toxics

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

Manori J. Silva *, Lee-Yang Wong, James L. Preau, Ella Samandar, Emmanuela Obi, Antonia M. Calafat and Julianne C. Botelho Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA 30341, USA; [email protected] (L.-Y.W.); [email protected] (J.L.P.); [email protected] (E.S.); [email protected] (E.O.); [email protected] (A.M.C.); [email protected] (J.C.B.) * Correspondence: [email protected]

 Received: 11 September 2019; Accepted: 4 October 2019; Published: 11 October 2019 

Abstract: We know little about the potential health risks from exposure to diisoheptyl phthalate (DiHpP), a 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.

Keywords: diisoheptyl phthalate; DiHpP ; exposure; oxidative metabolites

1. Introduction Diisoheptyl phthalate (DiHpP), an isomeric mixture of with branched and linear seven carbon backbones, is used commercially as a plasticizer in vinyl resins. DiHpP can be found in automotive, wire, cable, imitation leather, and flooring products [1]. DiHpP production ended in the European Union and United States in 2010 [2]. However, DiHpP may still be present in phthalate diester mixtures [1] that can be used in consumer products in the United States and European Union. As a result, human exposure to DiHpP can occur. Animal studies suggest potential adverse health effects from exposure to DiHpP [2–7]. In a 28-day repeated oral DiHpP dose toxicity test in male and female F344 rats, body weight gain was inhibited, but liver and kidney weights increased [8]. In a developmental toxicity study, female rats given DiHpP by oral gavage on gestational days 6–20 had increased resorptions and reduced fetal weight [7]. Metabolites of phthalates are often used as biomarkers of exposure [9]. To date, no human data on exposure to DiHpP have been reported. Hence, DiHpP biomonitoring to assess exposure in humans might prove to be valuable.

Toxics 2019, 7, 53; doi:10.3390/toxics7040053 www.mdpi.com/journal/toxics Toxics 2019, 7, 53 2 of 8

Oxidative metabolites form during metabolism of phthalates and are excreted in urine [10,11]. Toxics 2019, 7, x FOR PEER REVIEW 2 of 8 Population-based biomonitoring studies use oxidative metabolites of phthalates as biomarkers of exposure [9,12–Oxidative15]. Monohydroxyheptyl metabolites form during phthalate metabolism (MHHpP) of phthalates and monocarboxyhexyl and are excreted in urine phthalate [10,11]. (MCHxP), which formedPopulation-based as DiHpP metabolites,biomonitoring st wereudies identifieduse oxidative in metabolites urine of ratsof phthalates dosed withas biomarkers DiHpP of [16 ]. In our study, weexposure quantified [9,12–15]. three Monohydrox oxidativeyheptyl metabolites phthalate of (MHHpP) DiHpP toand better monocarboxyhexyl understand phthalate human exposure (MCHxP), which formed as DiHpP metabolites, were identified in urine of rats dosed with DiHpP to DiHpP.[16]. In our study, we quantified three oxidative metabolites of DiHpP to better understand human exposure to DiHpP. 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals and Equipment 2.1. Chemicals and equipment We bought MHHpP, mono-oxoheptyl phthalate (MOHpP), MCHxP (Figure1), d -MHHpP, We bought MHHpP, mono-oxoheptyl phthalate (MOHpP), MCHxP (Figure 1), d4-MHHpP, d4-4 d4-MOHpP,MOHpP, and dand4-MCHxP d4-MCHxP from from ADM ADM GermanyGermany (>95%). (>95%). We Webought bought high-performance high-performance liquid liquid chromatographychromatography (HPLC) (HPLC) grade grade acetonitrile, acetonitrile, water, and and methanol methanol (99.8%) (99.8%) from Honeywell from Honeywell Burdick & Burdick & JacksonJackson (Muskegon, (Muskegon, MI, MI, USA). USA).β -glucuronidaseβ-glucuronidase (Escherichia (Escherichia coli-K12) coli-K12) was purchased was purchased from Roche from Roche Biomedical (Mannheim, Germany). All chemicals and reagents were used without further Biomedicalpurification. (Mannheim, Germany). All chemicals and reagents were used without further purification.

