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TOXLET 8769 No. of Pages 7

Toxicology Letters xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.elsevier.com/locate/toxlet

Urinary metabolite excretion after oral dosage of bis(2-propylheptyl)

(DPHP) to five male volunteers – Characterization of suitable

biomarkers for human biomonitoring

a, b a b

Gabriele Leng *, Holger M. Koch , Wolfgang Gries , Andre Schütze ,

c b c

Angelika Langsch , Thomas Brüning , Rainer Otter

a

Currenta GmbH & Co. OHG, Institute of Biomonitoring, Leverkusen, Germany

b

Institute for Prevention and Occupational Medicine – Institute of the Ruhr-Universität Bochum, Bochum, Germany

c

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: Di(2-propylheptyl) phthalate (DPHP), a high molecular weight phthalate, is primarily used as a

Received 18 March 2014

in and vinyl chloride copolymers for technical applications, as a substitute for other

Received in revised form 30 May 2014

currently being scrutinized because of endocrine disrupting effects.

Accepted 23 June 2014

We determined urinary excretion fractions of three specific DPHP metabolites (mono-2-(propyl-6-

Available online xxx

hydroxy-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

Keywords:

50 mg labelled DPHP-d4 and subsequent urine sampling for 48 h. These excretion fractions are used to

Biomonitoring

back calculate external intakes from metabolite measurements in spot urine analysis. Following

Di(2-propylheptyl)phthalate

enzymatic hydrolysis to cleave possible conjugates, we determined these urinary metabolites by HPLC–

DPHP metabolites

– fi m

Urinary excretion NESI MS/MS with limits of quanti cation (LOQ) between 0.3 and 0.5 g/l.

–

Human volunteer study Maximum urinary concentrations were reached within 3 4 h post dose for all three metabolites;

– –

HPLC NESI MS/MS 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 di-

isodecyl phthalate (DIDP) metabolites is warranted (preferably by GC–MS) in order to distinguish DPHP

1 1 1

from general DIDP exposure. Palatinol , Hexamoll and DINCH are registered trademarks of BASF SE,

Germany.

ã 2014 The Authors. Published by Elsevier Ireland Ltd. All rights reserved.

* 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

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TOXLET 8769 No. of Pages 7

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1. Introduction collaborative project between the German Federal Ministry for

the Environment, Nature Conservation, Building and Nuclear

Di(2-propylheptyl) phthalate (DPHP), CAS No. 53,306-54-0, a Safety (BMUB) and the German Chemical Industry Association

REACH (Regulation (EC) No. 1907/2006) registered high molecular (VCI) evaluated a specific human biomonitoring method to

weight phthalate, is primarily used as a plasticizer in polyvinyl- determine exposure of the general population to DPHP using

chloride and vinyl chloride copolymers for technical applications. reliable and specific urinary biomarkers (Federal Ministry for the

1

DPHP, which is marketed under, e.g., the trade name “Palatinol Environment, 2010). We recently developed such a method for

1

10-P”, is produced by esterification of phthalic anhydride with a DINCH (di-isononyl-cyclohexane-1,2-dicarboxylate), a non-aro-

C10 alcohol consisting of 90% 2-propyl-heptanol and 10% 2-propyl- matic high molecular weight phthalate substitute mainly intended

4-methylhexanol or 2-propyl-5-methylhexanol. There are current- for sensitive applications such as toys, food contact materials and

ly two different C10 phthalates on the market. DPHP and di- medical devices (Koch et al., 2013a,b; Schütze et al., 2012, 2014).

isodecyl phthalate (DIDP) as described with the CAS No. 68,515-49- For DPHP, however, exposure needs to be distinguishable from

1: 1,2-benzenedicarboxylic acid, di-C9-11-branched alkyl esters, DIDP/DINP exposure. Previous exposure assessments based on

C10-rich. Another DIDP described by CAS No. 26,761-40-0 is no human biomonitoring have reported the cumulative exposure

longer produced in Europe and is not REACH registered. Further- (Kasper-Sonnenberg et al., 2012; Koch et al., 2009) to all phthalates

more, there are two C9 phthalates (di-isononyl phthalates, DINPs) containing C10 alkyl chains (DPHP, DINP, DIDP), because the

on the market: DINP1 (1,2-benzenedicarboxylic acid, di-C8-10- complex isomeric composition of DINP/DIDP interfered with the

branched alkyl esters, C9-rich, described with CAS No. 68,515-48-0 selective detection of the DPHP specific 2-propyl-heptyl based side

and DINP2 (di-isononyl phthalate) with CAS No. 28,553-12-0. chain metabolites.

