Human urinary excretion kinetics of the antimycotic climbazole: Biomonitoring of two new metabolites after oral and dermal dosage.

Climbazole is an antimycotic compound used in cosmetic products as a preservative or as an active ingredient in anti-dandruff (AD) formulations. In this study we provide human toxicokinetic data on climbazole. Using our previously published analytical method, we investigated the urinary excretion of two climbazole metabolites, (OH)2-climbazole and cx-OH-climbazole, for 48h after oral ingestion (n = 5, 49-77µg/kg bw) and for 72h after dermal application of either a climbazole-containing rinse-off AD shampoo or a leave-on hair tonic (n = 2×3). In total, 23.9% (18.0-33.4%) of the oral dose were excreted as the two abovementioned metabolites over 48h. In one volunteer, who used an over-the-counter phytopharmaceutical, metabolite excretion was about three times lower and we found influences on diastereoselectivity of (OH)2-climbazole formation using a modified analytical method. After dermal application, urinary concentration maxima occurred considerably later than after oral intake. The two different dermal exposure scenarios also revealed a relevance of exposure duration and product formulation on the systemic availability of climbazole. Back-calculated oral-dose-equivalent intakes from the dermal exposures showed a maximum climbazole intake of 18.5µg/kg bw/d after hair tonic use, or 6.6µg/kg bw/d after AD shampoo application.

A time trend of urinary 4-methylbenzylidene camphor metabolites in young adults from Germany.

4-methylbenzylidene camphor (4-MBC) is used as a UV-B filter in cosmetics. Two oxidized metabolites of 4-MBC - 3-(4-carboxybenzylidene)camphor (cx-MBC) and 3-(4-carboxybenzylidene)-6-hydroxycamphor (cx-MBC-OH) - were analyzed in 250 24-h urine samples from young adults in Germany. The samples were from the German Environmental Specimen Bank (ESB) and represented exposure in the years 1995, 2005, 2010, 2015 and 2019. A UHPLC-MS/MS method enabled the sensitive determination of both metabolites, with limits of quantification at 0.15 µg L-1 (cx-MBC) and 0.30 µg L-1 (cx-MBC-OH), respectively. A temporal trend of the internal exposure to 4-MBC was clearly noticeable. The metabolite cx-MBC was frequently quantifiable at the beginning of the period: in 70% of the samples in 1995, and 56% in 2005. After 2005, urinary concentrations and detection rates of cx-MBC dropped to reach very low levels. In 2015 and 2019, the detection rate was only 2% and 0%, respectively. A similar trend was observed for cx-MBC-OH, though overall, this metabolite was detected less often and at lower concentration levels than cx-MBC. Nowadays, measurable levels of urinary 4-MBC metabolites are an extremely rare occurrence in Germany. These trends are consistent with the history of 4-MBC use by the cosmetic industry. The highest measured individual concentration of 16.20 µg L-1 (in a sample of the year 2005) was still more than 30 times below the health-based guidance value (HBM-I). An investigation of the ratios between both metabolites uncovered several features of the 4-MBC metabolism which have been essentially overlooked until now. In particular, stereochemical aspects should be explored in future studies. As urine was collected in autumn/winter in Northwestern Germany, the 4-MBC metabolites measured probably do not arise from sunscreen products in a narrow sense. They rather may reveal the use of other skin care products containing 4-MBC for UV protection as an added feature.

A "dilute-and-shoot" column-switching UHPLC-MS/MS procedure for the rapid determination of branched nonylphenol in human urine: method optimisation and some fundamental aspects of nonylphenol analysis.

