthalate and phenol biomarkers with menarche in a multiethnic cohort of young girls.

To study potential environmental influences on puberty in girls, we investigated urinary biomarkers in relation to age at menarche. Phenols and phthalates were measured at baseline (6-8 years of age). Menarche was ascertained over 11 years for 1051 girls with menarche and biomarkers. Hazards ratios were estimated from Cox models adjusted for race/ethnicity and caregiver education (aHR, 95% confidence intervals [CI] for 5th vs 1st quintile urinary biomarker concentrations). 2,5-Dichlorophenol was associated with earlier menarche (aHR 1.34 [1.06-1.71]); enterolactone was associated with later menarche (aHR 0.82 [0.66-1.03]), as was mono-3-carboxypropyl phthalate (MCPP) (aHR 0.73 [0.59-0.91]); the three p-trends were <0.05. Menarche differed by 4-7 months across this range. Enterolactone and MCPP associations were stronger in girls with below-median body mass index. These analytes were also associated with age at breast development in this cohort. Findings from this prospective study suggest that some childhood exposures are associated with pubertal timing.

Prenatal phthalate biomarker concentrations and performance on the Bayley Scales of Infant Development-II in a population of young urban children.

INTRODUCTION: Evidence suggests prenatal phthalate exposures may have neurodevelopmental consequences. Our objective was to investigate prenatal exposure to phthalates and cognitive development in a cohort of young urban children. MATERIALS AND METHODS: We recruited pregnant women in New York City from 1998 to 2002 and measured concentrations of nine phthalate metabolites in urine collected in late pregnancy. We administered a neurodevelopmental screening instrument, the Bayley Scales of Infant Development II (BSID-II), to children who returned for follow-up at approximately 24 months (n=276). We estimated associations between phthalate metabolite concentrations in maternal urine and BSID-II indices (Mental Development Index (MDI), Psychomotor Development Index (PDI)). RESULTS: We observed no associations between phthalate metabolite concentrations and performance on the MDI or PDI in boys and girls combined. We did, however, observe evidence of effect measure modification by sex. We observed several negative associations between metabolite concentrations and both MDI and PDI scores among girls, suggesting poorer performance across multiple metabolites, with estimates equal to a 2-3 point decrease in score per ln-unit increase in creatinine-standardized metabolite concentration. Conversely, we observed multiple weakly positive associations among boys, equal to a 1-2 point increase in score per ln-unit increase in metabolite concentration. The strongest associations were for the metabolites mono-n-butyl phthalate, mono-isobutyl phthalate, monobenzyl phthalate, and mono(3-carboxylpropyl) phthalate (MCPP). CONCLUSIONS: Girls of mothers with higher urinary concentrations of MCPP and metabolites of dibutyl phthalates had lower MDI scores on the BSID-II. These same biomarker concentrations were often associated with improved scores among boys. We observed similar results for MnBP, MCPP, and MBzP on the PDI. Given the prevalence of phthalate exposures in reproductive aged women, the implications of potential neurotoxicity warrant further investigation.

Longitudinal Associations of Phthalate Exposures During Childhood and Body Size Measurements in Young Girls.

