Samples collected at visit 1 were analyzed at the National Center for Environmental Health laboratories at CDC for nine phthalate metabolites [n = 1,149; monoethylphthalate (MEP), mono-butyl phthalate, mono-iso-butyl phthalate, mono-benzyl phthalate, mono-3-carboxypropyl phthalate, mono-2-ethyl-5-carboxypentyl phthalate, mono-(2-ethyl-5-hydroxylhexyl) phthalate, mono-(2-ethyl-5-oxohexyl) phthalate, and mono-(2-ethylhexyl) phthalate (MEHP)], seven phenols (benzophenone-3, bisphenol A, 2,5-dichlorophenol, triclosan; n = 1,149; methyl-, butyl-, and propyl- parabens, n = 1,059), and three phytoestrogens (daidzein, genistein, enterolactone; n = 1,150). Parabens were not measured early in the study. At least one urinary biomarker measurement was available among 1,151 girls, 985 with breast stages. We substituted limit of detection ( for results below the LOD. Adjustment for urine dilution was accomplished using creatinine, to account for difference in sampling (spot specimens at MSSM and KPNC, early-morning samples at Cincinnati) and interindividual variation in urine dilution. We included log-creatinine in models using continuous log-biomarker variables, and we created quintile cut points from creatinine-corrected concentrations (micrograms per gram creatinine). As previously described, to reduce multiple comparisons we combined the phthalate metabolites into two groups that represent similar sources and similar biologic activity, low- (< 250 Da) and high-molecular-weight (> 250 Da) phthalate metabolites (low MWP and high MWP) [details in Supplemental Material, Table 2 (doi:10.1289/ehp.0901690)]. We expressed high MWP molar sum as MEHP (molecular weight 278) and the low MWP as MEP (molecular weight 194) so that units were the same as the other analytes (micrograms per liter). Similarly, a molar sum of the paraben metabolites was created (paraben sum) expressed as propyl paraben (molecular weight 180.2). Models with the individual phthalate and paraben metabolites were consistent with the molar sum variables. Results using di(2-ethylhexyl)phthalate (DEHP)-sum metabolites were almost identical to those for the high MWP, and they represented 75% ± 16% (mean ± SD) of the high MWP biomarkers. Therefore, only the latter models are presented.
Laboratory techniques and quality control protocols are identical to those reported previously in a pilot study (Wolff et al. 2007 (link)). Briefly, urine undergoes an automated cleanup with enzymatic deconjugation, followed by high-performance liquid chromatography-isotope dilution tandem mass spectrometry quantification (Kato et al. 2005 (link); Rybak et al. 2008 ; Ye et al. 2005 (link), 2006 ). In addition to the internal CDC quality control procedures, we incorporated approximately 10% masked quality control specimens (n = 101) from a single urine pool. The coefficients of variation (SD/mean concentration) were < 10% for 13 analytes and were between 10% and 21% for the remaining six biomarkers.
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