While DINP2 solely consists of C9 isomers DINP1 contains up to We used the method developed by Gries et al. (2012) to reliably

10% C10 isomers. Thus, the broad isomer distribution of DINP1 detect and quantify DHPH metabolites in the presence of other

(including C10 moieties) can also interfere with the analytical DIDP/DINP metabolites. Wittassek and Angerer, 2008 showed that

detection of both DIDP and DPHP. The lack of sufficient analytical DPHP is metabolized similarly to DEHP (Koch et al., 2004), i.e., the

separation of DINP and DIDP resulted in a group-TDI by EFSA (EFSA, monoester is formed by ester cleavage in a first step followed by

2005) for food contact applications (Commission Regulation (EU) extensive v and v-1 oxidation of the remaining single alkyl side

No. 10/2011). chain. A metabolism scheme of DPHP is presented in Fig. 1. The

The phthalates DINP, DPHP and DIDP are currently used as secondary, oxidized metabolites are the predominant metabolites.

substitutes for di-(2-ethylhexyl) phthalate (DEHP) which is listed The monoester MPHP is only a minor metabolite (<1% formed from

under REACH as a substance of very high concern (SVHC). Based on the parent compound and excreted with urine), which is typical for

their low volatility and low vapor pressure, the C10 phthalates all high molecular weight phthalates. The secondary metabolites

DPHP and DIDP are predominantly used in high temperature- have an added analytical benefit in that they are not subject to

resistant products such as electrical cables, carpet backing and car issues of sample contamination as described by Kato et al. (2004)

interiors, but they are also used for outdoor applications like and Schindler et al. (2014).

roofing membranes or tarpaulins (European Commission, 2003; We investigated renal excretion and metabolic conversion of

NICNAS, 2003, 2008; Wittassek, 2008). DPHP is currently not used DPHP by measuring three oxidized metabolites of the propylheptyl

in food contact. Because are not chemically bound in side-chain, mono(propyl-6-oxo-heptyl) phthalate (oxo-MPHP),

PVC products and thus can migrate out of these products, exposure mono(propyl-6-hydroxyheptyl) phthalate (OH-MPHP) and mono

of humans and the environment is possible. Therefore, a (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

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dosing of stable isotope (deuterium) labeled DPHP-d4 to five male 2.3. Standards

volunteers. The fraction of excreted metabolite is used to

determine conversion factors which enable the back calculation The study design is based on dosing of ring labeled DPHP-d4;

of the (daily) intake of DPHP (external dose) as described by Kohn therefore, the target substances were the respective d4-ring

et al. (2000) and David (2000). labeled metabolites. Ten milligram of each metabolite (OH-MPHP-

d4, oxo-MPHP-d4 or cx-MPHxP-d4) were weighed separately into

2. Materials and methods a 10 ml glass volumetric flask and diluted to volume with

acetonitrile (1000 mg/l). From these stock solutions, a multi-

2.1. Experimental design component starting solution was prepared by diluting 100 ml of

each in a 10 ml glass volumetric flask filled with acetonitrile. This

Di(2-propylheptyl) phthalate (DPHP) was orally dosed as ring- starting solution (10 mg/l) was further diluted for the preparation

deuterated DPHP-d4 to five healthy male volunteers, aged between of the working standards to achieve final standard concentrations

27 and 49 years, with body weights between 77 and 94 kg. The of 1 mg/l, 0.1 mg/l, 0.01 mg/l and 0.001 mg/l.

volunteers did not have any known occupational exposure to DPHP For the purpose of internal standardization, we used the non-

or to other plasticizers. Fifty milligram of DPHP-d4 was dissolved in labeled DPHP metabolite standards. Internal standard stock