Technical grade branched nonylphenol (NP) was determined in human urine by online solid phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (SPE-UHPLC-MS/MS). Prior to analysis, urine specimens were simply diluted and enzymatically deconjugated. The run time of the chromatography, including SPE and re-equilibration, was 9 min per injection. The enzymatic cleavage of NP conjugates was optimised with incurred sample material from a human metabolism study: the highest recoveries were obtained with ß-glucuronidase from E. coli K 12 in 0.1 M ammonium acetate at pH 6.5, within a minimal hydrolysis time of 30 to 60 min. Using sodium acetate instead of ammonium acetate led to systematically decreased recovery rates. The analytical method was validated regarding its precision (coefficients of variation: 2.9-7.4%), accuracy (relative recovery rates: 93-105%), robustness (relative recovery rates in individual urine matrices: 92-117%), selectivity, and limit of quantification (1.0 µg L-1). Fundamental aspects in the analysis of technical product mixtures such as NP, comprising various isomers and homologues, were considered. Validation results, an exposure scenario and the application of the procedure to real samples, show that it enables a rugged monitoring of NP exposures above, at, and significantly below health-based guidance values, corresponding to daily NP intakes in the low µg kg-1 d-1 range. On the other hand, background levels in non-specifically exposed populations cannot be detected with this method. Hence, while alternative approaches should be pursued for NP analysis at environmental trace level, the speed and simplicity of our method are ideal for high-throughput human biomonitoring in occupational medicine.

Human Metabolism and Urinary Excretion Kinetics of Nonylphenol in Three Volunteers after a Single Oral Dose.

Nonylphenol (NP) is an endocrine-disrupting anthropogenic chemical that is ubiquitous in the environment. Human biomonitoring data and knowledge on internal NP exposure are still sparse, and its human metabolism is largely unknown. Therefore, in this study, we investigated human metabolism and urinary excretion of NP. Three male volunteers received a single oral dose of 1 mg 13C6-labeled NP (10.6-11.7 µg/kg body weight). Consecutive full urine voids were collected for 48 h. A metabolite screening identified nine ring- and/or side chain-oxidized metabolites. We chose the most promising hits, the alkyl chain-oxidized metabolites hydroxy-NP (OH-NP) and oxo-NP, for quantitative investigation next to the parent NP. For this purpose, we newly synthesized specific n - 1-oxidized monoisomeric analytical standards. Quantification of the polyisomeric metabolites was performed via online-solid phase extraction-LC-MS/MS with stable isotope dilution using a previously published consensus method. Alkyl chain hydroxylation (OH-NP) constituted the major metabolism pathway representing 43.7 or 62.2% (depending on the mass transition used for quantification) of the NP dose excreted in urine. The urinary excretion fraction (FUE) for oxo-NP was 6.0 or 9.3%. The parent NP, quantified via an analogous isomeric 13C6-NP standard, represented 6.6%. All target analytes were excreted predominately as glucuronic acid conjugates. Excretion was rather quick, with concentration maxima in urine 2.3-3.4 h after dosing and biphasic elimination kinetics (elimination half-times first phase: 1.0-1.5 h and second phase: 5.2-6.8 h). Due to its high FUE and insusceptibility to external contamination (contrary to parent NP), OH-NP represents a robust and sensitive novel exposure biomarker for NP. The novel FUEs enable to robustly back-calculate the overall NP intakes from urinary metabolite levels in population samples for a well-informed cumulative exposure and risk assessment.

A validated LC-MS/MS method for the quantification of climbazole metabolites in human urine.

Climbazole is a preservative and an anti-dandruff ingredient with applications in various cosmetic products. The general population is therefore exposed to this chemical, and exposure monitoring is desirable. We have postulated a pathway for the human metabolism of climbazole, leading to two specific metabolites which can be excreted via urine. An analytical method for the determination of these metabolites in human urine was developed and validated. The sample preparation includes an enzymatic hydrolysis protocol. The measurement as such is based on online solid phase extraction (SPE), coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Intra- and inter-series coefficients of variation (CV) were determined in the concentration range from 1 µg/l to 100 µg/l with spiked pooled urine samples, and they were consistently below 15%, mostly below 10%. The corresponding accuracies (mean relative recovery rates) in spiked pooled urine varied from 97% to 103%. The robustness of the method was estimated by spiking individual urine samples. At 1 µg/l, the robustness was rather limited due to interfering matrix peaks in several samples, but excellent results were obtained at 10 µg/l and 100 µg/l, with CVs between 7% and 14% and accuracies from 101% to 110%. Matrix interferences often seemed to be associated with higher creatinine contents (≥2.0 g/l) of the samples. We subsequently applied the method to urine specimens from a human metabolism study involving documented climbazole exposures. We were able to identify and quantify the postulated metabolites in those real samples, thus validating our metabolism hypothesis. We also investigated the precision and accuracy of the enzymatic deconjugation with the real samples. The deconjugation step was found to be highly repeatable and largely quantitative. Both metabolites formed glucuronides, though varying fractions were also excreted in unconjugated (free) forms. Phase II conjugates other than glucuronides did not seem to be produced in significant amounts. With our method, both climbazole metabolites can be reliably quantified in the range between about 1.5 µg/l (depending on matrix interferences in individual samples) and at least 500 µg/l.

