Episode 147 | Anny Aurora, Niki - Surprise For Three (2018) 1080p | Knowing Brother Episode 89

Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring

Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring

G Model TOXLET 8769 No. of Pages 7 Toxicology Letters xxx (2014) xxx–xxx Contents lists available at ScienceDirect Toxicology Letters journal homep...

686KB Sizes 0 Downloads 0 Views

Recommend Documents

Plasma levels and urinary excretion of disodium cromoglycate after inhalation by human volunteers
The plasma levels, rate of urinary excretion, total urinary excretion and the amount deposited in the mouth were measure

Di-(2-propylheptyl) phthalate (DPHP) and its metabolites in blood of rats upon single oral administration of DPHP
Di-(2-propylheptyl) phthalate (DPHP) does not act as a reproductive toxicant or endocrine disruptor in contrast to other

Urinary excretion of ethylenethiourea in five volunteers on a controlled diet (multicentric study)
Urinary excretion of ethylenethiourea (ETU) was monitored for 8 days in a group of five male non-smoker volunteers on a

Urinary biomarkers of di-isononyl phthalate in rats
Commercial di-isononyl phthalate (DiNP) is a mixture of various branched-chain dialkyl phthalates mainly containing nine

Correction and comparability of phthalate metabolite measurements of Canadian biomonitoring studies (2007–2012)
•3 Biomonitoring studies were affected by inaccurate phthalate metabolite measurements.•Differences in the accuracy of t

Urinary monoamine metabolite excretion in disorders of movement
We have studied the urinary excretion of 1,4-methylhistamine (1,4-MeHm), 5-hydroxyindole-3-acetic acid (5-HIAA) and homo

Pregnancy urinary phthalate metabolite concentrations and gestational diabetes risk factors
•Higher 2nd trimester MEP concentrations were associated with a higher risk of IGT and excessive GWG•Higher 2nd trimeste

Urinary phthalate metabolite concentrations in relation to history of infertility and use of assisted reproductive technology
To examine urinary phthalate metabolite concentrations in pregnant women with planned pregnancies in relation to history

Human metabolism and excretion kinetics of the fragrance 7-hydroxycitronellal after a single oral or dermal dosage
7-Hydroxy-3,7-dimethyl-1-octanal, also known as 7-hydroxycitronellal (7-HC, CAS No. 107-75-5) is a synthetic fragrance w

Biomonitoring of urinary di(2-ethylhexyl) phthalate metabolites of mother and child pairs in South Korea
Di(2-ethylhexyl) phthalate (DEHP) is one of the common phthalate plasticizers used primarily in soft polyvinyl chloride,

G Model TOXLET 8769 No. of Pages 7

Toxicology Letters xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring Gabriele Leng a, *, Holger M. Koch b , Wolfgang Gries a , Andre Schütze b , Angelika Langsch c , Thomas Brüning b , Rainer Otter c a b c

Currenta GmbH & Co. OHG, Institute of Biomonitoring, Leverkusen, Germany Institute for Prevention and Occupational Medicine – Institute of the Ruhr-Universität Bochum, Bochum, Germany BASF SE, Ludwigshafen, Germany

H I G H L I G H T S

 Urinary excretion of three specific, secondary, oxidized metabolites (oxo-MPHP, OH-MPHP and cx-MPHxP) of DPHP was monitored following oral uptake by five volunteers.  Urinary elimination half-lives for these metabolites are between 6 and 8 h.  22.9% of the DPHP dose is excreted as one of the above three metabolites within 24 h, until 48 h post dose an additional 1–2% is excreted.  Based upon molar excretion fractions the DPHP intake of the general public and of workers can be calculated from urinary metabolite levels.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 18 March 2014 Received in revised form 30 May 2014 Accepted 23 June 2014 Available online xxx

Di(2-propylheptyl) phthalate (DPHP), a high molecular weight phthalate, is primarily used as a plasticizer in polyvinyl chloride and vinyl chloride copolymers for technical applications, as a substitute for other phthalates currently being scrutinized because of endocrine disrupting effects. We determined urinary excretion fractions of three specific DPHP metabolites (mono-2-(propyl-6hydroxy-heptyl)-phthalate (OH-MPHP), mono-2-(propyl-6-oxoheptyl)-phthalate (oxo-MPHP) and mono-2-(propyl-6-carboxy-hexyl)-phthalate (cx-MPHxP)) after oral dosing of five volunteers with 50 mg labelled DPHP-d4 and subsequent urine sampling for 48 h. These excretion fractions are used to back calculate external intakes from metabolite measurements in spot urine analysis. Following enzymatic hydrolysis to cleave possible conjugates, we determined these urinary metabolites by HPLC– NESI–MS/MS with limits of quantification (LOQ) between 0.3 and 0.5 mg/l. Maximum urinary concentrations were reached within 3–4 h post dose for all three metabolites; elimination half-lives were between 6 and 8 h. We identified oxo-MPHP as the major oxidized metabolite in urine representing 13.5  4.0% of the DPHP dose as the mean of the five volunteers within 48 h post dose. 10.7  3.6% of the dose was excreted as OH-DPHP and only 0.48  0.13% as cx-MPHxP. Thus, within 48 h, 24.7  7.6% of the DPHP dose was excreted as these three specific oxidized DPHP metabolites, with the bulk excreted within 24 h post dose (22.9  7.3%). These secondary, oxidized metabolites are suitable and specific biomarkers to determine DPHP exposure. In population studies, however, chromatographic separation of these metabolites from other isomeric diisodecyl phthalate (DIDP) metabolites is warranted (preferably by GC–MS) in order to distinguish DPHP from general DIDP exposure. Palatinol1, Hexamoll1 and DINCH1 are registered trademarks of BASF SE, Germany. ã 2014 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Keywords: Biomonitoring Di(2-propylheptyl)phthalate DPHP metabolites Urinary excretion Human volunteer study HPLC–NESI–MS/MS