0.25 ml of ethanol and mixed in an edible waffle cup with a solutions were prepared by dilution of 10 mg of OH-MPHP, oxo-

chocolate surface containing coffee or tea during breakfast. This MPHP or cx-MPHxP in 10 ml volumetric flasks with acetonitrile

resulted in doses for the five individuals of between 0.54 and (1000 mg/l). Starting solution A was prepared by diluting 100 ml of

0.66 mg/kg body weight. The DPHP dose was considerably below each of the three stock solutions into a 10 ml volumetric flask

the lowest NOAEL (no observed adverse effect level) for DPHP (BfR (10 mg/l) to the mark with acetonitrile. For the preparation of

Opinion No., 2011; Bhat et al., 2014) and comparable to the DINP solution B 1 ml of solution A was diluted in a 10 ml volumetric flask

1

(Koch and Angerer, 2007) or DINCH dose levels (Schütze et al., to its nominal volume with acetonitrile (1 mg/l).

2014) of previous human metabolism studies. The DPHP dose was

several orders of magnitude above exposure levels expected for the 2.4. Sample preparation

general population. Stable-isotope labeled DPHP-d4 was used to

exclude possible background exposures. Volunteers were dosed at Urine samples (or standards) were thawed and equilibrated to

the Institute for Prevention and Occupational Medicine of the room temperature. For enzymatic hydrolysis, 10 ml of b-glucuron-

German Social Accident Insurance, Institute of the Ruhr-Universi- idase and 20 ml of the internal standard solution in 200 ml 1 M

tät Bochum (IPA), frozen samples of urine were shipped to ammonium acetate buffer (pH 6.5) were added to 1000 ml of each

Currenta for quantification of the metabolites. The first urine sample and mixed. Samples were incubated at 37 C overnight.

samples were collected prior to dosage at 10:00 a.m. followed by Thereafter, all samples were acidified to pH 2 with hydrochloric

subsequent urine samples collected over 48 h post-dosing. The acid (37%) and extracted with tert-butylmethylether, mixed with a

volunteers recorded the time of the void of each sample. The urine vortex mixer for 10 min and centrifuged at 2200 g for 10 min at

volume of each individual sample was determined as the 10 C. The upper phase was aspirated with a Pasteur pipette and

difference between the weight of the filled and the empty placed into a glass test tube, and the samples were dried at 35 C

container. In all, we obtained 122 urine samples, i.e., between with nitrogen. All samples were re-dissolved in 200 ml of methanol

20 and 29 samples from each volunteer. The total 48 h urine for HPLC–MS/MS analysis. The creatinine concentration in each

volume ranged from 4133 to 8298 ml, depending on the volunteer. urine sample was measured according to the Jaffé method

All urinary samples were frozen at 18 C immediately after (Taussky, 1954).

delivery. The study was carried out in accordance with the code of

ethics of the World Medical Association (Declaration of Helsinki) 2.5. High performance liquid chromatography–tandem mass

and was approved by the ethical review board of the Medical spectrometry

Faculty of the Ruhr-University Bochum (Reg. No.: 4022-11). The

study design was presented to the volunteers in written form, and Chromatographic separation was performed on a Waters

all participants provided written informed consent. 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

2.2. Chemicals system (A: methanol, B: water and C: formic acid) was used to

separate the metabolites with the following conditions: at start,

Acetonitrile (supra solv), methanol (supra solv), glacial acetic 10 ml was injected onto the column with 10% A, 80% B and 10% C,

acid (p.a.) and hydrochloric acid 37% (p.a.) were purchased from flow was 0.2 ml/min and constant during the whole analysis which

Merck, Darmstadt, Germany. Ammonium acetate (p.a.) was lasted 25 min. Metabolites were separated by an increasing

purchased from Fluka, Taufkirchen, Germany. Formic acid (99%, methanol gradient, i.e., methanol (A) was increased from 10% to

ULC/MS) was purchased from Biosolve B.V., Valkenswaard, The 90% within 15 min while water (B) was reduced to 0%. Solvents A

Netherlands. Water from a millipore water cleaning system was

used and b-glucuronidase from Escherichia coli K12 was pur-

Table 1

chased from Roche, Mannheim, Germany. DPHP-d4 was provided

LC–MS/MS retention times, mass transitions and dwell times for the labeled and

by BASF SE. The following standards were synthesized at the

non-labeled DPHP metabolites.