Towards a systematic use of effect biomarkers in population and occupational biomonitoring.

Effect biomarkers can be used to elucidate relationships between exposure to environmental chemicals and their mixtures with associated health outcomes, but they are often underused, as underlying biological mechanisms are not understood. We aim to provide an overview of available effect biomarkers for monitoring chemical exposures in the general and occupational populations, and highlight their potential in monitoring humans exposed to chemical mixtures. We also discuss the role of the adverse outcome pathway (AOP) framework and physiologically based kinetic and dynamic (PBK/D) modelling to strengthen the understanding of the biological mechanism of effect biomarkers, and in particular for use in regulatory risk assessments. An interdisciplinary network of experts from the European chapter of the International Society for Exposure Science (ISES Europe) and the Organization for Economic Co-operation and Development (OECD) Occupational Biomonitoring activity of Working Parties of Hazard and Exposure Assessment group worked together to map the conventional framework of biomarkers and provided recommendations for their systematic use. We summarized the key aspects of this work here, and discussed these in three parts. Part I, we inventory available effect biomarkers and promising new biomarkers for the general population based on the H2020 Human Biomonitoring for Europe (HBM4EU) initiative. Part II, we provide an overview AOP and PBK/D modelling use that improved the selection and interpretation of effect biomarkers. Part III, we describe the collected expertise from the OECD Occupational Biomonitoring subtask effect biomarkers in prioritizing relevant mode of actions (MoAs) and suitable effect biomarkers. Furthermore, we propose a tiered risk assessment approach for occupational biomonitoring. Several effect biomarkers, especially for use in occupational settings, are validated. They offer a direct assessment of the overall health risks associated with exposure to chemicals, chemical mixtures and their transformation products. Promising novel effect biomarkers are emerging for biomonitoring of the general population. Efforts are being dedicated to prioritizing molecular and biochemical effect biomarkers that can provide a causal link in exposure-health outcome associations. This mechanistic approach has great potential in improving human health risk assessment. New techniques such as in silico methods (e.g. QSAR, PBK/D modelling) as well as 'omics data will aid this process. Our multidisciplinary review represents a starting point for enhancing the identification of effect biomarkers and their mechanistic pathways following the AOP framework. This may help in prioritizing the effect biomarker implementation as well as defining threshold limits for chemical mixtures in a more structured way. Several ex vivo biomarkers have been proposed to evaluate combined effects including genotoxicity and xeno-estrogenicity. There is a regulatory need to derive effect-based trigger values using the increasing mechanistic knowledge coming from the AOP framework to address adverse health effects due to exposure to chemical mixtures. Such a mechanistic strategy would reduce the fragmentation observed in different regulations. It could also stimulate a harmonized use of effect biomarkers in a more comparable way, in particular for risk assessments to chemical mixtures.

Metabolites of 4-methylbenzylidene camphor (4-MBC), butylated hydroxytoluene (BHT), and tris(2-ethylhexyl) trimellitate (TOTM) in urine of children and adolescents in Germany - human biomonitoring results of the German Environmental Survey GerES V (2014-2017).