Figure 1. Metabolites used to assess exposure to diisoheptyl phthalate. Only one isomer for each metaboliteFigure is shown. 1. Metabolites used to assess exposure to diisoheptyl phthalate. Only one isomer for each metabolite is shown. We used a QExactive plus Hybrid Quadrupole-Orbitrap mass spectrometer and Ultimate We used a QExactive plus Hybrid Quadrupole-Orbitrap mass spectrometer and Ultimate 3000 3000 high-performancehigh-performance chromatographychromatography system system (Therm (Thermoo Fisher Scientific, Fisher Scientific, Waltham, MA, Waltham, USA) for MA, USA) for samplesample analysis analysis and and Xcalibur Xcalibur 2.2 2.2 (Thermo Fisher Fisher Scientific) Scientific) for data for processing. data processing.

2.2. Subjects2.2. Subjects We collected urine anonymously from demographically diverse groups of U.S. male and female We collectedadults from urine Atlanta, anonymously GA, during 2000 from and demographically2018–2019 to study exposure diverse biomarkers groups to of environmental U.S. male and female adults fromchemicals. Atlanta, No GA, personal during information 2000 and from 2018–2019 the subjects towas study available. exposure Samples biomarkerswere collected tobetween environmental chemicals.8:00 No a.m. personal and 5:00 information p.m. and were from not the necessarily subjects first-morning was available. voids. Samples Same donors were may collected have between 8:00 a.m.contributed and 5:00 urine p.m. in anddifferent were collection not necessarily years, during first-morning different days, or voids. at different Same times donors of day. may have After collection, samples were stored at –70 °C until analysis. The Centers for Disease Control and contributedPrevention urine in(CDC) diff Institutionalerent collection Review years,Board approv duringed the di ffurineerent collection days, and or atanalysis. different A waiver times of day. After collection,of informed samples consent were was requested stored at under70 45C Code until of analysis. Federal Regulation The Centers (CFR) for46.116(d) Disease general Control and − ◦ Preventionrequirements (CDC) Institutional for informed Review consent. Board approved the urine collection and analysis. A waiver of informed consent was requested under 45 CFR 46.116(d) general requirements for informed consent. 2.3. Analytical Method 2.3. AnalyticalWe Method obtained the mass spectra for all three metabolites using analytical standards prepared in acetonitrile and optimized the fragmentation of the precursor ion for each metabolite (Table 1). We We obtainedadapted published the mass analytical spectra methods for allfor measuring three metabolites phthalate oxidative using metabolites analytical in urine standards [17,18]. prepared in acetonitrileBriefly, and urine optimized (0.1 mL) and the calibration fragmentation standards of (0.013–130 the precursor ng/mL) were ion forspiked each with metabolite an internal (Table1). standard solution (5–10 ng/mL) containing d4-MHHpP, d4-MOHpP, d4-MCHxP, and a buffered We adapted published analytical methods for measuring phthalate oxidative metabolites in urine [17,18]. solution of β-glucuronidase (Escherichia coli-K12; 25 μL, pH 6.5, 1 M ammonium acetate). The mixture Briefly, urinewas incubated (0.1 mL) at and37 °C calibrationfor a minimum standards of 120 min [17,18]. (0.013–130 The target ng analytes/mL) were in the spiked urine with were an internal standard solutionextracted using (5–10 on-line ng/mL) solid-phase containing extraction d4 -MHHpP,(Chromolith HighResolution d4-MOHpP, dRP-18,4-MCHxP, 4.6 mm andGuard a buffered solution of β-glucuronidase (Escherichia coli-K12; 25 µL, pH 6.5, 1 M ammonium acetate). The mixture was incubated at 37 ◦C for a minimum of 120 min [17,18]. The target analytes in the spiked urine were extracted using on-line solid-phase extraction (Chromolith HighResolution RP-18, 4.6 mm Guard Cartridge; Merck KGaA, Darmstadt, Germany) and chromatographically resolved using high-performance liquid chromatography (Thermo Scientific Betasil phenyl, 3 µm, 150 mm 2.1 mm × column) (Figure2). We then used high-resolution mass spectrometry in negative ion parallel reaction Toxics 2019, 7, 53 3 of 8 mode on a QExactive high-resolution mass spectrometer for quantification of the analytes (Table1). We used the lowest calibration level (0.013 ng/mL) as the limit of detection (LOD).

Table 1. Analytical parameters for the quantification of diisoheptyl phthalate metabolites.

