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International audience; BACKGROUND: Endocrine-disrupting effects of phthalates are understood primarily from in utero exposures within the fetal rat testis. Nevertheless, their path of action, dose-response character, and cellular target(s) within the fetal testis are not known. OBJECTIVES: In this study we investigated the effects of di-(2-ethylhexyl) phthalate (DEHP), mono-(2-ethylhexyl) phthalate (MEHP), and several of their metabolites on the development of organo-cultured testes from rat fetus. METHODS: We removed testes from 14.5-day-old rat fetuses and cultured them for 1-3 days with or without DEHP, MEHP, and the metabolites. RESULTS: DEHP (10(-5) M) produced a proandrogenic effect after 3 days of culture, whereas MEHP disrupted testis morphology and function. Leydig cells were the first affected by MEHP, with a number of them being inappropriately located within some seminiferous tubules. Additionally, we found a time- and dose-dependent reduction of testosterone. By 48 hr, gonocyte proliferation had decreased, whereas apoptosis increased. Sertoli cell number was unaffected, although some cells appeared vacuolated, and production of anti-Müllerian hormone decreased in a time- and dose-dependent manner. The derived metabolite mono-(2-ethyl-5-hydroxyhexyl) phthalate was the only one to cause deleterious effects to the rat fetal testis in vitro. CONCLUSION: We hope that this in vitro method will facilitate the study of different phthalate esters and other endocrine disruptors for direct testicular effects.

Based on the assumption that professional groups with frequent chemical exposure are at an increased risk for developing Multiple Chemical Sensitivity (MCS), a sample of 45 professional pest controllers was investigated.The examination of the pest controllers consisted of a physical and laboratory examination with urine screening for pyrethroid metabolites, a psychiatric interview, a neuropsychological test battery, and a chemical sensitivity questionnaire.Persistent or serious work related health problems and chemical sensitivity were not reported. In urine, cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA) was detected in 11%, 4-fluoro-3-phenoxybenzoic acid (F-PBA) in 7%. 3-phenoxybenzoic acid (3-PBA) exceeded the reference range in 9%, cis- and trans-3-(2,2-dichlorovinyl)-2,2-dimethyl-cyclopropane-1-carboxylic acid (Cl(2)CA) in 20%. Increased liver enzymes and blood count deviations were rather common. 38% had psychiatric disorders. With few exceptions, neuropsychological testing results were normal.The results do not support the hypothesis that work-related insecticide exposure promotes chemical sensitivity.

In May 2006, a serious environmental contamination with perfluorinated compounds (PFCs) became evident in a rural area of North Rhine-Westphalia (NRW) (Region Sauerland), Germany. In autumn 2006, we performed a human biomonitoring study in which a 4-8-fold increase in perfluorooctanoate (PFOA)-plasma concentrations of children, their mothers and men living in Arnsberg (District Hochsauerlandkreis, NRW) was observed compared with a reference population. The exposure was clearly related to the consumption of PFOA-contaminated tap water. However, there is no clear information on the duration of this contamination. The current investigation involves the analysis of PFCs in 30 blood samples of young adults (age 20-31 years) who had ever lived in the affected area. The samples were taken between 1977 and 2004 and stored at the German Environmental Specimen Bank for Human Tissues. Analyses of PFOA, perfluoroctanesulfonate (PFOS), perfluorohexanoate (PFHxA), perfluorohexanesulfonate (PFHxS), perfluoropentanoate (PFPA) and perfluorobutanesulfonate (PFBS) in blood plasma were performed by solid-phase extraction, HPLC and MS/MS detection. PFOA values (median, range) were 6.1, 1.7-40.7 microg/l, PFOS values were 18.8, 8.1-150.7 microg/l and PFHxS values were 1.7, 0.5-4.6 microg/l. The concentrations of PFHxA, PFPA and PFBS in plasma were all below limit of detection. Time-trend analysis showed that between 1977 and 2004 PFOA and PFOS levels remained fairly stable. PFOS and PFOA levels were in the range of current background levels of the general population. In contrast, PFHxS plasma levels have steadily increased since 1977. There was a close association between PFOS and PFOA-plasma levels. From this pilot study there are no indications for an increased exposure to PFCs of residents in Arnsberg in the years 1977-2004 prior to the contamination in 2006.

Concern about potential health impacts of low-level exposures to organ ophosphorus (OP) pesticides, bisphenol A (BPA), and phthalates among the general population is increasing. We measured levels of six dialkyl phosphate (DAP) metabolites of OP pesticides, a chlorpyrifos-specific metabolite (3,5,6-trichloro-2-pyridinol, TCPy), BPA, and 14 phthalate metabolites in urine samples of 100 pregnant women from the Generation R study, the Netherlands. The unadjusted and creatinine-adjusted concentrations were reported, and compared to National Health and Nutrition Examination Survey and other studies. In general, these metabolites were detectable in the urine of the women from the Generation R study and compared with other groups, they had relatively high-level exposures to OP pesticides and several phthalates but similar exposure to BPA. The median concentrations of total dimethyl (DM) metabolites was 264.0 nmol/g creatinine (Cr) and of total DAP was 316.0 nmol/g Cr. The median concentration of mono-ethyl phthalate (MEP) was 222.0 mu g/g Cr: the median concentrations of mono-isobutyl phthalate (MiBP) and mono-n-butyl phthalate (MnBP) were above 50 mu g/g Cr. The median concentrations of the three secondary metabolites of di-2-ethylhexyl phthalate (DEHP) were greater than 20 mu g/g Cr. The data indicate that the Generation R study population provides a wide distribution of selected environmental exposures. Reasons for the relatively high levels and possible health effects need investigation. (C) 2008 Elsevier Inc. All rights reserved.