The UV filter 4-methylbenzylidene camphor (4-MBC), used in cosmetics, the antioxidant butylated hydroxytoluene (BHT), used inter alia as a food additive and in cosmetics, and the plasticizer tris(2-ethylhexyl) trimellitate (TOTM), used mainly in medical devices as substitute for di-(2-ethylhexyl) phthalate (DEHP), are suspected to have endocrine disrupting effects. Human biomonitoring methods that allow for assessing the internal exposure of the general population to these substances were recently developed in a German cooperation to enhance the use of human biomonitoring. First-morning void urine samples from 3- to 17-year-old children and adolescents living in Germany were analysed for metabolites of 4-MBC (N = 447), BHT (N = 2091), and TOTM (N = 431) in the population-representative German Environmental Survey on Children and Adolescents 2014-2017 (GerES V). 4-MBC metabolites were found in quantifiable amounts only in single cases and exposure levels remained well below health-based guidance values. In contrast, ubiquitous exposure to BHT became evident with a geometric mean (GM) urinary concentration of the metabolite BHT acid of 2.346 µg/L (1.989 µg/gcreatinine) and a maximum concentration of 248 µg/L (269 µg/gcrea). The highest GM concentration was found in young children aged 3-5 years, yet no specific sources of exposure could be identified. Also, TOTM metabolites were found in quantifiable amounts only in very few samples. None of these findings could be related to previous hospital treatment or exposure via house dust. The presented results will be the basis to derive reference values for exposure of children and adolescents in Germany to BHT and will facilitate to identify changing exposure levels in the general population.

2-Mercaptobenzothiazole in urine of children and adolescents in Germany - Human biomonitoring results of the German Environmental Survey 2014-2017 (GerES V).

2-Mercaptobenzothiazole (2-MBT) is widely used as a vulcanisation accelerator and is contained in many products made from natural rubber, e.g. car tires. Additionally, it is used as a fungicide in paint or fibre. Systemically human exposure to 2-MBT can occur via dermal and oral uptake or inhalation. Locally, 2-MBT can cause skin sensitisation. The International Agency for Research on Cancer (IARC) classified 2-MBT as probably carcinogenic to humans. 516 urine samples of 3- to 17-year-old children and adolescents living in Germany were analysed for the concentration of 2-MBT in the population representative German Environmental Survey for Children and Adolescents 2014-2017 (GerES V). 2-MBT was quantified above the limit of quantification (LOQ) of 1.0 µg/L in 50% of the 516 samples analysed. The geometric mean of urinary 2-MBT concentration was 1.018 µg/L and 0.892 µg/gcreatinine, the arithmetic mean was 1.576 µg/L (1.351 µg/gcrea). The median concentration was below the LOQ. Analyses of subgroups revealed higher 2-MBT concentrations in children aged 3-5 years compared to 14- to 17-year-old adolescents. All urinary 2-MBT concentrations were well below the health-based guidance value HBM-I for children of 4.5 µg/L. Therefore, current exposure levels are - according to current knowledge - not of concern. For the first time, reference values can be derived for 2-MBT for children and adolescents in Germany. This will facilitate to recognise changing exposure levels in this population group in Germany and identification of unusually high exposures.

A biomonitoring study assessing the exposure of young German adults to butylated hydroxytoluene (BHT).

The antioxidant 2,6-di-tert-butyl-4-methylphenol (butylated hydroxytoluene, BHT) is used ubiquitously in food, cosmetics, pharmaceuticals, fuels, plastics, rubbers and many other products. Therefore, exposure of the general population to this substance is likely. We analyzed the BHT metabolite 3,5-di-tert-butyl-4-hydroxybenzoic acid ("BHT acid") in 24-h urine samples from the German Environmental Specimen Bank with the aim of gaining a better understanding of the internal burden of BHT in young nonspecifically exposed adults. The study population consisted of students between 20 and 29 years of age at the time of sampling, all from Halle/Saale in Central Germany. In total, 329 samples collected in the years 2000, 2004, 2008, 2012, 2015, and 2018 were measured by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). BHT acid was detected above the limit of quantification (0.2 µg/L) in 98% of the samples. The median of the measured concentrations was 1.06 µg/L and 1.24 µg/g creatinine respectively, the median of the daily excretion was 1.76 µg/24 h and - additionally normalized for body weight - 26.8 ng/24 h × kg bw respectively. The corresponding 90th percentiles were 3.28 µg/L, 3.91 µg/g creatinine, 5.05 µg/24 h, and 81.9 ng/24 h × kg bw. Medians of creatinine-corrected values were slightly higher in women than in men, while the opposite situation was observed for the volume concentrations and the 24-h excretion values (not corrected for body weight). Values simultaneously normalized both for 24-h excretion and body weight did not exhibit any significant differences between males and females, probably indicating a virtually identical magnitude of exposure for both genders. The background exposure of the investigated population was found to be largely constant since the year 2000, with only weak temporal trends at most. Daily intakes were estimated from excretion values and found to be largely below the acceptable daily intake (ADI) of BHT at 0.25 mg/kg bw: our worst-case estimate is a daily BHT intake of approximately 0.1 mg/kg bw at the 95th percentile level. However, these intake assessments rely on very limited quantitative data regarding human metabolism of BHT.

