A Novel Method for Calculating Potency-Weighted Cumulative

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A novel method for calculating potency-weighted cumulative phthalates exposure with implications for identifying racial/ethnic disparities among U.S. reproductive-aged women in NHANES 2001-2012 Julia R Varshavsky, Ami R Zota, and Tracey J. Woodruff Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00522 • Publication Date (Web): 31 Aug 2016 Downloaded from http://pubs.acs.org on August 31, 2016

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Environmental Science & Technology

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A novel method for calculating potency-weighted cumulative phthalates exposure

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with implications for identifying racial/ethnic disparities among U.S. reproductive-

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aged women in NHANES 2001-2012

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Julia R. Varshavsky*1, Ami R. Zota2, and Tracey J. Woodruff3

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1 Division

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California, Berkeley, Berkeley, CA, 94720, USA

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2 Department

of Environmental Health Sciences, School of Public Health, University of

of Environmental and Occupational Health, Milken Institute School of

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Public Health, George Washington University, Washington DC, 20052, USA

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3 Program

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Gynecology, and Reproductive Sciences, Institute for Health Policy Studies, University

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of California, San Francisco, San Francisco, California, 94143, USA

on Reproductive Health and the Environment, Department of Obstetrics,

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*Corresponding author:

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Julia Varshavsky, MPH Division of Environmental Health Sciences School of Public Health University of California, Berkeley 50 University Hall, Room 221 Berkeley, CA 94720 Email: [email protected]; Phone: 510.384.0169

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TOC Abstract Art

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Abstract

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affect reproduction and development. Multiple anti-androgenic phthalates

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exposure during fetal development can have greater impacts than individual

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exposure; thus, the National Academy of Sciences (NAS) recommends them for

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cumulative assessment. Using National Health and Nutrition Examination Survey

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data (NHANES, 2001-2012), we developed a potency-weighted sum of daily

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intake (∑androgen-disruptor; µg/kg/day) of di-n-butyl phthalate (DnBP), diisobutyl

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phthalate (DiBP), butyl benzyl phthalate (BBzP), and di(2-ethylhexyl) phthalate

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(DEHP) based on NAS recommendations, and included diethyl phthalate (DEP)

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and diisononyl phthalate (DiNP) in additional metrics (2005-2012). We compared

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racial/ethnic differences in ∑androgen-disruptor among 2842 reproductive-aged

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women. In sensitivity analyses, we assessed the influence of potency

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assumptions, alternate urine dilution adjustment methods, and weighting

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phthalate metabolites directly rather than daily intake estimates of parent

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compounds. We found that DEHP contributed most to ∑androgen-disruptor (48-

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64%), and that ∑androgen-disruptor decreased over time. Black women

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generally had higher cumulative exposures than white women, although the

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magnitude and precision of the difference varied by model specification. Our

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approach provides a blueprint for combining chemical exposures linked to

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common adverse outcomes, and should be considered in future exposure, risk,

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and epidemiological studies.

Phthalates are ubiquitous chemicals linked to hormonal disruptions that

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INTRODUCTION

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of health outcomes. Fetal phthalate exposures cause a group of male

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reproductive problems known as the “phthalate syndrome” in animals, which

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includes birth defects of the testes and penis (e.g. cryptorchidism, hypospadias),

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low sperm count, and infertility.1 Human studies support an association between

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developmental phthalate exposures and male reproductive effects.2-6 Certain

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phthalates, such as di-n-butyl phthalate (DnBP), diisobutyl phthalate (DiBP), butyl

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benzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP), and diisononyl

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phthalate (DiNP), exert toxicity primarily through androgen disruption.1

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Phthalates esters are hormonally active chemicals linked to a wide range

Phthalate exposures are ubiquitous due to their widespread use in myriad

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consumer and personal care products.7, 8 Biomonitoring studies show the U.S.