* Corresponding author at: Currenta GmbH & Co. OHG, Institute of Biomonitoring, Chempark Leverkusen, Building L 9, 51368 Leverkusen, Germany. Tel.: +49 2143065679; fax: +49 2143021307. E-mail address: [email protected] (G. Leng). http://dx.doi.org/10.1016/j.toxlet.2014.06.035 0378-4274/ ã 2014 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035

G Model TOXLET 8769 No. of Pages 7

2

G. Leng et al. / Toxicology Letters xxx (2014) xxx–xxx

1. Introduction Di(2-propylheptyl) phthalate (DPHP), CAS No. 53,306-54-0, a REACH (Regulation (EC) No. 1907/2006) registered high molecular weight phthalate, is primarily used as a plasticizer in polyvinylchloride and vinyl chloride copolymers for technical applications. DPHP, which is marketed under, e.g., the trade name “Palatinol1 10-P”, is produced by esterification of phthalic anhydride with a C10 alcohol consisting of 90% 2-propyl-heptanol and 10% 2-propyl4-methylhexanol or 2-propyl-5-methylhexanol. There are currently two different C10 phthalates on the market. DPHP and diisodecyl phthalate (DIDP) as described with the CAS No. 68,515-491: 1,2-benzenedicarboxylic acid, di-C9-11-branched alkyl esters, C10-rich. Another DIDP described by CAS No. 26,761-40-0 is no longer produced in Europe and is not REACH registered. Furthermore, there are two C9 phthalates (di-isononyl phthalates, DINPs) on the market: DINP1 (1,2-benzenedicarboxylic acid, di-C8-10branched alkyl esters, C9-rich, described with CAS No. 68,515-48-0 and DINP2 (di-isononyl phthalate) with CAS No. 28,553-12-0. While DINP2 solely consists of C9 isomers DINP1 contains up to 10% C10 isomers. Thus, the broad isomer distribution of DINP1 (including C10 moieties) can also interfere with the analytical detection of both DIDP and DPHP. The lack of sufficient analytical separation of DINP and DIDP resulted in a group-TDI by EFSA (EFSA, 2005) for food contact applications (Commission Regulation (EU) No. 10/2011). The phthalates DINP, DPHP and DIDP are currently used as substitutes for di-(2-ethylhexyl) phthalate (DEHP) which is listed under REACH as a substance of very high concern (SVHC). Based on their low volatility and low vapor pressure, the C10 phthalates DPHP and DIDP are predominantly used in high temperatureresistant products such as electrical cables, carpet backing and car interiors, but they are also used for outdoor applications like roofing membranes or tarpaulins (European Commission, 2003; NICNAS, 2003, 2008; Wittassek, 2008). DPHP is currently not used in food contact. Because plasticizers are not chemically bound in PVC products and thus can migrate out of these products, exposure of humans and the environment is possible. Therefore, a

collaborative project between the German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association (VCI) evaluated a specific human biomonitoring method to determine exposure of the general population to DPHP using reliable and specific urinary biomarkers (Federal Ministry for the Environment, 2010). We recently developed such a method for DINCH1 (di-isononyl-cyclohexane-1,2-dicarboxylate), a non-aromatic high molecular weight phthalate substitute mainly intended for sensitive applications such as toys, food contact materials and medical devices (Koch et al., 2013a,b; Schütze et al., 2012, 2014). For DPHP, however, exposure needs to be distinguishable from DIDP/DINP exposure. Previous exposure assessments based on human biomonitoring have reported the cumulative exposure (Kasper-Sonnenberg et al., 2012; Koch et al., 2009) to all phthalates containing C10 alkyl chains (DPHP, DINP, DIDP), because the complex isomeric composition of DINP/DIDP interfered with the selective detection of the DPHP specific 2-propyl-heptyl based side chain metabolites. We used the method developed by Gries et al. (2012) to reliably detect and quantify DHPH metabolites in the presence of other DIDP/DINP metabolites. Wittassek and Angerer, 2008 showed that DPHP is metabolized similarly to DEHP (Koch et al., 2004), i.e., the monoester is formed by ester cleavage in a first step followed by extensive v and v-1 oxidation of the remaining single alkyl side chain. A metabolism scheme of DPHP is presented in Fig. 1. The secondary, oxidized metabolites are the predominant metabolites. The monoester MPHP is only a minor metabolite (<1% formed from the parent compound and excreted with urine), which is typical for all high molecular weight phthalates. The secondary metabolites have an added analytical benefit in that they are not subject to issues of sample contamination as described by Kato et al. (2004) and Schindler et al. (2014). We investigated renal excretion and metabolic conversion of DPHP by measuring three oxidized metabolites of the propylheptyl side-chain, mono(propyl-6-oxo-heptyl) phthalate (oxo-MPHP), mono(propyl-6-hydroxyheptyl) phthalate (OH-MPHP) and mono (propyl-6-carboxyhexyl)- phthalate (cx-MPHxP) following oral