Hexamoll® DINCH and DPHP metabolites in urine of children and adolescents in Germany. Human biomonitoring results of the German Environmental Survey GerES V, 2014-2017.

The production and use of the plasticisers Hexamoll® DINCH (di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate) and DPHP (di-(2-propylheptyl) phthalate) have increased after both chemicals were introduced into the market in the early 2000s as substitutes for restricted high molecular weight phthalates. During the population representative German Environmental Survey (GerES) of Children and Adolescents (GerES V, 2014-2017), we collected urine samples and measured the concentrations of DINCH and DPHP metabolites in 2228 and in a subsample of 516 participants, respectively. We detected DINCH and DPHP metabolites in 100% and 62% of the 3-17 years old children and adolescents, respectively. Geometric means of DINCH metabolites were 2.27 µg/L for OH-MINCH, 0.93 µg/L for oxo-MINCH, 1.14 µg/L for cx-MINCH and 3.47 µg/L for DINCH (Σ of OH-MINCH + cx-MINCH). Geometric means of DPHP metabolites were 0.30 µg/L for OH-MPHP, 0.32 µg/L for oxo-MPHP and 0.64 µg/L for DPHP (Σ of OH-MPHP + oxo-MPHP). The 3-5 years old children had almost 3-fold higher DINCH biomarkers levels than adolescents (14-17 years). Higher concentrations of DPHP biomarkers among young children only became apparent after creatinine adjustment. Urinary levels of DINCH but not of DPHP biomarkers were associated with the levels of the respective plasticisers in house dust. When compared to HBM health-based guidance values, we observed no exceedance of the HBM-I value of 1 mg/L for DPHP (Σ of OH-MPHP + oxo-MPHP). However, 0.04% of the children exceeded the health based guidance value HBM-I of 3 mg/L for DINCH (Σ of OH-MINCH + cx-MINCH). This finding shows that even a less toxic replacement of restricted chemicals can reach exposures in some individuals, at which, according to current knowledge, health impacts cannot be excluded with sufficient certainty. In conclusion, we provide representative data on DINCH and DPHP exposure of children and adolescents in Germany. Further surveillance is warranted to assess the substitution process of plasticisers, and to advise exposure reduction measures, especially for highly exposed children and adolescents. Providing the results to the European HBM Initiative HBM4EU will support risk assessment and risk management not only in Germany but also in Europe.

Internal exposure of young German adults to di(2-propylheptyl) phthalate (DPHP): Trends in 24-h urine samples from the German Environmental Specimen Bank 1999-2017.

Di(2-propylheptyl) phthalate (DPHP) is used as a substitute for high molecular weight phthalates like di(2-ethylhexyl) phthalate (DEHP) which were subjected to authorization under REACh in 2015. An earlier study on the time trend of exposure in human 24-h urine samples from the German Environmental Specimen Bank has revealed that metabolites of DPHP emerged in 2009 and 2012 (Schütze et al., 2015). In order to better assess a potential trend and the present state of exposure to DPHP, we now measured 180 urine samples from the German Environmental Specimen Bank, 60 per year, collected in 2011, 2014 and 2017, randomized and blinded before analysis. Together with the previously analyzed samples, data for a total of 480 samples covering 19 years from 1999 to 2017 was thus generated. We were able to show that DPHP exposure of the studied population, university students from Münster (Northwestern Germany), has remained essentially constant since 2011, after a rapid increase starting around 2009. Even so, urinary metabolite concentrations were always in the low ppb or sub-ppb range, indicating that DPHP exposure of the general population is substantially lower than for other modern plasticizers, and far below levels currently regarded as critical. DPHP is a plasticizer which is mostly used in non-sensitive applications with little probability of close contact to humans. Still, we observed how temporal trends of DPHP exposure largely follow trends of DPHP consumption in the Western European market. Our results hence demonstrate the potential of biomonitoring to sensitively detect the effects of industrial product strategy on the environment, even when biomarkers are present only at trace level.