BACKGROUND: Phthalates are environmental chemicals that may play a role in the development of obesity. Few studies have investigated longitudinal associations between postnatal phthalate exposures and subsequent anthropometric measurements in children. METHODS: We collected data as part of The Breast Cancer and Environment Research Program at three US sites. A total of 1,239 girls, aged 6-8 years, were enrolled in 2004-2007. We categorized baseline phthalate exposures, assessed from creatinine-corrected urinary concentrations of low-molecular weight phthalate metabolites, as low, <78; medium, 78 to <194; and high, ≥194 µg/g creatinine and of high-molecular weight phthalates as low, <111; medium, 111-278; and high, ≥278 µg/g creatinine. Anthropometric measurements were collected through 2012 (n = 1,017). Linear mixed effects regression estimated how baseline low and high-molecular weight phthalate concentrations related to changes in girls' body mass index (BMI), height, and waist circumference at ages 7-13 years. RESULTS: Low-molecular weight phthalates were positively associated with gains in BMI and waist circumference. Predicted differences in BMI and waist circumference between girls with high versus low concentrations of low-molecular weight phthalates increased from 0.56 (95% confidence interval [CI]: -0.02, 1.1) to 1.2 kg/m (95% CI: 0.28, 2.1) and from 1.5 (95% CI: -0.38, 3.3) to 3.9 cm (95% CI: 1.3, 6.5), respectively. High-molecular weight phthalates were negatively associated with height but only among girls who were normal weight at baseline (BMI ≤ 85th percentile). CONCLUSION: Phthalates, specifically low-molecular weight phthalates, have small but detectable associations with girls' anthropometric outcomes. Low-molecular weight phthalates showed stronger associations than other types of phthalates.

Identification of potential biomarkers of exposure to diundecyl phthalate.

Diundecyl phthalate (DUP) is a high production volume chemical used as a plasticizer in polyvinyl chloride and other plastics. Specific biomarkers of DUP would be useful for human exposure assessment. To identify such biomarkers, we investigated the in vitro metabolism of DUP with human liver microsomes using online solid phase extraction coupled to HPLC-mass spectrometry. Using high resolution mass spectrometry, we conclusively confirmed the structures of four DUP specific metabolites: monoundecyl phthalate (MUP), mono-hydroxyundecyl phthalate (MHUP), mono-oxoundecyl phthalate (MOUP), and mono-carboxydecyl phthalate (MCDP). We also used high resolution mass spectrometry to isolate MCDP and MHUP from co-eluting isobaric metabolites of diisononyl phthalate (i.e., monocarboxyisononyl phthalate) and diisododecyl phthalate (i.e., monohydroxyisododecyl phthalate), respectively, that could not be separated with low resolution tandem mass spectrometry. To evaluate the potential usefulness of the newly identified DUP metabolites as exposure biomarkers, we analyzed 36 human urine samples by high resolution mass spectrometry. We detected MHUP and MCDP in >83% of the samples; median concentrations were 0.21ng/mL and 0.36ng/mL, respectively. MOUP was detected only in 14% of the samples analyzed, and MUP was not detected. All three metabolites eluted as peak clusters likely because of the presence of multiple oxidation sites and multiple isomers in DUP technical mixtures. Taken together, these findings suggest that with the appropriate mass spectrometry quantification techniques, MHUP and MCDP may serve as suitable biomarkers for assessing background exposure to DUP.

Quantification of tetrabromo benzoic acid and tetrabromo phthalic acid in rats exposed to the flame retardant Uniplex FPR-45.

The first withdrawal of certain polybrominated diphenyl ethers flame retardants from the US market occurred in 2004. Since then, use of brominated non-PBDE compounds such as bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB) in commercial formulations has increased. Assessing human exposure to these chemicals requires identifying metabolites that can potentially serve as their biomarkers of exposure. We administered by gavage a dose of 500 mg/Kg bw of Uniplex FRP-45 (>95 % BEH-TEBP) to nine adult female Sprague-Dawley rats. Using authentic standards and mass spectrometry, we positively identified and quantified 2,3,4,5-tetrabromo benzoic acid (TBBA) and 2,3,4,5-tetrabromo phthalic acid (TBPA) in 24-h urine samples collected 1 day after dosing the rats and in serum at necropsy, 2 days post-exposure. Interestingly, TBBA and TBPA concentrations correlated well (R (2) = 0.92). The levels of TBBA, a known metabolite of EH-TBB, were much higher than the levels of TBPA both in urine and serum. Because Uniplex FRP-45 was technical grade and EH-TBB was present in the formulation, TBBA likely resulted from the metabolism of EH-TBB. Taken together, our data suggest that TBBA and TBPA may serve as biomarkers of exposure to non-PBDE brominated flame retardant mixtures. Additional research can provide useful information to better understand the composition and in vivo toxicokinetics of these commercial mixtures.