Urinary MS/MS Scan Collision Energy Parent Chemical Internal Standard Metabolite (Native) (eV a) Toxics 2019, 7, x FOR PEER REVIEW 3 of 8 Diisoheptyl MHHpP d4-MHHpP 279.1/121.03 15 phthalate MOHpP d -MOHpP 277.1/121.03 16 Cartridge; Merck KGaA, Darmstadt, Germany)4 and chromatographically resolved using high- (DHpP) MCHxP d -MCHxP 293.1/145.09 16 performance liquid chromatography (Thermo4 Scientific Betasil phenyl, 3 μm, 150 mm × 2.1 mm a Collisioncolumn) energy(Figure applied2). We then in QExactiveused high-resolution high-resolution mass spectrometry mass spectrometer in negative inparallel ion parallel reaction reaction mode. mode on a QExactive high-resolution mass spectrometer for quantification of the analytes (Table 1). We used the lowest calibration level (0.013 ng/mL) as the limit of detection (LOD).

Figure 2. ChromatographicFigure 2. Chromatographic separation separation and and mass spectr spectraa of diisoheptyl of diisoheptyl phthalate phthalate metabolites. metabolites.

Table 1. Analytical parameters for the quantification of diisoheptyl phthalate metabolites. We also compared the urinary MHHpP concentrations to the hydroxylated metabolites of dibutyl Parent Chemical Urinary Metabolite Internal Standard MS/MS Scan (Native) Collision Energy (eVa) phthalate, ,MHHpP and di-2-ethylhexyld4-MHHpP phthalate279.1/121.03 concentrations in human 15 urine reported Diisoheptyl phthalate MOHpP d4-MOHpP 277.1/121.03 16 in the 2015–2016(DHpP) National Health and Nutrition Examination Survey [19]. MCHxP d4-MCHxP 293.1/145.09 16 We used SASa Collision (version energy applied 9.4;SAS in QExactive Institute high-resolut Inc.,ion Cary, mass spectrometer NC, USA) in parallel to perform reaction mode. statistical analyses. For metabolite concentrationsWe also compared belowthe urinary the MHHpP LOD, weconcentr imputedations to a valuethe hydroxylated equal to metabolites the LOD dividedof by the square root ofdibutyl 2 [20 phthalate,]. Statistical diisobutyl significance phthalate, and was di-2-e setthylhexyl at Pearson phthalate correlation concentrations coe in ffihumancient urine (p) < 0.05. reported in the 2015–2016 National Health and Nutrition Examination Survey [19]. 3. Results and DiscussionWe used SAS (version 9.4; SAS Institute Inc., Cary, NC, USA) to perform statistical analyses. For metabolite concentrations below the LOD, we imputed a value equal to the LOD divided by the DiHpPsquare has been root of used 2 [20]. inStatistical vinyl significance , includingwas set at Pearson flooring, correlation but coefficient no data (p are) < 0.05. available on human exposure. In3. Results this proof-of-concept and Discussion study, we report the urinary concentrations of three oxidative metabolites of DiHpPDiHpP has (MHHpP, been used in MOHpP, vinyl plastics, and including MCHxP). flooring, The but urineno data wasare available collected on human during 2000 and 2018–2019 fromexposure. convenience In this proof-of-concept samples of study, U.S. adultswe report not the knownurinary concentrations to be occupationally of three oxidative exposed. We used high-resolutionmetabolites mass of spectrometry DiHpP (MHHpP, to MOHpP, resolve and analytes MCHxP). from The urine isobaric was collected interferences, during 2000 which and allowed us 2018–2019 from convenience samples of U.S. adults not known to be occupationally exposed. We to quantify theused threehigh-resolution metabolites mass atspectrometry sub-parts to perresolve billion analytes concentrations. from isobaric interferences, Because DiHpPwhich consists of multiple isomersallowed withus to quantify similar the physical three metabolites and chemical at sub-parts properties,per billion concentrations. metabolites Because were DiHpP eluted as broad HPLC peaksconsists with of similar multiple mass isomers spectrometric with similar physical transitions, and chemical as properties, reported metabolites previously were foreluted other isomeric as broad HPLC peaks with similar mass spectrometric transitions, as reported previously for other phthalate mixturesisomeric phthalate [21,22]. mixtures [21,22]. Table2 lists geometric means (GM), select percentile concentrations, and detection frequencies of

DiHpP metabolites in urine. We detected MCHxP in all the urine samples tested. MHHpP was also detected in the 2018–2019 (100%) and 2000 (96%) samples. MOHpP was detected less frequently, at 57% in the 2018–2019 samples and 92% in the 2000 samples. The frequent detection of these metabolites in the 2018–2019 samples suggests recent exposure to DiHpP. Toxics 2019, 7, 53 4 of 8

Table 2. Selected percentiles of urinary concentrations (95% CI) of three oxidative metabolites (ng/mL) of diisoheptyl phthalate in a convenience sample of U.S. adults.