New specific and sensitive biomonitoring methods for chemicals of emerging health relevance.

In this publication the challenges to cope for the aim to obtain innovative biomonitoring methods in our laboratory are visualized for di(2-propylheptyl)phthalate, 2-mercaptobenzothiazole, 3,5-di-tert-butyl-4-hydroxytoluene, 4-nonylphenol, 4-tert-octylphenol, 3-(4-methylbenzylidene)camphor, 4,4'-methylene diphenyl diisocyanate, and Hexabromocyclododecane. For these substances new specific markers were explored based on animal or human kinetic data with urine being the preferred matrix compared to blood. The determination of these markers was complex in all cases, because the sample preparation as well as the detection by high performance liquid chromatography, capillary gas chromatography coupled to tandem mass spectrometers or high resolution mass spectrometry should enable the lowest possible detection limit by use of minimal biological sample volumes. To get a first hint of a possible background level, the analytical methods were applied to urine samples of about 40 persons for each chemical. For Di(2-propylheptyl)phthalate and 2-Mercaptobenzothiazole first results are presented from population biomonitoring.

New human biomonitoring methods for chemicals of concern-the German approach to enhance relevance.

In Germany strong efforts have been made within the last years to develop new methods for human biomonitoring (HBM). The German Federal Ministry for the Environment, Nature Conservation, Building and Nuclear Safety (BMUB) and the German Chemical Industry Association e. V. (VCI) cooperate since 2010 to increase the knowledge on the internal exposure of the general population to chemicals. The projects aim is to promote human biomonitoring by developing new analytical methods Key partner of the cooperation is the German Environment Agency (UBA) which has been entrusted with the scientific coordination. Another key partner is the "HBM Expert Panel" which each year puts together a list of chemicals of interest to the project from which the Steering Committee of the project choses up to five substances for which method development will be started. Emphasis is placed on substances with either a potential health relevance or on substances to which the general population is potentially exposed to a considerable extent. The HBM Expert Panel also advises on method development. Once a method is developed, it is usually first applied to about 40 non-occupationally exposed individuals. A next step is applying the methods to different samples. Either, if the time trend is of major interest, to samples from the German Environmental Specimen Bank, or, in case exposure sources and distribution of exposure levels in the general population are the focus, the new methods are applied to samples from children and adolescents from the population representative 5th German Environmental Survey (GerES V). Results are expected in late 2018. This article describes the challenges faced during method development and solutions found. An overview presents the 34 selected substances, the 14 methods developed and the 7 HBM-I values derived in the period from 2010 to mid 2016.

Occurrence of chlorinated and brominated dioxins/furans, PCBs, and brominated flame retardants in blood of German adults.

Persistent organic pollutants are widespread in the environment, and are associated with a particular health and ecological concern. The human body burden of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDDs/Fs), polybrominated dibenzo-p-dioxins and dibenzofurans (PBDDs/Fs), polychlorinated biphenyls (PCBs), polybrominated diphenylether (PBDEs), and hexabromocyclododecanes (HBCDs) was determined. Blood samples were collected in Germany, originating from 42 randomly selected subjects between 20 and 68 years old. The median (95th percentile) concentrations, expressed as WHO2005-TEQ for PCDD/PCDFs and dioxin-like PCBs, were 6.2 (19.1) pg/g l.w. and 4.1 (8.8) pg/g l.w., respectively. PBDDs/Fs were found with a median of 2.8 pgTEQ/g l.w. and a 95th percentile of 8.7 pgTEQ/g l.w. (using similar interim TEF values as for PCDDs/Fs) On a median basis, the contribution of PCDD/Fs, dioxin-like PCBs, and PBDDs/Fs to total TEQ were 47%, 31%, and 21%, respectively. The sum of the 6 non-dioxin-like PCBs exhibited a median of 267ng/g l.w. and a 95th percentile of 834ng/g l.w. The median value for the sum of six tetra- to hepta-PBDE congeners was 1.7ng/g l.w. (95th percentile: 4.9ng/g l.w.). BDE 209 was the most abundant congener with a median of 1.8ng/g l.w. HBCDs were only found in some samples, and concentrations ranged between the limit of detection (5ng/g l.w.) and the limit of quantification (16ng/g l.w.). Results for PBDEs and HBCDs are comparable to other European studies. Our study demonstrated that the body burden of PCDD/Fs and PCBs declined continously since the last three decades, but exposure may exceed precautionary guideline levels.