Paired Serum and Urine Concentrations of Biomarkers of Diethyl Phthalate, Methyl Paraben, and Triclosan in Rats.

BACKGROUND: Exposure to environmental chemicals, including phthalates and phenols such as parabens and triclosan, is ubiquitous within the U.S. general population. OBJECTIVE: This proof-of-concept rodent study examined the relationship between oral doses of three widely used personal care product ingredients [diethyl phthalate (DEP), methyl paraben (MPB), and triclosan] and urine and serum concentrations of their respective biomarkers. METHODS: Using female Sprague-Dawley rats, we carried out two rounds of experiments with oral gavage doses selected in accordance with no observed adverse effect levels (NOAELs) derived from previous studies: 1,735 (DEP), 1,050 (MPB), 50 (triclosan) mg/kg/day. Administered doses ranged from 0.005 to 173 mg/kg/day, 10-100,000 times below the NOAEL for each chemical. Controls for the MPB and triclosan experiments were animals treated with olive oil (vehicle) only; controls for the DEP serum experiments were animals treated with the lowest doses of MPB and triclosan. Doses were administered for 5 days with five rats in each treatment group. Urine and blood serum, collected on the last day of exposure, were analyzed for biomarkers. Relationships between oral dose and biomarker concentrations were assessed using linear regression. RESULTS: Biomarkers were detected in all control urine samples at parts-per-billion levels, suggesting a low endemic environmental exposure to the three chemicals that could not be controlled even with all of the precautionary measures undertaken. Among the exposed animals, urinary concentrations of all three biomarkers were orders of magnitude higher than those in serum. A consistently positive linear relationship between oral dose and urinary concentration was observed (R2 > 0.80); this relationship was inconsistent in serum. CONCLUSIONS: Our study highlights the importance of carefully considering the oral dose used in animal experiments and provides useful information in selecting doses for future studies. CITATION: Teitelbaum SL, Li Q, Lambertini L, Belpoggi F, Manservisi F, Falcioni L, Bua L, Silva MJ, Ye X, Calafat AM, Chen J. 2016. Paired serum and urine concentrations of biomarkers of diethyl phthalate, methyl paraben, and triclosan in rats. Environ Health Perspect 124:39-45; http://dx.doi.org/10.1289/ehp.1409586.

Development of urine standard reference materials for metabolites of organic chemicals including polycyclic aromatic hydrocarbons, phthalates, phenols, parabens, and volatile organic compounds.

Two new Standard Reference Materials (SRMs), SRM 3672 Organic Contaminants in Smokers' Urine (Frozen) and SRM 3673 Organic Contaminants in Non-Smokers' Urine (Frozen), have been developed in support of studies for assessment of human exposure to select organic environmental contaminants. Collaborations among three organizations resulted in certified values for 11 hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs) and reference values for 11 phthalate metabolites, 8 environmental phenols and parabens, and 24 volatile organic compound (VOC) metabolites. Reference values are also available for creatinine and the free forms of caffeine, theobromine, ibuprofen, nicotine, cotinine, and 3-hydroxycotinine. These are the first urine Certified Reference Materials characterized for metabolites of organic environmental contaminants. Noteworthy, the mass fractions of the environmental organic contaminants in the two SRMs are within the ranges reported in population survey studies such as the National Health and Nutrition Examination Survey (NHANES) and the Canadian Health Measures Survey (CHMS). These SRMs will be useful as quality control samples for ensuring compatibility of results among population survey studies and will fill a void to assess the accuracy of analytical methods used in studies monitoring human exposure to these organic environmental contaminants.

Identification of di-2-ethylhexyl terephthalate (DEHTP) metabolites using human liver microsomes for biomonitoring applications.

Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of the plasticizer di-2-ethylhexyl phthalate (DEHP), is used in food packaging and medical devices, among other applications, and is a potential replacement for DEHP and other ortho-phthalate plasticizers. Identifying sensitive and specific biomarkers of DEHTP is necessary to assess humans' background exposure to DEHTP. Using mass spectrometry, we investigated the metabolism of DEHTP by human liver microsomes to identify in vitro DEHTP metabolites. We unequivocally identified terephthalic acid (TPA) and mono-2-ethylhydroxyhexyl terephthalate (MEHHTP), using authentic standards, and tentatively identified mono-2-ethylhexyl terephthalate (MEHTP) and two other oxidative metabolites of DEHTP: mono-2-ethyloxohexyl terephthalate (MEOHTP), and mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) from their mass spectrometry fragmentation patterns. We also evaluated the formation of in vitro metabolites of DEHP. DEHTP and DEHP produced similar metabolites, but their metabolite profiles differed considerably. DEHTP metabolized to form TPA, a metabolite of several terephthalates, as the major in vitro metabolite, followed by MEHTP, MEHHTP, MEOHTP and MECPTP. MEHTP, MEHHTP, MEOHTP and MECPTP, which are specific metabolites of DEHTP, may be suitable biomarkers for assessing exposure to DEHTP. Nonetheless, data on the urinary excretion fraction and temporal stability of these metabolites, among other considerations, are needed to demonstrate their utility as exposure biomarkers.

Exploring the potential association between brominated diphenyl ethers, polychlorinated biphenyls, organochlorine pesticides, perfluorinated compounds, phthalates, and bisphenol A in polycystic ovary syndrome: a case-control study.

BACKGROUND: Polycystic Ovary Syndrome (PCOS) is an endocrine-metabolic disorder that affects approximately 6-10% of women of child-bearing age. Although preliminary studies suggest that certain pollutants may act as endocrine disruptors in animals, little is known about their potential association with PCOS. The objective of this case-control pilot study is to determine whether women with PCOS have higher concentrations of specific environmental contaminants compared to women who have not developed PCOS. METHODS: Fifty-two PCOS case-patients (diagnosed using the National Institutes of Health 1990 definition) and 50 controls were recruited in 2007-2008, from an urban academic medical center in Los Angeles, CA. Brominated diphenyl ethers, polychlorinated biphenyls (PCBs), organochlorine pesticides, and perfluorinated compounds (PFCs) were measured in serum, and phthalates metabolites and bisphenol A (BPA) in urine. RESULTS: PCOS case-patients had significantly higher geometric mean (GM) serum concentrations of two PFCs: perfluorooctanoate (PFOA) (GMcases = 4.1 µg/L, GMcontrols = 2.3 µg/L; p = 0.001) and perfluorooctane sulfonate (PFOS) (GMcases = 8.2 µg/L, GMcontrols = 4.9 µg/L; p = 0.01), and lower urinary concentrations of monobenzyl phthalate (mBzP) (GMcases = 7.5 µg/g creatinine, GMcontrols = 11.7 µg/g creatinine; p = 0.02). Logistic regression, controlling for body mass index, age and race, identified an increased likelihood of PCOS in subjects with higher serum concentrations of PFOA and PFOS (adjusted-ORs = 5.8-6.9, p < 0.05), and with lower urine concentrations of mBzP and mono-n-butyl phthalate (mBP) (aORs = 0.14-0.25, p < 0.05). CONCLUSIONS: Our data suggest that PCOS case-patients may differ from controls in their environmental contaminant profile. PCOS subjects had higher serum concentrations of two PFCs, PFOA and PFOS, and lower urine concentrations of mBP and mBzP. Future studies are needed to confirm these preliminary findings and determine if these chemicals or their precursors may have a role in the pathogenesis of PCOS.

Dietary predictors of urinary environmental biomarkers in young girls, BCERP, 2004-7.

