André Schütze

Entering markets and bodies: increasing levels of the novel plasticizer Hexamoll® DINCH® in 24 h urine samples from the German Environmental Specimen Bank.

DINCH (diisononylcyclohexane-1,2-dicarboxylate) was introduced into the world market in 2002 as a non-aromatic plasticizer and phthalate substitute. We analyzed 300 urine samples (24 h voids) of the German Environmental Specimen Bank (ESB for Human tissues, ESB Hum) for specific DINCH metabolites by on-line HPLC-MS/MS with isotope dilution quantification. Urine samples of the ESB Hum were from the years 1999, 2003, 2006, 2009 and 2012, chosen to investigate the appearance and a possible trend of DINCH exposure since its market introduction. No DINCH metabolites were detected in the 1999 and 2003 samples. From 2006 on, the percentage of samples with DINCH metabolites above the LOQ increased significantly over the years (7% in 2006, 43% in 2009 and 98% in 2012). The cyclohexane-1,2-dicarboxylic acid-mono(hydroxy-isononyl) ester (OH-MINCH) was the predominant metabolite. Median (and 95th percentile) concentrations (in μg/l) increased from 0.75, p<0.001). The median (95th percentile) DINCH intake in 2012 was calculated to be 0.14 (1.07)μg/kg body weight/day which is considerably below daily intakes currently deemed tolerable. DINCH is regarded to have a preferred toxicological profile over certain anti-androgenic phthalates. The continuation of DINCH measurements in the ESB Hum and other human biomonitoring studies like the German Environmental Survey (GerES) allows tracking the development of DINCH body burdens, the distribution of exposure levels and daily intakes, providing basic data for future toxicological assessment and further epidemiological studies.

Quantification of biomarkers of environmental exposure to di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) in urine via HPLC–MS/MS

Di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH) is a major substitute for some high molecular weight phthalates that adversely affect reproductive function. Like for the phthalates a broad exposure of the population has to be expected. We postulated the DINCH monoester (MINCH) and secondary oxidized metabolites (OH-MINCH, cx-MINCH and oxo-MINCH) as human metabolites and possible biomarkers of DINCH exposure. We developed an on-line HPLC-MS/MS method for their determination in human urine. Identification was performed with authentic standard substances and quantification via isotope dilution. The analytical method is highly selective and sensitive with limits of quantification (LOQ) between 0.05 μg/l and 0.1 μg/l. In a pilot study with 22 volunteers from the general German population oxidized DINCH metabolites were found in above 80% of the samples. OH-MINCH was most abundant (mean 0.71 μg/l; maximum 3.69 μg/l) followed by cx-MINCH (0.61 μg/l; 2.82 μg/l) and oxo-MINCH (0.33 μg/l; 1.05 μg/l). All three oxidized metabolites correlated strongly among each other (ρ ≥ 0.76). MINCH was detected in one sample only and has to be regarded a weak marker of exposure. With this analytical method we are able to perform human metabolism studies to provide metabolic conversion factors and to investigate the extent of DINCH exposure in the general population.

Non-phthalate plasticizers in German daycare centers and human biomonitoring of DINCH metabolites in children attending the centers (LUPE 3)

Plasticizers have been widely used for decades as additives in diverse applications, including consumer and building products, toys, cables, and floorings. Due to toxicological concerns and restrictions of different dialkyl ortho-phthalates, other plasticizers have been increasingly used in recent years. Therefore, di-isononyl cyclohexane-1,2-dicarboxylate (DINCH), di(2-ethylhexyl) terephthalate (DEHT), di(2-ethylhexyl) adipate (DEHA), acetyl tri-n-butyl citrate (ATBC), and trioctyl trimellitate (TOTM) plasticizer levels in indoor air and dust samples from 63 daycare centers in Germany were measured. Moreover, the urine samples of 208 children who attend 27 of these facilities were analyzed for the presence of four DINCH metabolites. DINCH, DEHT, and DEHA were present in indoor air with median values of 108 ng/m(3), 20 ng/m(3), and 34 ng/m(3), respectively. Median values of 302 mg/kg for DINCH, 49 mg/kg for DEHA, 40 mg/kg for DEHT, and 24 mg/kg ATBC were found in dust. In the urine samples, the three secondary metabolites of DINCH were observed with median values (95th percentiles) of 1.7 μg/l (10.0 μg/l) for OH-MINCH, 1.5 μg/l (8.0 μg/l) for oxo-MINCH, and 1.1 μg/l (6.1 μg/l) for cx-MINCH. Overall, these metabolite levels are orders of magnitude lower than the current HBM I values set by the German Human Biomonitoring Commission. Using general exposure assumptions, the intake resulting from dust ingestion and inhalation is low for children. The total daily DINCH intake calculated from biomonitoring data was 0.5 μg/kg b.w. using median values and 9.8 μg/kg b.w. as the maximum value. At present, non-phthalate plasticizers, especially DINCH, can be found in considerable amounts in dust samples from daycare centers and as DINCH metabolites in the urine of children. In relation to previous studies, the concentrations of DINCH in dust and urine have an increasing time trend. Compared with tolerable daily intake values, the total daily intake of DINCH reached only 1% of its maximum value to date; however, due to its increased use, higher exposure of DINCH is expected in the future.