Rapid and sensitive LC-MS-MS determination of 2-mercaptobenzothiazole, a rubber additive, in human urine.

2-Mercaptobenzothiazole (MBT) is one of the most important vulcanization accelerators in the industrial production of rubber, especially car tires. Given its wide use in household articles and industrial rubber products it has a high potential to migrate into the environment. Humans can be exposed by dermal, oral, or inhalative routes. Incorporated MBT is excreted in urine, mainly as conjugates to glucuronide, sulfate, and mercapturic acid. On the basis of these facts MBT has been selected as a substance of high interest in the large scale 10-year German project on human biomonitoring (HBM); a cooperation between the German Federal Ministry for the Environment (BMUB) and the German Chemical Industry Association (VCI) with the objective of developing new analytical methods for relevant chemicals. The presented method was developed to determine MBT in human urine to reliably investigate the internal human MBT dose. Total MBT is measured after enzymatic hydrolysis followed by application of high-pressure liquid chromatography tandem mass spectrometry (HPLC-MS-MS) in positive-electrospray-ionization mode (ESI+) using isotope-dilution quantification. High sample throughput could be obtained by use of the column-switching technique. Optimization yielded an analytical method with a low and reproducible limit of detection (LOD) of 0.4 µg L(-1) and a limit of quantification (LOQ) of 1 µg L(-1), and low relative standard deviations in the range 1.6-5.8 %. A small biomonitoring study covering unexposed humans and occupationally exposed workers was performed to establish the feasibility and reliability of the method. MBT was found in only one urine sample from the unexposed humans, at a value of 10.8 µg MBT per liter, whereas it was found in all samples from the tested workers at values of up to 6210 µg MBT per liter.

Chromosomal alterations in exfoliated urothelial cells from bladder cancer cases and healthy men: a prospective screening study.

BACKGROUND: Chromosomal instability in exfoliated urothelial cells has been associated with the development of bladder cancer. Here, we analyzed the accumulation of copy number variations (CNVs) using fluorescence in situ hybridization in cancer cases and explored factors associated with the detection of CNVs in tumor-free men. METHODS: The prospective UroScreen study was designed to investigate the performance of UroVysion™ and other tumor tests for the early detection of bladder cancer in chemical workers from 2003-2010. We analyzed a database compiling CNVs of chromosomes 3, 7, and 17 and at 9p21 that were detected in 191,434 exfoliated urothelial cells from 1,595 men. We assessed the accumulation of CNVs in 1,400 cells isolated from serial samples that were collected from 18 cancer cases up to the time of diagnosis. A generalized estimating equation model was applied to evaluate the influence of age, smoking, and urine status on CNVs in cells from tumor-free men. RESULTS: Tetrasomy of chromosomes 3, 7 and 17, and DNA loss at 9p21 were the most frequently observed forms of CNV. In bladder cancer cases, we observed an accumulation of CNVs that started approximately three years before diagnosis. During the year prior to diagnosis, cells from men with high-grade bladder cancer accumulated more CNVs than those obtained from cases with low-grade cancer (CNV < 2: 7.5% vs. 1.1%, CNV > 2: 16-17% vs. 9-11%). About 1% of cells from tumor-free men showed polysomy of chromosomes 3, 7, or 17 or DNA loss at 9p21. Men aged ≥50 years had 1.3-fold more cells with CNVs than younger men; however, we observed no further age-related accumulation of CNVs in tumor-free men. Significantly more cells with CNVs were detected in samples with low creatinine concentrations. CONCLUSIONS: We found an accumulation of CNVs during the development of bladder cancer starting three years before diagnosis, with more altered cells identified in high-grade tumors. Also, a small fraction of cells with CNVs were exfoliated into urine of tumor-free men, mainly exhibiting tetraploidy or DNA loss at 9p21. Whether these cells are preferentially cleared from the urothelium or are artifacts needs further exploration.

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.