BACKGROUND: Exposures of children to phthalates, parabens, and bisphenol-A (BPA) are of concern because of their hormonal potential. These agents are found in a wide range of foods and packaging. We investigated whether intake of certain foods predict exposures to these chemicals in young girls. METHODS: Among 1101 girls (6-8 years at enrollment) from the Breast Cancer and Environment Research Program (BCERP) study, we measured urinary exposure biomarkers for phthalates, parabens, and BPA and assessed dietary intake using 24-h recall 2-4 times. We examined the average daily servings of major and minor food groups categorized as 0 to <0.5, 0.5 to <1 and ≥ 1 servings per day. Items included dairy, eggs, fats, fish, fruit, single grains, meat, non-poultry meats, pasta, poultry and vegetables. Covariate-adjusted least squares geometric means and 95% confidence intervals of creatinine-corrected phthalate and phenol metabolite concentrations in urine were calculated in relation to food intake. RESULTS: Grains, flour and dry mixes and total fish consumption were positively associated with BPA and the sum of four di-2-ethylhexylphthalate (DEHP) urinary metabolite concentrations. Non-fresh vegetables and poultry were both positively associated with BPA and paraben urinary concentrations. Fats, oils and poultry consumption were positively associated with BPA. Whole-fat dairy consumption was associated with ΣDEHP. CONCLUSIONS: Some foods may contribute to child exposures to certain chemicals, and this may constitute modifiable means to reduce these environmental exposures.

In vitro metabolites of di-2-ethylhexyl adipate (DEHA) as biomarkers of exposure in human biomonitoring applications.

Di-2-ethylhexyl adipate (DEHA) is a common plasticizer used in food packaging. At high doses, DEHA can cause adverse health effects in rats. Although the potential for human exposure to DEHA is high, no DEHA specific biomarkers are identified for human biomonitoring. Using human liver microsomes, we investigated the in vitro phase I metabolism of DEHA and its hydrolytic metabolite mono-2-ethylhexyl adipate (MEHA) and, for comparison purposes, of the analogous di-2-ethylhexyl phthalate (DEHP) and its hydrolytic metabolite mono-2-ethylhexyl phthalate. We unequivocally identified MEHA, a DEHA specific biomarker, and adipic acid, a nonspecific biomarker, using authentic standards. On the basis of their mass spectrometric fragmentation patterns, we tentatively identified two other DEHA specific metabolites: mono-2-ethylhydroxyhexyl adipate (MEHHA) and mono-2-ethyloxohexyl adipate (MEOHA), analogous to the oxidative metabolites of DEHP. Interestingly, although adipic acid was the major in vitro metabolite of DEHA, the analogous phthalic acid was not the major in vitro metabolite of DEHP. Our preliminary data for 144 adults with no known exposure to DEHA suggests that adipic acid is also the main in vivo urinary metabolite, while MEHA, MEHHA, and MEOHA are only minor metabolites. Therefore, the use of these specific metabolites for assessing the exposure of DEHA may be limited to highly exposed populations.

Environmental exposure to the plasticizer 1,2-cyclohexane dicarboxylic acid, diisononyl ester (DINCH) in U.S. adults (2000-2012).

1,2-Cyclohexane dicarboxylic acid, diisononyl ester (DINCH) is a complex mixture of nine carbon branched-chain isomers. It has been used in Europe since 2002 as a plasticizer to replace phthalates such as di(2-ethylhexyl)phthalate (DEHP) and diisononyl phthalate (DINP). Urinary concentrations of the oxidative metabolites of DINCH, namely cyclohexane-1,2-dicarboxylic acid-monocarboxy isooctyl ester (MCOCH); cyclohexane-1,2-dicarboxylic acid-mono(oxo-isononyl) ester (MONCH); and cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (MHNCH), can potentially be used as DINCH exposure biomarkers. The concentrations of MCOCH, MONCH and MHNCH were measured by online solid phase extraction-high performance liquid chromatography-tandem mass spectrometry in urine collected in 2000 (n=114), 2001 (n=57), 2007 (n=23), 2009 (n=118), 2011 (n=94) and 2012 (n=121) from convenience groups of anonymous U.S. adult volunteers with no known DINCH exposure. None of the DINCH metabolites were detected in samples collected in 2000 and 2001. Only one sample collected in 2007 had measureable concentrations of DINCH metabolites. The detection rate for all three metabolites increased from 2007 to 2012. The presence of oxidative metabolites of DINCH in urine suggests that these oxidative metabolites can be used as DINCH biomarkers for exposure assessment even at environmental exposure levels.

Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: a 2-step standardization method based on regression residuals.

BACKGROUND: Environmental epidemiology and biomonitoring studies typically rely on biological samples to assay the concentration of non-persistent exposure biomarkers. Between-participant variations in sampling conditions of these biological samples constitute a potential source of exposure misclassification. Few studies attempted to correct biomarker levels for this error. We aimed to assess the influence of sampling conditions on concentrations of urinary biomarkers of select phenols and phthalates, two widely-produced families of chemicals, and to standardize biomarker concentrations on sampling conditions. METHODS: Urine samples were collected between 2002 and 2006 among 287 pregnant women from Eden and Pélagie cohorts, from which phthalates and phenols metabolites levels were assayed. We applied a 2-step standardization method based on regression residuals. First, the influence of sampling conditions (including sampling hour, duration of storage before freezing) and of creatinine levels on biomarker concentrations were characterized using adjusted linear regression models. In the second step, the model estimates were used to remove the variability in biomarker concentrations due to sampling conditions and to standardize concentrations as if all samples had been collected under the same conditions (e.g., same hour of urine collection). RESULTS: Sampling hour was associated with concentrations of several exposure biomarkers. After standardization for sampling conditions, median concentrations differed by--38% for 2,5-dichlorophenol to +80 % for a metabolite of diisodecyl phthalate. However, at the individual level, standardized biomarker levels were strongly correlated (correlation coefficients above 0.80) with unstandardized measures. CONCLUSIONS: Sampling conditions, such as sampling hour, should be systematically collected in biomarker-based studies, in particular when the biomarker half-life is short. The 2-step standardization method based on regression residuals that we proposed in order to limit the impact of heterogeneity in sampling conditions could be further tested in studies describing levels of biomarkers or their influence on health.

Identification of potential biomarkers of exposure to di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), an alternative for phthalate plasticizers.

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is used as an alternative for some phthalate plasticizers. In rats, DINCH mostly eliminates in feces as cyclohexane-1,2-dicarboxylic acid (CHDA), mono isononyl ester (MINCH) or in urine as CHDA. However, CHDA is not a specific biomarker of DINCH and measuring MINCH in feces is impractical. To identify additional potential biomarkers, we administered DINCH (500 mg/kg body weight) in a single subcutaneous (SC) or oral dose to four adult female Sprague-Dawley rats. We collected 24-h urine samples before dosing (to be used as controls) and 24-h and 48-h after dosing, and serum at necropsy after 48 h. We positively identified and accurately quantified CHDA and cyclohexane-1,2-dicarboxylic [corrected] acid, mono hydroxyisononyl ester (MHNCH) using authentic standards. Moreover, we tentatively identified MINCH and 12 oxidative metabolites, including 4 cyclohexane ring oxidation products, based on their mass spectrometric-fragmentation patterns. CHDA and MHNCH levels were higher in the urine collected 24 h after oral than SC administration. By contrast, 48-h after dosing, CHDA urinary levels were similar regardless of the exposure route. We detected all but two of the urine metabolites also in serum. Levels of CHDA and MHNCH in serum were lower than in the two post-dose urine collections. Our results suggest that several urinary oxidative metabolites, specifically CHDA, mono oxoisononyl ester and MHNCH may be used as specific biomarkers of DINCH exposure in humans.