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

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-6-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 50mg 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 μg/l. Maximum urinary concentrations were reached within 3-4h post dose for all three metabolites; elimination half-lives were between 6 and 8h. 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 24h 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 from general DIDP exposure. Palatinol(®), Hexamoll(®) and DINCH(®) are registered trademarks of BASF SE, Germany.

Bis-(2-propylheptyl)phthalate (DPHP) metabolites emerging in 24h urine samples from the German Environmental Specimen Bank (1999-2012).

Bis-(2-propylheptyl)-phthalate (DPHP) has been introduced as a substitute for other high molecular weight phthalates primarily used in high temperature applications (e.g. cable wires, roofing membranes). The aim of this study was to investigate how the increased usage of DPHP is reflected in urine samples collected over the last 14 years and to evaluate the current extent of exposure. We analyzed 300 urine samples (24h voids) from the German Environmental Specimen Bank collected in the years 1999, 2003, 2006, 2009 and 2012, 60 samples per year, from 30 male and 30 female volunteers (age: 20-30 years) for three specific, secondary oxidized DPHP metabolites (with hydroxy, oxo and carboxy modifications of the alkyl side chain). We determined DPHP metabolites with a previously developed GC-HRMS method, enabling us to unambiguously distinguish DPHP metabolites from co-eluting, structurally isomeric di-iso-decyl phthalate (DIDP) metabolites. All samples were blinded before analysis. We detected no DPHP metabolites in urine samples from the years 1999, 2003 and 2006. Thereafter, detection rates increased from 3.3% in 2009 to 21.7% in 2012. Mono-oxo-propylheptylphthalate (oxo-MPHP) was the most abundant metabolite, with concentrations between <LOQ and 0.96μg/l. Extrapolating from oxo-MPHP concentrations in the 24h urine samples we calculated a maximum daily DPHP intake of 0.32μg/kg body weight. Our results show that the general German population is increasingly exposed to DPHP. However, exposure is considerably lower than for DIDP or other high molecular weight phthalates. Future measurements will enable us to monitor the development of DPHP exposure and advise risk management steps, if warranted.

Rapid determination of N-acetyl-4-aminophenol (paracetamol) in urine by tandem mass spectrometry coupled with on-line clean-up by two dimensional turbulent flow/reversed phase liquid chromatography

N-Acetyl-4-aminophenol (NAAP) is the major urinary metabolite of aniline. The general population is known to be ubiquitously exposed to aniline through various sources. Furthermore, NAAP, known under the trade name paracetamol (resp. acetaminophen), is one of the most commonly used over-the-counter analgesics. Recent studies suggest anti-androgenic properties of NAAP. Although NAAP has been used as a pain reliever over decades and its role in aniline metabolism is well known there is a lack of internal exposure data both in environmental and occupational settings. To determine the internal NAAP exposure of the general population, workers exposed to aniline and users of paracetamol we developed a fast on-line HPLC-MS/MS method with isotope dilution quantification of NAAP after enzymatic hydrolysis of its conjugates in urine. We achieved minimal sample pretreatment through on-line extraction and enrichment of the analyte by turbulent flow chromatography on a Waters Oasis HLB phase followed by back-flush transfer onto the analytical column. The limit of quantification (LOQ) was 0.75 μg/L. In a pilot study, urine samples of 21 volunteers, not occupationally exposed to aniline, were analyzed for NAAP. NAAP was detected in all samples in a wide concentration range between 8.7 μg/L and 22100 μg/L (median 85.7 μg/L). The highest concentration was measured in a volunteer who took paracetamol one day ago. Half of the volunteers quoted to either never have taken paracetamol or at least not during several weeks before the study. Therefore, other routes of exposure than direct use of paracetamol, like aniline or paracetamol contaminated foodstuff, leading to the NAAP excretions have to be taken into account.