Urinary Percentile Geometric Frequency of Collection Year N Metabolite 25th 50th 75th 90th Mean, ng/mL Detection (%) 2018–2019 205 0.25 (0.19, 0.29) 0.55 (0.4, 0.72) 1.71 (1.31, 2.2) 4.88 (3.39, 7.07) 0.59 (0.5, 0.7) 100 MHHpP 2000 144 0.16 (0.13, 0.21) 0.44 (0.34, 0.57) 1.04 (0.87, 1.53) 1.99 (1.7, 2.4) 0.38 (0.31, 0.46) 96 0.02 (

TheThe highest highest concentrations concentrations were were for MCHxP, for MCHxP, followed followed by MHHpP by MHHpPand MOHpP. and However, MOHpP. However,MCHxP is MCHxP not a DiHpP-specific is not a DiHpP-specific metabolite. metabolite.MCHxP can also MCHxP be produced can also by be other produced high-molecular- by other high-molecular-weightweight phthalates [23], phthalateswhereas MHHpP [23], whereas and MOHp MHHpPP are and specific MOHpP biomarkers are specific for DiHpP. biomarkers The GM for DiHpP.(95% CI) The concentrations GM (95% CI) of concentrations MOHpP (0.19 of (0.16, MOHpP 0.24) (0.19vs. 0.03 (0.16, (0.03, 0.24) 0.04) vs. 0.03ng/mL (0.03, for 0.04)2000 ngand/mL 2018– for 20002019, and respectively) 2018–2019, and respectively) MCHxP (2.0 and (1.66, MCHxP 2.43) (2.0vs. 1.31 (1.66, (1.15, 2.43) 1.50) vs. 1.31ng/mL (1.15, for1.50) 2000 ngand/mL 2018–2019, for 2000 andrespectively) 2018–2019, were respectively) higher in samples were higher collected in samples in 2000 collected than in 2018–2019 in 2000 than (Table in 2018–2019 2). In contrast, (Table 2the). InGM contrast, concentrations the GM concentrations of MHHpP were of MHHpPhigher in were samples higher collected in samples in 2018–2019 collected (0.59 in 2018–2019 (0.50, 0.70) (0.59 ng/mL) (0.50, 0.70)than ngin /2000mL) than(0.38 in(0.31, 2000 0.46) (0.38 ng/mL), (0.31, 0.46) perhaps ng/mL), because perhaps of differences because of diinff theerences formulations in the formulations before and beforeafter manufacturing and after manufacturing changes for changesDiHpP in for the DiHpP United in States. the United Although, States. the Although, production the of productionDiHpP was of DiHpP was discontinued in 2010 [1], the use of DiHpP in C6–C8,C7–C9, and C6–C10 and other discontinued in 2010 [1], the use of DiHpP in C6–C8, C7–C9, and C6–C10 and other commercial commercialphthalate mixtures phthalate [1] mixtures might have [1] mightcontributed have contributedto exposure to in exposure later years. in later years. AsAs expected,expected, thethe correlationcorrelation analysisanalysis showedshowed statisticallystatistically significantsignificant correlationscorrelations ((pp << 0.001)0.001) between the log10-transformed concentrations of MHHpP and MOHpP (Figure3). The correlation between the log10-transformed concentrations of MHHpP and MOHpP (Figure 3). The correlation waswas moremore significantsignificant inin samplessamples collectedcollected inin 20002000 (correlation(correlation coecoefficientfficient (r),(r), (95%(95% CI)CI) == 0.940.94 (0.91,(0.91, 0.95))0.95)) thanthan in samples collected collected in in 2018–2019 2018–2019 (r (,r (95%, (95% CI) CI) = 0.59= 0.59 (0.49, (0.49, 0.67)) 0.67)) (Figure (Figure 3).3 To). Toexplain explain this thisfinding, finding, we wehypothesize hypothesize that that phthalate phthalate formulations formulations used used before before the the 2010 2010 ending ending of DiHpPDiHpP productionproduction inin thethe USAUSA maymay havehave includedincluded aa largerlarger percentagepercentage ofof DiHpPDiHpP isomersisomers withwith straight-chainstraight-chain C-backbone,C-backbone, wherewhere oxidationoxidation toto formform MOHpPMOHpP cancan readilyreadily occur.occur. UrinaryUrinary concentrationsconcentrations ofof MHHpPMHHpP alsoalso correlated with those those of of MCHxP MCHxP (r (, r(95%, (95% CI) CI) = 0.87= 0.87 (0.83, (0.83, 0.91), 0.91), 0.49 0.49(0.38, (0.38, 0.59) 0.59)for 2000 for and 2000 2018– and 2018–2019,2019, respectively respectively (p < 0.001)) (p < 0.001)) (Figure (Figure 3), suggesting3), suggesting DiHpP DiHpP as the as primary the primary source source for MCHxP for MCHxP in these in thesesamples. samples.