Fig. 1. Proposed human metabolism of DPHP, based upon its major 2-propylheptyl alkyl-chain isomer. The 2-propyl-heptyl side chain makes up about 90% the DPHP side chains; the remainder is made up of 2-propyl-4-methylhexyl and 2-propyl-5-methylhexyl side chains. The stars (*) depict the positions of the deuterium label.

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035

G Model TOXLET 8769 No. of Pages 7

G. Leng et al. / Toxicology Letters xxx (2014) xxx–xxx

dosing of stable isotope (deuterium) labeled DPHP-d4 to five male volunteers. The fraction of excreted metabolite is used to determine conversion factors which enable the back calculation of the (daily) intake of DPHP (external dose) as described by Kohn et al. (2000) and David (2000). 2. Materials and methods 2.1. Experimental design Di(2-propylheptyl) phthalate (DPHP) was orally dosed as ringdeuterated DPHP-d4 to five healthy male volunteers, aged between 27 and 49 years, with body weights between 77 and 94 kg. The volunteers did not have any known occupational exposure to DPHP or to other plasticizers. Fifty milligram of DPHP-d4 was dissolved in 0.25 ml of ethanol and mixed in an edible waffle cup with a chocolate surface containing coffee or tea during breakfast. This resulted in doses for the five individuals of between 0.54 and 0.66 mg/kg body weight. The DPHP dose was considerably below the lowest NOAEL (no observed adverse effect level) for DPHP (BfR Opinion No., 2011; Bhat et al., 2014) and comparable to the DINP (Koch and Angerer, 2007) or DINCH1 dose levels (Schütze et al., 2014) of previous human metabolism studies. The DPHP dose was several orders of magnitude above exposure levels expected for the general population. Stable-isotope labeled DPHP-d4 was used to exclude possible background exposures. Volunteers were dosed at the Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr-Universität Bochum (IPA), frozen samples of urine were shipped to Currenta for quantification of the metabolites. The first urine samples were collected prior to dosage at 10:00 a.m. followed by subsequent urine samples collected over 48 h post-dosing. The volunteers recorded the time of the void of each sample. The urine volume of each individual sample was determined as the difference between the weight of the filled and the empty container. In all, we obtained 122 urine samples, i.e., between 20 and 29 samples from each volunteer. The total 48 h urine volume ranged from 4133 to 8298 ml, depending on the volunteer. All urinary samples were frozen at 18  C immediately after delivery. The study was carried out in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) and was approved by the ethical review board of the Medical Faculty of the Ruhr-University Bochum (Reg. No.: 4022-11). The study design was presented to the volunteers in written form, and all participants provided written informed consent. 2.2. Chemicals Acetonitrile (supra solv), methanol (supra solv), glacial acetic acid (p.a.) and hydrochloric acid 37% (p.a.) were purchased from Merck, Darmstadt, Germany. Ammonium acetate (p.a.) was purchased from Fluka, Taufkirchen, Germany. Formic acid (99%, ULC/MS) was purchased from Biosolve B.V., Valkenswaard, The Netherlands. Water from a millipore water cleaning system was used and b-glucuronidase from Escherichia coli K12 was purchased from Roche, Mannheim, Germany. DPHP-d4 was provided by BASF SE. The following standards were synthesized at the Institut für Dünnschichttechnologie e.V. (IDM), Teltow, Germany: mono-2-(propyl-6-hydroxy-heptyl)-phthalate (OH-MPHP), mono2-(propyl-6-oxo-heptyl)-phthalate (oxo-MPHP), mono-2-(propyl6-carboxy-hexyl)- phthalate (cx-MPHxP), mono-2-(propyl-6-hydroxy-heptyl)-phthalate-d4 ring deuterated (OH-MPHP-d4), mono-2-(propyl-6-oxo-heptyl)-phthalate-d4 ring deuterated (oxo-MPHP-d4), and mono-2-(propyl-6-carboxy-hexyl)-phthalate-d4 ring deuterated (cx-MPHxP-d4). The purity of all compounds was determined by 1H-NMR and was 95%.