Determination of metabolites of di(2-ethylhexyl) terephthalate (DEHTP) in human urine by HPLC-MS/MS with on-line clean-up.

Di(2-ethylhexyl) terephthalate (DEHTP) is used as a substitute for ortho-phthalate based plasticizers like di(2-ethylhexyl) phthalate (DEHP) which are discussed and regulated due to their reproductive toxicity. We developed a fast and rugged method to quantify side chain oxidized monoesters of DEHTP in human urine, namely 5OH-MEHTP, 5oxo-MEHTP, 2cx-MMHTP and 5cx-MEPTP. Sample preparation was kept simple with enzymatic deconjugation and a two column assembly for on-line sample clean up. Metabolites were identified with authentic standards and quantified via isotope dilution LC-MS/MS. The limit of quantification was 0.2μg/L for 5cx-MEPTP and 5oxo-MEHTP, 0.3μg/L for 5OH-MEHTP and 0.4μg/L for 2cx-MMHTP. Accuracy (relative recovery: 95.8-111%) and precision (relative standard deviation: <7%) were highly acceptable. In a pilot biomonitoring study with 34 volunteers (aged 25-61 (median 42), 20 female and 14 male) not known to be occupationally exposed to DEHTP, we could detect 5cx-MEPTP above the limit of quantification in 94% of the samples (median: 0.9μg/L, maximum: 38.7μg/L). The other metabolites investigated were detected at a lower rate and at lower concentration levels (5oxo-MEHTP: 21%, maximum: 1.8μg/L; 5OH-MEHTP: 18%, maximum: 3.4μg/L; 2cx-MMHTP: 9%, maximum: 0.9μg/L). All target analytes can be regarded as promising and specific urinary biomarkers for DEHTP exposure. With this method we provide a basis for quantitatively investigating the human metabolism of DEHTP and for performing exposure and risk assessments in the general population and the working environment.

Phthalate metabolites in 24-h urine samples of the German Environmental Specimen Bank (ESB) from 1988 to 2015 and a comparison with US NHANES data from 1999 to 2012

The German Environmental Specimen Bank (ESB) continuously collects 24-h urine samples since the early 1980s in Germany. In this study we analyzed 300 urine samples from the years 2007 to 2015 for 21 phthalate metabolites (representing exposure to 11 parent phthalates) and combined the data with two previous retrospective measurement campaigns (1988 to 2003 and 2002 to 2008). The combined dataset comprised 1162 24-h urine samples spanning the years 1988 to 2015. With this detailed set of human biomonitoring data we describe the time course of phthalate exposure in Germany over a time frame of 27 years. For the metabolites of the endocrine disrupting phthalates di(2-ethylhexyl) phthalate (DEHP), di-n-butyl phthalate (DnBP) and butylbenzyl phthalate (BBzP) we observed a roughly ten-fold decline in median metabolite levels from their peak levels in the late 1980s/early 1990s compared to most recent levels from 2015. Probably, bans (first enacted in 1999) and classifications/labelings (enacted in 2001 and 2004) in the European Union lead to this drop. A decline in di-isobutyl phthalate (DiBP) metabolite levels set in only quite recently, possibly due to its later classification as a reproductive toxicant in the EU in 2009. In a considerable number of samples collected before 2002 health based guidance values (BE, HBM I) have been exceeded for DnBP (27.2%) and DEHP (2.3%) but also in recent samples some individual exceedances can still be observed (DEHP 1.0%). A decrease in concentration for all low molecular weight phthalates, labelled or not, was seen in the most recent years of sampling. For the high molecular weight phthalates, DEHP seems to have been substituted in part by di-isononyl phthalate (DiNP), but DiNP metabolite levels have also been declining in the last years. Probably, non-phthalate alternatives increasingly take over for the phthalates in Germany. A comparison with NHANES (National Health and Nutrition Examination Survey) data from the United States covering the years 1999 to 2012 revealed both similarities and differences in phthalate exposure between Germany and the US. Exposure to critical phthalates has decreased in both countries with metabolite levels more and more aligning with each other, but high molecular weight phthalates substituting DEHP (such as DiNP) seem to become more important in the US than in Germany.