100 2018-2019 2018-2019 1

10

0.1 1

0.01 0.1 Urinary concentration of MOHpP, ng/mL MOHpP, of concentration Urinary Urinary concentration of MCHxP, ng/mL MCHxP, of concentration Urinary 0.001 0.01 0.0 0.0 0.1 1.0 10.0 100.0 0.0 0.0 0.1 1.0 10.0 100.0

Urinary concentration of MHHpP, ng/mL Urinary concentration of MHHpP, ng/mL

10 100 2000 2000

1 10

0.1 1

0.01 0.1 Urinary concentration of MOHpP, ng/mL MOHpP, of concentration Urinary Urinary concentrationUrinary of ng/mL MCHxP, 0.001 0.01 0.0 0.1 1.0 10.0 0.0 0.1 1.0 10.0 Urinary concentration of MHHpP, ng/mL Urinary concentration of MHHpP, ng/mL

Figure 3. Correlation analyses of urinary concentrations of the metabolites of diisoheptyl phthalate Figure 3. Correlation analyses of urinary concentrations of the metabolites of diisoheptyl phthalate (only the concentrations above the limits of detection are shown). (only the concentrations above the limits of detection are shown). The detection of DiHpP metabolites among a diverse group of U.S. adults suggests exposure The detection of DiHpP metabolites among a diverse group of U.S. adults suggests exposure to to DiHpP in the United States. However, the concentrations of the metabolites were lower than DiHpP in the United States. However, the concentrations of the metabolites were lower than most most other phthalate metabolites detected in human urine reported from the 2015–2016 National other phthalate metabolites detected in human urine reported from the 2015–2016 National Health Health and Nutrition Examination Survey (Figure4)[ 19]. These pilot data suggest that the DiHpP and Nutrition Examination Survey, suggesting comparatively low exposures to DiHpP (Figure 4) [19]. These pilot data suggest that the DiHpP metabolites (MHHpP, MOHpP, and MCHxP) can serve as biomarkers of exposure to DiHpP in large-scale biomonitoring studies.

Toxics 2019, 7, 53 6 of 8 metabolites (MHHpP, MOHpP, and MCHxP) can serve as biomarkers of exposure to DiHpP in large-scaleToxics 2019, 7, biomonitoringx FOR PEER REVIEW studies. 7 of 8