3

2.3. Standards The study design is based on dosing of ring labeled DPHP-d4; therefore, the target substances were the respective d4-ring labeled metabolites. Ten milligram of each metabolite (OH-MPHPd4, oxo-MPHP-d4 or cx-MPHxP-d4) were weighed separately into a 10 ml glass volumetric flask and diluted to volume with acetonitrile (1000 mg/l). From these stock solutions, a multicomponent starting solution was prepared by diluting 100 ml of each in a 10 ml glass volumetric flask filled with acetonitrile. This starting solution (10 mg/l) was further diluted for the preparation of the working standards to achieve final standard concentrations of 1 mg/l, 0.1 mg/l, 0.01 mg/l and 0.001 mg/l. For the purpose of internal standardization, we used the nonlabeled DPHP metabolite standards. Internal standard stock solutions were prepared by dilution of 10 mg of OH-MPHP, oxoMPHP or cx-MPHxP in 10 ml volumetric flasks with acetonitrile (1000 mg/l). Starting solution A was prepared by diluting 100 ml of each of the three stock solutions into a 10 ml volumetric flask (10 mg/l) to the mark with acetonitrile. For the preparation of solution B 1 ml of solution A was diluted in a 10 ml volumetric flask to its nominal volume with acetonitrile (1 mg/l). 2.4. Sample preparation Urine samples (or standards) were thawed and equilibrated to room temperature. For enzymatic hydrolysis, 10 ml of b-glucuronidase and 20 ml of the internal standard solution in 200 ml 1 M ammonium acetate buffer (pH 6.5) were added to 1000 ml of each sample and mixed. Samples were incubated at 37  C overnight. Thereafter, all samples were acidified to pH 2 with hydrochloric acid (37%) and extracted with tert-butylmethylether, mixed with a vortex mixer for 10 min and centrifuged at 2200 g for 10 min at 10  C. The upper phase was aspirated with a Pasteur pipette and placed into a glass test tube, and the samples were dried at 35  C with nitrogen. All samples were re-dissolved in 200 ml of methanol for HPLC–MS/MS analysis. The creatinine concentration in each urine sample was measured according to the Jaffé method (Taussky, 1954). 2.5. High performance liquid chromatography–tandem mass spectrometry Chromatographic separation was performed on a Waters Alliance HPLC System equipped with a Zorbax Eclipse Plus C18 column (2.1 mm  150 mm  3.5 mm (Agilent)) at 30  C. A tertiary system (A: methanol, B: water and C: formic acid) was used to separate the metabolites with the following conditions: at start, 10 ml was injected onto the column with 10% A, 80% B and 10% C, flow was 0.2 ml/min and constant during the whole analysis which lasted 25 min. Metabolites were separated by an increasing methanol gradient, i.e., methanol (A) was increased from 10% to 90% within 15 min while water (B) was reduced to 0%. Solvents A

Table 1 LC–MS/MS retention times, mass transitions and dwell times for the labeled and non-labeled DPHP metabolites. Metabolite

cx-MPHxP cx-MPHxP-d4 OH-MPHP OH-MPHP-d4 oxo-MPHP oxo-MPHP-d4

Rt

Parent

(min)

(m/z)

19.83 19.81 20.15 20.13 19.65 19.60

335.16 339.12 321.13 325.16 319.12 323.15

Daughter

Dwell time (S)

187.04 187.04 121.02 125.04 121.02 124.98

0.1 0.1 0.1 0.1 0.1 0.1

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035

G Model TOXLET 8769 No. of Pages 7

4

G. Leng et al. / Toxicology Letters xxx (2014) xxx–xxx

Table 2 Within-day and between-day precision data. Analyte

Within-day precision (n = 8)

cx-MPHxP-d4

OH-MPHP-d4

oxo-MPHP-d4

Between-day precision (n = 5)

Conc. (mg/l)

Recov. (%)

R.S.D. (%)

Conc. (mg/l)

Recov. (%)

R.S.D. (%)

1 10 100

109 108 108

4.6 3.2 1.5



– 111 106

– 3.9 10.2

1 10 100

108 101 103

2.2 3.8 2.4



– 102 103

– 1.1 1.7

1 10 100

100 97 101

6.5 2.8 5.3



– 99 99

– 4.9 2.9

10 100

10 100

10 100

(90%) and B (0%) were kept constant for 2 min and then a gradient was used to reach 10% A and 80% B at 18 min. These conditions were kept for 7 min until 25 min when the analysis was finished. C was kept constant at 10% during the analysis. The mass spectrometric detection and quantification was performed on a Waters Quattro Ultima MS/MS with negative electrospray ionization (NESI) in MRM mode. Mass transitions are depicted in Table 1. Further details are given in Gries et al. (2012). 2.5.1. Calibration and quantification Calibration was carried out by spiking 1 ml of water with concentrations ranging from 0.1 mg/l to 5000 mg/l of each deuterated standard. All calibration samples were analyzed as described in the sample section. Due to the high dynamic range of the HPLC–MS/MS a calibration range up to 5000 mg/l of each metabolite can be obtained in case a quadratic curve fit is used (coefficient of correlation better than 0.99 for each analyte). The wide calibration range was desirable for the determination of some

B: post - dose sample

A: pre - dose sample Relative abundancy [%]

100

100

OH-MPHP (ISTD)

OH-MPHP (ISTD)

OH-MPHP-d4 (91.4 µg/l) OH-MPHP-d4 (n.d.) 0

16.00

18.00

20.00

22.00

24.00

Relative abundancy [% %]