Evaluation of exposure to phthalate esters and DINCH in urine and nails from a Norwegian study population

Phthalate esters (PEs) and 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) used as additives in numerous consumer products are continuously released into the environment, leading to subsequent human exposure which might cause adverse health effects. The human biomonitoring approach allows the detection of PEs and DINCH in specific populations, by taking into account all possible routes of exposure (e.g. inhalation, transdermal and oral) and all relevant sources (e.g. air, dust, personal care products, diet). We have investigated the presence of nine PE and two DINCH metabolites and their exposure determinants in 61 adult residents of the Oslo area (Norway). Three urine spots and fingernails were collected from each participant according to established sampling protocols. Metabolite analysis was performed by LC-MS/MS. Metabolite levels in urine were used to back-calculate the total exposure to their corresponding parent compound. The primary monoesters, such as monomethyl phthalate (MMP, geometric mean 89.7ng/g), monoethyl phthalate (MEP, 104.8ng/g) and mono-n-butyl phthalate (MnBP, 89.3ng/g) were observed in higher levels in nails, whereas the secondary bis(2-ethylhexyl) phthalate (DEHP) and DINCH oxidative metabolites were more abundant in urine (detection frequency 84-100%). The estimated daily intakes of PEs and DINCH for this Norwegian population did not exceed the established tolerable daily intake and reference doses, and the cumulative risk assessment for combined exposure to plasticizers with similar toxic endpoints indicated no health concerns for the selected population. We found a moderate positive correlation between MEP levels in 3 urine spots and nails (range: 0.56-0.68). Higher frequency of personal care products use was associated with greater MEP concentrations in both urine and nail samples. Increased age, smoking, wearing plastic gloves during house cleaning, consuming food with plastic packaging and eating with hands were associated with higher levels in urine and nails for some of the metabolites. In contrast, frequent hair and hand washing was associated with lower urinary levels of monoisobutyl phthalate (MiBP) and mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), respectively.

Human metabolism and excretion kinetics of the fragrance lysmeral after a single oral dosage

2-(4-tert-Butylbenzyl)propionaldehyde, also known as lysmeral, lilial or lily-aldehyde (CAS No 80-54-6) is a synthetic fragrance used in a variety of consumer products like perfumes, after shave lotions, cosmetics and others. Due to its broad application, lysmeral was selected for the development of a biomonitoring method for the general population within the frame of the cooperation project of the Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI). The project also comprises the identification of suitable biomarkers of exposure in human urine as well as basic toxicokinetic data after defined, experimental exposure. For this purpose, 5 healthy subjects were orally dosed once with 5.26mg lysmeral. Urine was collected immediately before and for 48h after administration of the fragrance. The lysmeral metabolites lysmerol, lysmerylic acid, hydroxylated lysmerylic acid and 4-tert-butylbenzoic acid (TBBA) were determined in all urine samples by a newly developed UPLC-MS/MS (ultra-high pressure liquid chromatography combined with tandem mass spectrometry) method. Peak excretion for all metabolites occurred between 2 and 5h after oral application, with the primary metabolites (lysmerol and lysmerylic acid) being excreted about 1h earlier than the secondary metabolites (hydroxylated lysmerylic acid and TBBA). More than 90% of all measured lysmeral metabolites were excreted after 12h, with the renal excretion being virtually complete after 48h. After this time period, TBBA, lysmerol, lysmerylic acid and hydroxyl-lysmerylic acid represent on average 14.3, 1.82, 0.20 and 0.16%, respectively, of the dose administered. In total, the 4 metabolites determined represent about 16.5% of the dose. With the conversion factors derived from the controlled human study, we estimated median exposure doses for lysmeral in a group of 40 human volunteers from the general population of approximately 140-220μg per day. In conclusion, the lysmeral metabolites lysmerol, lysmerylic acid, hydroxyl-lysmerylic acid and TBBA in urine are suitable biomarkers of exposure and can be applied, either single or in any combination, for biomonitoring of the general population.