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population is exposed to multiple phthalates simultaneously.9-12 Animal studies

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demonstrate that phthalate mixtures result in higher male reproductive risk than

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individual phthalates, especially during fetal development.13-15 Human studies

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also find higher risks from multiple phthalates.2, 5 In 2008, the National Academy

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of Sciences (NAS) recommended phthalates and other anti-androgens for

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cumulative risk assessment based on their ability to cause common adverse

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outcomes (i.e. changes in testosterone concentration during development) rather

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than shared mechanisms of action (i.e. how testosterone concentration is

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disrupted).1 More recent mixture studies confirm NAS findings and further

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highlight the need to consider the joint effects of co-occurring phthalates.16-19


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However, advancements in the epidemiology of phthalate mixtures are limited by

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current methods for characterizing cumulative exposures.20

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Identifying high-risk subpopulations for cumulative exposure warrants examination since individual phthalate profiles may not accurately represent the

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distribution of overall phthalate burden. Individual phthalate exposures have been

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shown to vary by race/ethnicity and socioeconomic status (SES), often in

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opposing directions.9, 21-24 For example, DnBP and BBzP are higher in low SES

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groups, while DEHP exposure is lower in the same subpopulation.22, 23

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Characterizing cumulative exposure inequalities may help elucidate health

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disparities and identify solutions that better protect those at increased risk of

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exposure and disease.25

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In this paper, we develop a potency-weighted metric of androgen-

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disrupting phthalates using 2008 NAS recommendations and examine

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demographic differences in cumulative phthalates exposure among U.S.

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reproductive-aged women. We focus on women of reproductive age because in

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utero development has been identified as the most sensitive window for

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phthalate toxicity,1 and previous work suggests that phthalate exposures among

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reproductive-aged and pregnant women are similar12 but often distinct from other

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subpopulations.26

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MATERIALS AND METHODS

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Study Population. We pooled data from six cycles (2001-2012) of the National

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Health and Nutrition Examination Survey (NHANES), a nationally representative



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survey and physical examination of the civilian, non-institutionalized U.S.

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population conducted by the U.S. Centers for Disease Control (CDC)

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(http://www.cdc.gov/nchs/nhanes.htm). The study population was limited to

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females aged 15-44 years (n=3480). Of these, we further excluded 544 women

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who self-identified as a race/ethnicity other than non-Hispanic white, non-

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Hispanic black, or Mexican American, who together comprise 86% of the total

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population. We also excluded participants who did not have at least one

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phthalate metabolite measurement (n=94), resulting in a final sample size of

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2842 participants.

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Phthalate Metabolite Measurements. In each NHANES survey cycle, phthalate

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metabolites are measured in one-third of survey participants. Analytical methods

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are described elsewhere.27 Briefly, spot urine samples are collected as part of the

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NHANES medical examination and analyzed at the CDC’s National Center for

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Environmental Health (Atlanta, GA). Phthalate metabolites are quantified using

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high performance liquid chromatography coupled with tandem mass

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spectrometry.27, 28 Laboratory files were downloaded from the NHANES website

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in March 2015 and included necessary impurity corrections for some previously

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used analytical standards.29 Not all phthalate metabolites are measured in every

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NHANES cycle, and limits of detection (LOD) for each metabolite can vary by

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cycle. Thus, we used the maximum LOD for each phthalate metabolite and

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substituted concentrations below the LOD with a value equal to the LOD divided

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by the square root of two.11, 30


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Cumulative Exposure Metric. To characterize cumulative exposure to androgen-

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disrupting phthalates, we calculated a potency-weighted aggregate sum

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(∑androgen-disruptor) of four phthalates (DnBP, DiBP, BBzP, and DEHP) that

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are recognized as anti-androgenic by the NAS and whose metabolites were

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measured in every cycle between 2001-2012 (Figure 1; model 1). The primary

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hydrolytic metabolites for DnBP, DiBP, BBzP, and DEHP, respectively, are

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mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), mono-benzyl

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phthalate (MBzP), and mono-(2-ethylhexyl) phthalate (MEHP). MEHP further

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metabolizes to oxidative metabolites: mono-(2-ethyl-5-oxohexyl) phthalate

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(MEOHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), and mono-(2-

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ethyl-5-carboxypentyl) phthalate (MECPP).