100

16.00

18.00

20.00

22.00

24.00

100

oxo-MPHP (ISTD)

oxo-MPHP ((ISTD))

oxo-MPHP-d4 (122.9 µg/l) oxo-MPHP-d4 (n d ) (n.d.) 0

16.00

18.00

20.00

22.00

24.00

0

16.00

18.00

20.00

cx-MPHxP (ISTD)

24.00

cx-MPHxP (ISTD)

cx-MPHxP-d4 (10.6 µg/l)

cx-MPHxP-d4 (n.d.) 0

22.00

100

100

Relative abundan ncy [%]

0

16 00 16.00

18 00 18.00

20 00 20.00

22 00 22.00

24.00 00

0

16 00 16.00

18.00 00

20 00 20.00

22 00 22.00

24 00 24.00

Time [min] Fig. 2. Chromatograms of a representative pre-dose (A) and post-dose (B) urine sample with extracted ion chromatograms for the three oxidized DPHP metabolites. The dotted line represents the non-labeled internal standards, the bold line the D4-labeled metabolites generated by the oral dose of D4-labeled DPHP.

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035

G Model TOXLET 8769 No. of Pages 7

G. Leng et al. / Toxicology Letters xxx (2014) xxx–xxx

high metabolite concentrations expected in this dosing study. Samples with concentrations above the calibration range were analyzed again after sample dilution with water. The calibration curves were obtained by plotting the quotient of the peak areas of the target deuterated analytes and the corresponding unlabeled internal standards against the standard concentrations. 2.5.2. Quality control and validation As quality control samples were not available, they had to be prepared in the laboratory with spiked urine samples to cover different concentration ranges (1 mg/l, 10 mg/l or 100 mg/l of each labeled metabolite). One millilitre aliquots of these control samples were stored frozen at 18  C. Two samples with either 10 or 100 mg/l concentration of each deuterated standard were analyzed during the analysis sequences for each volunteer on five different days to determine between day precision data. The within-day precision was obtained by analyzing pooled urine samples in three concentrations of each deuterated standard as described above. These samples were analyzed eight times in a row and all samples were quantified against the calculated calibration curve. Moreover, the background of unlabeled DIDP/DPHP metabolites in the samples was tested in several experiments. As there was no significant interfering DIDP/DPHP background observed in the dosing samples (DIDP/DPHP metabolite levels were consistent below 2 mg/l), the samples were spiked with 200 mg/l of each unlabeled DPHP metabolite as internal standards. Quality control data (relative recovery, precision), depicted in Table 2, was acceptable and comparable to that of Gries et al. (2012).

5

with spiked internal standard concentrations at 200 mg/l, the omnipresent but low background exposure to DIDP/DPHP did not interfere with the study design. Elimination kinetics could be monitored and specific metabolic conversion factors could be established. In the chromatograms of Fig. 2B, additional peaks with same fragmentation patterns as the propylheptyl derived oxidized standards emerged, albeit at different retention times. These peaks most likely originate from the minor alkyl chain isomers of DPHP (2-propyl-4-methylhexyl or 2-propyl-5-methylhexyl side chain) and/or from oxidative modifications other than in the v- or v-1position. All further quantitative data are based on the sole integration of the specific propylheptyl derived oxidized isomer peaks present as analytical standard substances. The elimination of these specific DPHP metabolites in urine over time (48 h) for the five volunteers is shown in Fig. 3A (in mg/l), B (in mg/g creatinine) and C (absolute amount in mg), calculated for 6 h increments. All forms of presentation clearly depict the rapid appearance of all three DPHP metabolites in urine after oral dosage.

2.5.3. Limit of detection (LOD) and limit of quantification (LOQ) Detection limits were calculated according to the calibration curve method (DIN 32645) by use of the six lowest calibration points. LODs were 0.1 mg/l for cx-MPHxP-d4 and 0.2 mg/l for OHMPHP-d4 and oxo-MPHP-d4. The corresponding LOQs were 0.3 mg/ l, 0.5 mg/l and 0.5 mg/l for cx-MPHxP-d4, OH-MPHP-d4 and oxoMPHP-d4, respectively. 2.6. Statistical analysis Statistical analysis was carried out using Microsoft Excel 2010. Exponential regression modeling was used to calculate exponential functions for decreasing metabolite levels after cmax. C(t) is the time dependent concentration, whereas C0 is the maximum concentration. K is the metabolite specific renal excretion constant. Dt represents the time beginning from cmax till the end of sample collection. CðtÞ ¼ C 0  expðkDtÞ Furthermore, metabolic half-time is given by the natural logarithm of two over k (according to Clark and Smith, 1986). 3. Results The LOQs of the HPLC–MS/MS method applied were sufficiently low to quantify the d4-ring-labelled DPHP metabolites in all postdose urine samples obtained from this dosing study. LC–MS/MS chromatograms in Fig. 2A and B illustrate the appearance of the d4ring-labeled oxidized DPHP metabolites in the post-dose urine samples. In the pre-dose urine samples, no d4-ring-labelled DPHP metabolites could be detected. As explained above, we used nonlabeled propylheptyl derived DPHP metabolite standards for internal standardization. In some urine samples, a background trace level of isomeric, oxidized (non-labelled) DIDP metabolites was visible, but at levels much lower than the spiked DPHP standards (maximum concentrations of 2 mg/l, not shown). Thus,

Fig. 3. Elimination of the three oxidized DPHP metabolites in urine over time (48 h) for the five volunteers is shown in Fig. 3A (in mg/L), B (in mg/g creatinine) and C (absolute amount in mg), calculated for 6 h increments. The bold boxes represent mean values calculated over all five volunteers; the bars depict the ranges among the five volunteers.