Institut für Dünnschichttechnologie e.V. (IDM), Teltow, Germany:

Metabolite Rt Parent Daughter Dwell time

mono-2-(propyl-6-hydroxy-heptyl)-phthalate (OH-MPHP), mono-

2-(propyl-6-oxo-heptyl)-phthalate (oxo-MPHP), mono-2-(propyl- (min) (m/z) (S)

6-carboxy-hexyl)- phthalate (cx-MPHxP), mono-2-(propyl-6-hy-

cx-MPHxP 19.83 335.16 187.04 0.1

droxy-heptyl)-phthalate-d4 ring deuterated (OH-MPHP-d4), cx-MPHxP-d4 19.81 339.12 187.04 0.1

mono-2-(propyl-6-oxo-heptyl)-phthalate-d4 ring deuterated OH-MPHP 20.15 321.13 121.02 0.1

OH-MPHP-d4 20.13 325.16 125.04 0.1

(oxo-MPHP-d4), and mono-2-(propyl-6-carboxy-hexyl)-phthal-

oxo-MPHP 19.65 319.12 121.02 0.1

ate-d4 ring deuterated (cx-MPHxP-d4). The purity of all com-

oxo-MPHP-d4 19.60 323.15 124.98 0.1

1

pounds was determined by H-NMR and was 95%.

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

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(90%) and B (0%) were kept constant for 2 min and then a gradient

Table 2

was used to reach 10% A and 80% B at 18 min. These conditions

Within-day and between-day precision data.

were kept for 7 min until 25 min when the analysis was finished. C

Analyte Within-day precision (n = 8) Between-day precision (n = 5)

was kept constant at 10% during the analysis. The mass

fi

Conc. Recov. R.S.D. Conc. Recov. R.S.D. spectrometric detection and quanti cation was performed on a

m m

( g/l) (%) (%) ( g/l) (%) (%) Waters Quattro Ultima MS/MS with negative electrospray ioniza-

cx-MPHxP-d4 1 109 4.6 – – – tion (NESI) in MRM mode. Mass transitions are depicted in Table 1.

10 108 3.2 10 111 3.9

Further details are given in Gries et al. (2012).

100 108 1.5 100 106 10.2

2.5.1. Calibration and quantification

OH-MPHP-d4 1 108 2.2 – – –

10 101 3.8 10 102 1.1 Calibration was carried out by spiking 1 ml of water with

100 103 2.4 100 103 1.7 concentrations ranging from 0.1 mg/l to 5000 mg/l of each

deuterated standard. All calibration samples were analyzed as

oxo-MPHP-d4 1 100 6.5 – – –

described in the sample section. Due to the high dynamic range of

10 97 2.8 10 99 4.9

the HPLC–MS/MS a calibration range up to 5000 mg/l of each

100 101 5.3 100 99 2.9

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

A: pre-dose sample B: post- dose sample

100 100

OH-MPHP (ISTD) OH-MPHP (ISTD)

OH-MPHP-d4 (91.4 µg/l)

abundancy [%] OH-MPHP -d4 (n.d.)

Relative Relative 0 0

16.00 18.00 20.00 22.00 24.00 16.00 18.00 20.00 22.00 24.00 100 100

]

oxo -MPHP (ISTD) oxo-MPHP (ISTD) %

oxo-MP HP-d4

(12 2.9 µg/l) oxo-MPHP -d4

lative abundancylative [ (n.d.)

0 0

16.00 18.00 20.00 22.00 24.00 16.00 18.00 20.00 22.00 24.00

100 100

cx-MPHxP (ISTD) cx-MPHxP (ISTD) cy [%] Re

n

cx-MPHxP -d4

cx-MPHxP-d4 (10.6 µg/l) (n.d.)