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To calculate unitless relative potency factors (RPFs) for the four

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phthalates, we used NAS phthalate-specific benchmark doses (BMDs)

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(mg/kg/day), which are statistically derived doses related to a predefined change

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from controls in benchmark response (BMR) (in this case a 5% reduction in

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testosterone concentration, or a BMR equal to 5%) (Table 1).1, 31 BMDs were

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used rather than the lower limit of a one-sided 95% confidence interval (CI) on

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the BMD (BMDL) because we are comparing across chemicals.31 In the NAS

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analysis, DnBP had the highest potency, or lowest BMD. Therefore, we derived

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RPFs by dividing the DnBP reference BMD by that of each phthalate.

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RPF i = (BMD Reference / BMD i ); i = individual phthalate (Eq. 1)



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Because BMDs are based on phthalate doses administered to laboratory animals

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and not metabolite concentrations, we estimated the daily intake of parent

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phthalate compounds (μg/kg/day) from measured urinary metabolites (ng/mL =

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μg/L) using a pharmacokinetic modeling equation adapted from previous

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studies.32, 33

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where ME i is the urinary concentration of metabolite per gram of creatinine (μg/g)

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for each phthalate, CE is the creatinine excretion rate normalized by body weight

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(mg/kg/day), F UE,i is the molar fraction of urinary excreted metabolite related to

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parent compound for each phthalate (unitless), 1000 is a conversion factor (i.e.

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mg/g), and MW p and MW m are the molecular weights of the parent phthalates

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and metabolites, respectively.

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Daily Intake i = (ME i x CE) x (MW p ) (Eq. 2) (F UE,i x 1000) x (MW m )

Although creatinine excretion rates vary across racial/ethnic groups, we

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initially assumed a uniform creatinine excretion rate of 18 mg/kg/day34 for all

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participants. We calculated creatinine-adjusted concentrations (ME) for each

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participant by dividing their measured urinary concentrations (μg/L) by their

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measured urinary creatinine concentration (g/L). The fractional urinary excretion

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values, or F UE , are 0.69, 0.69, and 0.73 for DnBP, DiBP, and BBzP,

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respectively.35, 36 The values for MEHP, MEHHP, and MEOHP are 0.062, 0.149,

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and 0.109, respectively. MECPP was not included as a DEHP metabolite for

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2001-2012 analyses because it was not measured in all survey cycles, but it was

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included in 2005-2012 analyses with an F UE of 0.132.37 Potency-weighted daily

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intake estimates of ∑androgen-disruptor, expressed in μg/kg/day, were then 8 ACS Paragon Plus Environment

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calculated by summing the products of RPFs and daily intakes for each phthalate

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(See Figure S1 for sample calculations).

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∑androgen-disruptor = ∑(Daily Intake i x RPF i ) (Eq. 3)

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In addition to our original metric (Figure 1; model 1), we calculated a

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second version of ∑androgen-disruptor by adding diethyl phthalate (DEP) and

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DiNP to model 1 (Figure 1; model 2a). While DEP does not exhibit anti-

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androgenic properties in laboratory studies,35 several epidemiologic studies

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report an association between MEP and male reproductive health endpoints,

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although findings are inconsistent.3, 5, 6 Therefore, we assumed DEP to have a

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low but non-zero potency equal to one order of magnitude less than the least

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potent phthalate in our model (RPF = 1/10 multiplied by 0.24, or 0.024). Mono-

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ethyl phthalate (MEP) is the primary metabolite of DEP, and DEP was assumed

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to have the same F UE as DnBP.32 We also added DiNP to the metric since it has

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been recognized as an anti-androgenic phthalate.1, 35 Since DiNP’s primary

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metabolite, mono(carboxy-isooctyl) phthalate (MCOP), was not measured before

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2005, model 2a was examined in 2005-2012 participants only (N=1723). DiNP

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was assumed to be 2.3 times less potent than DEHP.18 We used an F UE value of

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0.099 for MCOP.37

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Data Analysis. We conducted analyses using SAS software, Version 9.4 (SAS

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Institute Inc., Cary, NC). Since we combined multiple cycles of data, we

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calculated new sample population weights according to NHANES analytical

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guidelines.38 All analyses were adjusted for sample population weights and the



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NHANES clustered sample design. We defined statistical significance at p

A Novel Method for Calculating Potency-Weighted Cumulative

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