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035

G Model TOXLET 8769 No. of Pages 7

6

G. Leng et al. / Toxicology Letters xxx (2014) xxx–xxx

Table 3 Elimination half-lives and times of maximum urinary excretion for the three oxidized DPHP metabolites after oral dosage (calculated from the five volunteers). Parameter

Mean tmax  SD

Mean t1/2  SD

(h) oxo-MPHP-d4 OH-MPHP-d4 cx-MPHxP-d4

3.65  1.31 3.65  1.31 4.05  1.39

6.51  1.64 6.87  1.63 8.16  0.67

SD: standard deviation.

Table 4 Molar urinary excretion fractions in % of oral dose (mean  standard deviation) for five volunteers). Time

cx-MPHxP-d4

(h)

(%)

0–24 0–48

0.42  0.11 0.48  0.13

OH-MPHP-d4

oxo-MPHP-d4

9.91  3.45 10.70  3.61

12.61  3.90 13.52  4.04

P

of 3 metab.

22.94  7.33 24.70  7.64

Both OH-MPHP and oxo-MPHP are clearly the predominant metabolites over cx-MPHxP which is excreted at considerably lower concentrations. All metabolites are excreted rather rapidly and steadily over the 48 h investigated. However, at 48 h post-dose, all three metabolites were still detectable. Based upon the creatinine corrected elimination curve (Fig. 3B), all three metabolites seem to follow a one-phasic elimination pattern. Times of maximum urinary excretion for the three oxidized DPHP metabolites and elimination half-lives calculated from the individual data of each of the five volunteers are depicted in Table 3. Molar excretion fractions in percent of the oral dose were calculated by using the respective molecular weights of the metabolites cx-MPHxP-d4 (340.39 g/mol), OH-MPHP-d4 (326.40 g/ mol), and oxo-MPHP-d4 (324.39 g/mol). Total excreted amount was estimated as the sum of all samples taken per volunteer and taking into account all three metabolites (see Table 4). Oxo-MPHP is the most abundant metabolite, representing in the mean over the five volunteers 13.5% of the oral DPHP dose in urine after 48 h, closely followed by OH-MPHP (10.7%). Cx-MPHxP (0.5%) is regarded as a minor metabolite. All three oxidized metabolites represent about 25% of the dose excreted in urine within 48 h. 4. Discussion Wittassek and Angerer (2008) reported the first results on human DPHP metabolism, when the senior author ingested a single DPHP dose of 98 mg during breakfast. In their pilot study they reported that after 61 h around 34% of the applied dose was excreted with urine as oxidized metabolites (including approx. 1% as the simple monoester). Taking into account that they included other metabolites with oxidative modifications and that their sampling time was longer, their data are consistent with the data of the study reported here. The data obtained for DPHP in this study is also consistent with human metabolism data for other high molecular weight phthalates like DEHP and DINP (Koch et al., 2005, 2007; Anderson et al., 2011; Kessler et al., 2012). Similar elimination half-lives were also calculated for all DPHP metabolites (6.51–8.16 h) compared with DEHP and DINP. They are in good accordance to the respective metabolite half-lives of DINP (4–8 h; Anderson et al., 2011) and DEHP (4.6–6.6 h; Kessler et al., 2012). For DEHP, the three main, oxidized metabolites excreted in urine represent about 38.6–57.8% of the oral dose, depending on

the study; for DINP, the three main oxidized metabolites excreted in urine represent about 29.8–37.5% of the dose, depending on the study. In all these studies, it was shown that an increasing alkyl chain length of the plasticizer results in a decreased formation of the simple monoester. Thus, for high molecular weight plasticizers, the simple monoester is not a relevant urinary metabolite. Furthermore, since the simple monoester is prone to external contamination, the oxidized metabolites have to be regarded as the most suitable biomarkers for monitoring exposure to high molecular weight phthalates in urine (Koch and Calafat, 2009). The metabolic conversion factors established in this study for DPHP based on the five male volunteers allow a reliable back calculation from urinary DPHP metabolite levels to external exposure, and thus enable a solid risk assessment of the human body burden for the general public as well as for individuals occupationally exposed. A reliable back-calculation to DPHP exposure, however, can only be performed, if the above secondary, oxidized DPHP metabolites are chromatographically separated from the oxidized metabolites of DIDP/DINP that are generally present in urine samples of the general population, due to the omnipresent DIDP/DINP exposures. Gries et al. (2012) have recently published a GC–HRMS methodology that can unambiguously and reliably quantify these oxidized DPHP metabolites, even in the presence of high DIDP/DINP body burdens. For 40 random spot urine samples, they reported a maximum urinary concentration of 0.93 mg/l oxo-MPHP. Most of the currently available human biomonitoring data (summarized e.g., in Wittassek et al., 2007, 2011; Koch and Calafat, 2009; Kasper-Sonnenberg et al., 2012) do not distinguish between oxidized C10 metabolites of DIDP/DINP and DPHP due to the limited chromatographic resolution of the HPLC–MS methodology applied. The C10-metabolite levels from these studies, however, indicate a cumulative C10-phthalate exposure (DINP/DIDP and DPHP) that is considerably higher than that for DPHP alone. Future studies using differential integration of specific DPHP metabolites next to the cumulative measurement of C10-phthalate metabolites have to confirm this finding. Conflict of interest None for all authors except for A. Langsch and R. Otter who both are employed by BASF SE, a producer of DPHP. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements The study was carried out as part of a ten-year project on human biomonitoring. The project is a cooperation agreed in 2010 between the Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the Verband der chemischen Industrie e.V. (German Chemical Industry Association – VCI); it is administered by the Federal Environment Agency (UBA). The study aims to characterize suitable biomarkers for human biomonitoring and to develop a new analytical method based upon these biomarkers and was funded by the German Chemicals Industry. Experts from government authorities, industry and science accompany the project in selecting substances and developing methods. References Anderson, W.A.C., Castle, L., Hird, S., Jeffery, J., Scotter, M.J., 2011. A twenty-volunteer study using deuterium labelling to determine the kinetics and fractional