6

Geometric mean 5 Median

4

3

2

1 Urinary hydroxy metabolite, ng/mL 0 MHBP MHiBP MHHpP MEHHP

Figure 4. GeometricGeometric mean and median median concentrations of the hydroxy metabolites of (DBP), diisobutyl phthalate (DiBP), and di-2-ethylhexyl phthalate (DEHP) in urine samples from (DBP), diisobutyl phthalate (DiBP), and di-2-ethylhexyl phthalate (DEHP) in urine samples from the the 2015–2016 National Health and Nutrition Examination Survey (NHANES) compared with 2015–2016 National Health and Nutrition Examination Survey compared with MHHpP (current MHHpP (2018–2019 current study). Monohydroxybutyl phthalate (MHBP)-metabolite of DBP; study). Monohydroxybutyl phthalate (MHBP)-metabolite of DBP; monohydroxy-isobutyl phthalate monohydroxy-isobutyl phthalate (MHiBP)-metabolite of DiBP; mono-2-ethyl-5-hydroxyhexyl phthalate (MHiBP)-metabolite of DiBP; mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP)-metabolite of (MEHHP)-metabolite of DEHP. DEHP. 4. Disclaimer 4. Disclaimer The use of trade names is for identification purposes only and does not constitute endorsement by the U.S.The Department use of trade of names Health is andfor identification Human Services purp oroses the Centersonly and for does Disease not constitute Control and endorsement Prevention. Theby the findings U.S. Department and conclusions of Health in this and report Human are those Services of the or authors the Centers and do for not Disease necessarily Control represent and thePrevention. views of The the findings Centers forand Disease conclusions Control in this and report Prevention. are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. Author Contributions: Conceptualization, methodology and validation, M.J.S.; formal data analysis, L.-Y.W., sampleReferences analysis M.J.S., J.L.P., E.O., and E.S.; investigation, writing, review and editing, M.J.S., J.C.B., and A.M.C. Funding: This research received no external funding. 1. CPSC. CPSC staff toxicity review of 17 phthalates for consideration by the chronic hazard advisory panel - Conflicts2010. of Available Interest: Authorsonline: https://cpsc.gov/s3fs declare no conflict of-public/CPSCStaffToxicity17Phthalates.pdf interest. (accessed on 10 October 2019). References2. CPSC. Toxicity review of diisoheptyl phthalate (dihp). Available online: 1. https://www.cpsc.gov/Global/Research-and-Statistics/Technical-CPSC. CPSC Staff Toxicity Review of 17 Phthalates for Consideration by the Chronic Hazard Advisory Reports/Chemical/PhthalatPanel - 2010. Available online:es/ToxicityReviewOfDiHP.pdf https://cpsc.gov/s3fs-public (accessed/CPSCSta on 11ffToxicity17Phthalates.pdf September 2019). (accessed on 3. 10Hannas, October B.R.; 2019). Lambright, C.S.; Furr, J.; Howdeshell, K.L.; Wilson, V.S.; Gray, L.E. Dose-response 2. assessmentCPSC. Toxicity of fetal Review testosterone of Diisoheptyl production Phthalate and (dihp).gene expression Available levels online: in https: rat testes//www.cpsc.gov following in/Global utero/ exposureResearch-and-Statistics to diethylhexyl/Technical-Reports phthalate, diisobutyl/Chemical phthal/ate,Phthalates diisoheptyl/ToxicityReviewOfDiHP.pdf phthalate, and diisononyl (accessed phthalate. on 11Toxicol. September Sci. 2011 2019)., 123, 206–216. 3.4. Jin,Hannas, M.L.; B.R.; Dewa, Lambright, Y.; Kawai, C.S.; M.; Furr, Nishimura, J.; Howdeshell, J.; Saeg K.L.;usa, Wilson, Y.; Kemmochi, V.S.; Gray, L.E.S.; Harada, Dose-response T.; Shibutani, assessment M.; Mitsumori,of fetal testosterone K. Induction production of liver andpreneoplastic gene expression foci in f344 levels rats in subj ratected testes to following 28-day oral in uteroadministration exposure toof diheptyldiethylhexyl phthalate phthalate, and its diisobutyl in vivo genotoxic phthalate, potential. diisoheptyl Toxicology phthalate, 2009 and, 264 diisononyl, 16–25. phthalate. Toxicol. Sci. 5. Nakane,2011, 123 F.;, 206–216. Kunieda, [CrossRef M.; Shimizu,][PubMed S.; Kobayashi,] Y.; Akane, H.; Akie Y.; Saito, A,; Noguchi, M.; Kadota, T.; 4. Mitsumori,Jin, M.L.; Dewa, K. Twenty-six-week Y.; Kawai, M.; oral Nishimura, toxicity of J.; diheptyl Saegusa, phthalate Y.; Kemmochi, with special S.; Harada, emphasis T.; on Shibutani, its induction M.; ofMitsumori, liver proliferative K. Induction lesions of liverin male preneoplastic F344 rats. J.foci Toxicol. in f344 Sci.rats 2012 subjected;37(3):527-37. to 28-day oral administration of 6. Lambright,diheptyl phthalate C.; Howdeshell, and its in vivoK.; Furr, genotoxic J.; Gray, potential. L.; WilsToxicologyon, V. In 2009utero, 264exposure, 16–25. to [CrossRef di-isoheptyl][PubMed phthalate] (DIHP) reduces testicular testosterone (T) production in fetal sprague dawley (SD) rats. Biol. Reprod. 2008, 547. 7. McKee, R.H.; Pavkov, K.L.; Trimmer, G.W.; Keller, L.H.; Stump, D.G. An assessment of the potential developmental and reproductive toxicity of di-isoheptyl phthalate in rodents. Reprod. Toxicol. 2006, 21, 241– 252.

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