Biomonitoring following a chemical incident with acrylonitrile and ethylene in 2008.

The analytical determination of hemoglobin adducts was used as an effective biomonitoring tool after a fire outbrake at a chemical plant close to Cologne/Germany in 2008. More than 1000 people (e.g. fire-men, police officers, and workers) were potentially exposed to acrylonitrile and ethylene. Air monitoring in the surrounding was performed, and acrylonitrile was measured in concentrations up to 20 ppm, the mean value being 7 ppm (time range: 8 h). As many people were concerned about their individual body burden, biomonitoring was recommended for all people involved. 816 persons took advantage of this opportunity and came for blood sampling to the occupational health department of our company. Regarding the lifespan of erythrocytes up to 3 months, it was possible to analyze hemoglobin adducts of acrylonitrile and ethylene during and after the accident. In case of acrylonitrile the hemoglobin adduct N-(2-cyanoethyl) valine and regarding ethylene, N-(2-hydroxyethyl) valine was determined. As a result, the body burden was in nearly all cases within our internal adduct reference values (CyEtVal<15 µg/L blood or <612 pmol/g globin; HyEtVal<15 µg/L blood or 646 pmol/g globin). In about 1% of the cases, the adduct concentrations were slightly above these reference values. This means that the body burden measured by biomonitoring turned out to be far lower than the one expected from the air data. Therefore, following chemical incidents, in case biomonitoring is meaningful, it is highly recommended beside of air monitoring.

Screening for bladder cancer with urinary tumor markers in chemical workers with exposure to aromatic amines.

PURPOSE: To validate urinary markers for the early detection of bladder cancer (BC) in chemical workers. METHODS: UroScreen was conducted as a validation study for tumor markers within the frame of a health surveillance program of the German Social Accident Insurance for active or retired workers with former exposure to aromatic amines. From 2003 to 2010, 1,609 men took part in voluntary annual screens. Cytology, the quantitative NMP22(®) assay, and UroVysion™ were applied to 7,091 urine samples. RESULTS: Fifteen out of 21 tumors were detected following test positivity. The UroVysion/NMP22 panel detected 14 out of 21 tumors versus 8 tumors with cytology alone (sensitivity 66.7 vs. 44.4 %, specificity 94.5 vs. 98.5 %). The sensitivity of the panel increased to 85.7 % in samples collected ≤12 months before diagnosis and when papillomas were excluded, compared to 58.3 % with cytology. About 3 % of NMP22 tests were false-positive. UroVysion results overlapped with cytology due to the preselection of atypical cells. NMP22 was less and UroVysion more frequently positive in diluted urine samples. Leukocytes confounded NMP22 but not UroVysion. The low incidence of BC in this study population yielded low positive predictive values of the markers and high costs per tumor detected with screening. CONCLUSIONS: UroVysion in combination with NMP22 detected more cases than cytology alone, at the expense of a lower specificity. High costs per detected case resulted from a lower BC incidence than in the past when levels of occupational exposure to aromatic amines were higher. Currently, it cannot be recommended to apply these markers for screening in asymptomatic workers. The increase in sensitivity is not balanced by the high costs of UroVysion and the false-positive tests of NMP22.

Reliable quantitation of ß-hydroxyethoxyacetic acid in human urine by an isotope-dilution GC-MS procedure.

An analytical method for the determination of ß-hydroxyethoxyacetic acid (HEAA), the main urinary metabolite of 1,4-dioxane was developed and validated. The presented method involves liquid-liquid extraction of HEAA from the urine samples, followed by silylation and subsequent analytical separation and detection using GC-MS. The method is characterized by its simple and fast sample preparation in combination with a robust chromatography. The use of isotope dilution analysis enables an efficient compensation of matrix related effects and analyte losses due to sample workup. The excellent reliability and reproducibility of the method is demonstrated by the good accuracy and precision data. Within-day precision and day-to-day precision ranged from 0.6 to 1.2% and 1.5 to 2.6%, respectively. The mean relative recovery of the method was found to be 98-101%. The LOD and LOQ of HEAA were determined to be 0.2mg/L and 0.6mg/L, respectively. In summary, the presented analytical method is well suited to be used for routine biomonitoring of occupational exposure to 1,4-dioxane.