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035

G Model TOXLET 8769 No. of Pages 7

G. Leng et al. / Toxicology Letters xxx (2014) xxx–xxx excretion of primary and secondary urinary metabolites of di-2-ethylhexylphthalate and di-iso-nonylphthalate. Food Chem. Toxicol. 49, 2022–2029 http:// dx.doi.org/10.1016/j.fct.2011.05.013. BfR Opinion No., 004/2012 of 28 June 2011, http://www.bfr.bund.de/cm/349/dphpdetected-in-toys-bfr-assessing-the-risk-of-the-softener.pdf. Bhat, V.S., Durham, J.L., Ball, G.L., English, J.C., 2014. Derivation of an oral reference dose (RfD) for the plasticizer, di-(2-propylheptyl) phthalate (Palatinol1 10-P), (in press). Clark, B., Smith, D.A. (Eds.), 1986. An Introduction to Pharmacokinetics. second ed. Blackwell Scientific, Oxford. Commission Regulation (EU) No 10/2011, Official Journal of the European Union L 12/1, 2011. David, R.M., 2000. Exposure to phthalate esters. Environ. Health Perspect. 108, A440. DIN 32645, 1994. Chemische Analytik, Nachweis-, Erfassungs- und Bestimmungsgrenze. German Institute of Standardisation. EFSA, 2005. Opinion of the Scientific Panel on Food Additives, flavourings, processing aids and materials in contact with food (AFC) on a request from the commission related to di-isodecyl phthalate (DIDP) for use in food contact materials, question no. EFSA-Q -2003-195 Adopted on 30 July 2005. European Commission, 2003. In: Munn, S.J., Allanou, R., Aschberger, K., Berthault, F., de Bruijn, J., Musset, C., O’Connor, S., Pakalin, S., Pellegrini, G., Scheer, S., Vegro, S. (Eds.), EUR 20785EN, European Union Risk Assessment Report, 1,2-Benzenedicarboxylic Acid, Di-C9-11-branched alkyl esters, C10-rich and Di-“isodecyl” Phthalate (DIDP), Volume 36. Luxembourg: Office for Official Publications of the European Communities. Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety, 2010. Press release No. 068/10, http://www.bmub.bund.de/en/bmub/ press-and-speeches/current-press-releases/detailansicht-en/artikel/environment-ministry-and-chemical-industry-association-start-of-cooperation-inhuman-biomonitoring/?tx_ttnews%5BbackPid%5D=1892&cHash=17bb2c3b58a4a9aec63190a69db9813d downloaded (07.03.14.). Gries, W., Ellrich, D., Küpper, K., Ladermann, B., Leng, G., 2012. Analytical method for the sensitive determination of major di-(2-propylheptyl)-phthalate metabolites in human urine. J. Chrom. B 908, 128–136 http://dx.doi.org/10.1016/j. jchromb.2012.09.019. Kasper-Sonnenberg, M., Koch, H.M., Wittsiepe, J., Wilhelm, M., 2012. Levels of phthalate metabolites in urine among mother–child-pairs – results from the Duisburg birth cohort study, Germany. Int. J. Hyg. Environ. Health 215, 373–382 doi:http://dx.doi.org/10.1016/j.ijheh.2006.11.008. Kato, K., Silva J:, M., Reidy, J.A., Hurtz, D., Malek, N.A., Needham, L.L., Nakazawa, H., Barr, D.B., Calafat, A.M., 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. Kessler, W., Numtip, W., Völkel, W., Seckin, E., Csanády, G.A., Pütz, C., Klein, D., Fromme, H., Filser, J.G., 2012. Kinetics of di(2-ethylhexyl) phthalate (DEHP) and mono(2-ethylhexyl) phthalate in blood and of DEHP metabolites in urine of male volunteers after single ingestion of ring-deuterated DEHP. Toxicol. Appl. Pharmacol. 264, 284–291. Koch, H.M., Bolt, H.M., Angerer, J., 2004. Di(2-ethylhexyl) phthalate (DEHP) metabolites in human urine and serum after a single oral dose of deuteriumlabelled DEHP. Arch. Toxicol. 78, 123–130. Koch, H.M., Bolt, H.M., Preuss, R., Angerer, J., 2005. New metabolites of di(2ethylhexyl)phthalate (DEHP) in human urine and serum after single oral doses