Relative abunda 0 0

16.00 18. 00 20.00 22.00 24. 00 16.00 18. 00 20.00 22.00 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

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high metabolite concentrations expected in this dosing study. with spiked internal standard concentrations at 200 mg/l, the

Samples with concentrations above the calibration range were omnipresent but low background exposure to DIDP/DPHP did not

analyzed again after sample dilution with water. The calibration interfere with the study design. Elimination kinetics could be

curves were obtained by plotting the quotient of the peak areas of monitored and specific metabolic conversion factors could be

the target deuterated analytes and the corresponding unlabeled established. In the chromatograms of Fig. 2B, additional peaks with

internal standards against the standard concentrations. same fragmentation patterns as the propylheptyl derived oxidized

standards emerged, albeit at different retention times. These peaks

2.5.2. Quality control and validation most likely originate from the minor alkyl chain isomers of DPHP

As quality control samples were not available, they had to be (2-propyl-4-methylhexyl or 2-propyl-5-methylhexyl side chain)

prepared in the laboratory with spiked urine samples to cover and/or from oxidative modifications other than in the v- or v-1-

different concentration ranges (1 mg/l, 10 mg/l or 100 mg/l of each position. All further quantitative data are based on the sole

labeled metabolite). One millilitre aliquots of these control integration of the specific propylheptyl derived oxidized isomer

samples were stored frozen at 18 C. Two samples with either peaks present as analytical standard substances.

10 or 100 mg/l concentration of each deuterated standard were The elimination of these specific DPHP metabolites in urine

analyzed during the analysis sequences for each volunteer on five over time (48 h) for the five volunteers is shown in Fig. 3A (in mg/l),

different days to determine between day precision data. The B (in mg/g creatinine) and C (absolute amount in mg), calculated for

within-day precision was obtained by analyzing pooled urine 6 h increments. All forms of presentation clearly depict the rapid

samples in three concentrations of each deuterated standard as appearance of all three DPHP metabolites in urine after oral dosage.

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).

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 OH-

MPHP-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 oxo-

MPHP-d4, respectively.

2.6. Statistical analysis

Statistical analysis was carried out using Microsoft Excel 2010.

Exponential regression modeling was used to calculate exponen-

tial 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 C0 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 post-

dose urine samples obtained from this dosing study. LC–MS/MS

chromatograms in Fig. 2A and B illustrate the appearance of the d4-

ring-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 non-

labeled propylheptyl derived DPHP metabolite standards for

Fig. 3. Elimination of the three oxidized DPHP metabolites in urine over time (48 h)

internal standardization. In some urine samples, a background

for the five volunteers is shown in Fig. 3A (in mg/L), B (in mg/g creatinine) and C

trace level of isomeric, oxidized (non-labelled) DIDP metabolites

(absolute amount in mg), calculated for 6 h increments. The bold boxes represent

was visible, but at levels much lower than the spiked DPHP

mean values calculated over all five volunteers; the bars depict the ranges among

m

standards (maximum concentrations of 2 g/l, not shown). Thus, 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

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

Table 3

the study; for DINP, the three main oxidized metabolites excreted

Elimination half-lives and times of maximum urinary excretion for the three

in urine represent about 29.8–37.5% of the dose, depending on the

oxidized DPHP metabolites after oral dosage (calculated from the five volunteers).

study. In all these studies, it was shown that an increasing alkyl

Parameter Mean t SD Mean t SD

max 1/2 chain length of the plasticizer results in a decreased formation of

(h) the simple monoester. Thus, for high molecular weight plasticizers,

the simple monoester is not a relevant urinary metabolite.

oxo-MPHP-d4 3.65 1.31 6.51 1.64

Furthermore, since the simple monoester is prone to external

OH-MPHP-d4 3.65 1.31 6.87 1.63

cx-MPHxP-d4 4.05 1.39 8.16 0.67 contamination, the oxidized metabolites have to be regarded as the

most suitable biomarkers for monitoring exposure to high

SD: standard deviation.

molecular weight phthalates in urine (Koch and Calafat, 2009).