7

of deuterium-labelled DEHP. Arch. Toxicol. 79, 367–376 doi:http://dx.doi.org/ 10.1007/s00204-004-0642-4. Koch, H.M., Müller, J., Angerer, J., 2007. Determination of secondary, oxidised di-isononylphthalate (DINP) metabolites in human urine representative for the exposure to commercial DINP plasticizers. J. Chrom. B 847, 114–125 doi:http:// dx.doi.org/10.1016/j.jchromb.2006.09.044. Koch, H.M., Angerer, J., 2007. Di-iso-nonylphthalate (DINP) metabolites in human urine after a single oral dose of deuterium-labelled DINP. Int. J. Hyg. Environ. Health 210, 9–19. Koch, H.M., Calafat, A.M., 2009. Human body burdens of chemicals used in plastic manufacture. Phil. Trans. R. Soc. B. 364, 2063–2078. Koch, H.M., Schütze, A., Pälmke, C., Angerer, J., Brüning, T., 2013a. Metabolism of the phthalate substitute diisononyl-cyclohexane-1,2-dicarboxylate (DINCH) in humans after single oral doses. Arch. Toxicol. 87, 799–806 http://dx.doi.org/ 10.1007/s00204-012-0990-4. Koch, H.M., Lorber, M., Christensen, K.L.Y., Pälmke, C., Koslitz, S., Brüning, T., 2013b. Identifying sources of phthalate exposure with human biomonitoring: results of a 48 h fasting study with urine collection and personal activity patterns. Int. J. Hyg. Environ. Health 216, 672–681. Kohn, M.C., Parham, F., Masten, S.A., Portier, C.J., Shelby, M.D., Brock, J.W., Needham, L.L., 2000. Human exposure estimates for phthalates. Environ. Health Perspect. 108, A440–A442. National industrial chemicals notification and assessment scheme (NICNAS), 2003. Full public report, 1,2-benzenedicarboxylic acid, bis(2-propylheptyl) ester, (Palatinol 10-P), http://www.nicnas.gov.au/__data/assets/pdf_file/0014/10391/ STD1054FR.pdf, downloaded (07.03.14.). National industrial chemicals notification and assessment scheme (NICNAS), 2008. Full public report, diisodecyl phthalate, http://www.nicnas.gov.au/__data/ assets/pdf_file/0010/4969/DIDP-hazard-assessment.pdf, downloaded (07.03.2014.). Schindler, B.K., et al., 2014. The European COPHES/DEMOCOPHES project: towards transnational comparability and reliability of human biomonitoring results. Int. J. Hyg. Environ. Health http://dx.doi.org/10.1016/j.ijheh.2013.12.002. Schütze, A., Pälmke, C., Angerer, J., Weiss, T., Brüning, T., Koch, H.M., 2012. Quantification of biomarkers of environmental exposure to di(isononyl) cyclohexane-1,2-dicarboxylate (DINCH) in urine via LC/LC–MS/MS. J. Chrom. B. 895–896, 123–130 http://dx.doi.org/10.1016/j.jchromb.2012.03.030. Schütze, A., Kolossa-Gehring, M., Apel, P., Brüning, T., Koch, H.M., 2014. Entering markets and bodies: increasing levels of the novel plasticizer DINCH1 in 24 h urine samples from the German Environmental Specimen Bank. Int. J. Hyg. Environ. Health 217, 421–426 http://dx.doi.org/10.1016/j.ijheh.2013.08.004. Taussky, H.H., 1954. A micro colorimetric determination of creatinine in urine by the Jaffé reaction. J. Biol. Chem. 208, 853–861. Wittassek, M., Angerer, J., 2008. Phthalates: metabolism and exposure. Int. J. Androl. 31, 131–138. Wittassek, M., Wiesmüller, G.A., Koch, H.M., Eckard, R., Dobler, L., Müller, J., Angerer, J., Schlüter, C., 2007. Internal phthalate exposure over the last two decades – a retrospective human biomonitoring study. Int. J. Hyg. Environ. Health 210, 319– 333. Wittassek, M., Koch, H.M., Angerer, J., Brüning, T., 2011. Assessing exposure to phthalates – the human biomonitoring approach. Mol. Nutr. Food Res. 55, 7–31 doi:http://dx.doi.org/10.1002/mnfr.201000121.

Please cite this article in press as: Leng, G., et al., Urinary metabolite excretion after oral dosage of bis(2-propylheptyl) phthalate (DPHP) to five male volunteers – Characterization of suitable biomarkers for human biomonitoring, Toxicol. Lett. (2014), http://dx.doi.org/10.1016/j. toxlet.2014.06.035