The metabolic conversion factors established in this study for

fi

Table 4 DPHP based on the ve male volunteers allow a reliable back

Molar urinary excretion fractions in % of oral dose (mean standard deviation) for

calculation from urinary DPHP metabolite levels to external

five volunteers).

exposure, and thus enable a solid risk assessment of the human

P

Time cx-MPHxP-d4 OH-MPHP-d4 oxo-MPHP-d4 of 3 metab. body burden for the general public as well as for individuals

occupationally exposed. A reliable back-calculation to DPHP

(h) (%)

exposure, however, can only be performed, if the above secondary,

0–24 0.42 0.11 9.91 3.45 12.61 3.90 22.94 7.33

oxidized DPHP metabolites are chromatographically separated

0–48 0.48 0.13 10.70 3.61 13.52 4.04 24.70 7.64

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 unambigu-

Both OH-MPHP and oxo-MPHP are clearly the predominant ously and reliably quantify these oxidized DPHP metabolites, even

metabolites over cx-MPHxP which is excreted at considerably in the presence of high DIDP/DINP body burdens. For 40 random

lower concentrations. All metabolites are excreted rather rapidly spot urine samples, they reported a maximum urinary concentra-

and steadily over the 48 h investigated. However, at 48 h post-dose, tion of 0.93 mg/l oxo-MPHP. Most of the currently available human

all three metabolites were still detectable. Based upon the biomonitoring data (summarized e.g., in Wittassek et al., 2007,

creatinine corrected elimination curve (Fig. 3B), all three 2011; Koch and Calafat, 2009; Kasper-Sonnenberg et al., 2012) do

metabolites seem to follow a one-phasic elimination pattern. not distinguish between oxidized C10 metabolites of DIDP/DINP

Times of maximum urinary excretion for the three oxidized and DPHP due to the limited chromatographic resolution of the

DPHP metabolites and elimination half-lives calculated from the HPLC–MS methodology applied. The C10-metabolite levels from

fi

individual data of each of the ve volunteers are depicted in these studies, however, indicate a cumulative C10-phthalate

Table 3. exposure (DINP/DIDP and DPHP) that is considerably higher than

Molar excretion fractions in percent of the oral dose were that for DPHP alone. Future studies using differential integration of

calculated by using the respective molecular weights of the specific DPHP metabolites next to the cumulative measurement of

metabolites cx-MPHxP-d4 (340.39 g/mol), OH-MPHP-d4 (326.40 g/ C10-phthalate metabolites have to confirm this finding.

mol), and oxo-MPHP-d4 (324.39 g/mol). Total excreted amount

was estimated as the sum of all samples taken per volunteer and

Conflict of interest

taking into account all three metabolites (see Table 4). Oxo-MPHP

is the most abundant metabolite, representing in the mean over

None for all authors except for A. Langsch and R. Otter who both

the five volunteers 13.5% of the oral DPHP dose in urine after 48 h,

are employed by BASF SE, a producer of DPHP.

closely followed by OH-MPHP (10.7%). Cx-MPHxP (0.5%) is

regarded as a minor metabolite. All three oxidized metabolites

Transparency document

represent about 25% of the dose excreted in urine within 48 h.

The Transparency document associated with this article can be

4. Discussion

found in the online version.

Wittassek and Angerer (2008) reported the first results on

Acknowledgements

human DPHP metabolism, when the senior author ingested a

single DPHP dose of 98 mg during breakfast. In their pilot study

The study was carried out as part of a ten-year project on

they reported that after 61 h around 34% of the applied dose was

human biomonitoring. The project is a cooperation agreed in 2010

excreted with urine as oxidized metabolites (including approx. 1%

between the Federal Ministry for the Environment, Nature

as the simple monoester). Taking into account that they included

Conservation, Building and Nuclear Safety (BMUB) and the

other metabolites with oxidative modifications and that their

Verband der chemischen Industrie e.V. (German Chemical Industry

sampling time was longer, their data are consistent with the data of

Association – VCI); it is administered by the Federal Environment

the study reported here.

Agency (UBA). The study aims to characterize suitable biomarkers

The data obtained for DPHP in this study is also consistent with

for human biomonitoring and to develop a new analytical method

human metabolism data for other high molecular weight

based upon these biomarkers and was funded by the German

phthalates like DEHP and DINP (Koch et al., 2005, 2007; Anderson

Chemicals Industry. Experts from government authorities, indus-

et al., 2011; Kessler et al., 2012). Similar elimination half-lives were

try and science accompany the project in selecting substances and

also calculated for all DPHP metabolites (6.51–8.16 h) compared

developing methods.

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

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