Health protection of persons exposed to chemical substances is achieved by limitation and supervision of the concentrations a) of substances in the air (Environmental Monitoring, EM) and b) of substances and metabolites in body fluids (Biological Monitoring, BM). Both strategies belong to the sphere of responsibility of the analytical chemist. EM and BM show requirements which are different in some characteristic facts among them

Toxicological analysis in blood and urine for biological monitoring of occupationally exposed persons must be carried out in the Federal Republic of Germany under an internal and external quality control scheme. The results of many years of experiences with 5 commercially available control specimens (1 blood, 1 serum, 3 urine samples) from three companies for internal quality assurance are presented. Precision and accuracy were evaluated by comparison of our results with the data of the initial period (within day precision of the methods) and the assigned values. Over years the commercially available control specimens show good comparable results. Except in a few cases, there was a good agreement between our results and the assigned values. An expected worldwide increase of the importance of biological monitoring makes the availability of control specimens for everyday use a prerequisite in effective internal quality control. This is especially valid for the determination of organic compounds, e.g. solvents and pesticides in blood and urine samples.

Phthalates are used as plasticizers in PVC plastics. As the phthalate plasticizers are not chemically bound to PVC, they can leach, migrate or evaporate into indoor air and atmosphere, foodstuff, other materials, etc. Consumer products containing phthalates can result in human exposure through direct contact and use, indirectly through leaching into other products, or general environmental contamination. Humans are exposed through ingestion, inhalation, and dermal exposure during their whole lifetime, including intrauterine development. This paper presents an overview on current risk assessments done by expert panels as well as on exposure assessment data, based on ambient and on current human biomonitoring results. Some phthalates are reproductive and developmental toxicants in animals and suspected endocrine disruptors in humans. Exposure assessment via modelling ambient data give hints that the exposure of children to phthalates exceeds that in adults. Current human biomonitoring data prove that the tolerable intake of children is exceeded to a considerable degree, in some instances up to 20-fold. Very high exposures to phthalates can occur via medical treatment, i.e. via use of medical devices containing DEHP or medicaments containing DBP phthalate in their coating. Because of their chemical properties exposure to phthalates does not result in bioaccumulation. However, health concern is raised regarding the developmental and/or reproductive toxicity of phthalates, even in environmental concentrations.

In regulatory toxicology, the dose-response relationship between occupational exposure and biomarkers is of importance in setting threshold values. We analyzed the relationships between occupational exposure to polycyclic aromatic hydrocarbons (PAH) and various biomarkers of internal exposure and DNA damage with data from 284 highly exposed male workers. Personal exposure to phenanthrene and other PAHs was measured during shift and correlated with the sum of 1−, 2+9−, 3−, and 4-hydroxyphenanthrenes in post-shift urine. PAHs and hydroxyphenanthrenes were associated with DNA damage assessed in WBC as 8-oxo-7,8-dihydro-2′-deoxyguanosine/106 dGuo and strand breaks by Comet assay as Olive tail moment. Hydroxyphenanthrenes correlated with phenanthrene (Spearman rs = 0.70; P < 0.0001). No correlations could be found between strand breaks and exposure (rs = 0.01, P < 0.0001 for PAHs; rs = −0.03, P = 0.68 for hydroxyphenanthrenes). Correlations with 8-oxo-7,8-dihydro-2′-deoxyguanosine/106 dGuo were weakly negative (rs = −0.22, P = 0.004 for PAHs) or flat (rs = −0.08, P = 0.31 for hydroxyphenanthrenes). Linear splines were applied to assess the relationships between the log-transformed variables. All regression models were adjusted for smoking and type of industry. For hydroxyphenanthrenes, 51.7% of the variance could be explained by phenanthrene and other predictors. Up to 0.77 μg/m3 phenanthrene, no association could be found with hydroxyphenanthrenes. Above that point, hydroxyphenanthrenes increased by a factor of 1.47 under a doubling of phenanthrene exposure (slope, 0.56; 95% confidence interval, 0.47-0.64). Hydroxyphenanthrenes may be recommended as biomarker of occupational PAH exposure, whereas biomarkers of DNA damage in blood did not show a dose-response relation to PAH exposure. (Cancer Epidemiol Biomarkers Prev 2007;16(9):1863–73)

Perfluorinated compounds (PFC) are a large group of chemicals produced for several decades and widely used for many industrial and consumer applications. Human Biomonitoring studies reveal a background exposure of the general population to perfluorooctanoic acid (PFOA) and pefluorooctane sulfonate (PFOS) in many parts of the world. Reference values for PFOS and PFOA in the German population are currently not available. However, the data of three PFC human biomonitoring studies are taken as basis for deriving a preliminary reference value. The first two studies were performed in southern Germany with 105 (sampling period 2003-2004) and 356 adults (sampling period 2005). The third study was performed in North Rhine-Westphalia (sampling period October and November 2006) in connection with the high PFOA contamination of drinking water in the Sauerland region. Non PFOA exposed control groups comprised of 80 children and 153 females from Siegen and 103 men from Brilon. The whole study which could be taken as a basis for PFOS reference considerations comprised of 170 children, 317 females and 204 men. Though the studies are not representative for the German population, they provide at present the best available data basis for deriving reference values. The 95th percentile values of the studies were used and the following preliminary reference values are recommended: PFOA, 10microg/l for all groups; for PFOS 10microg/l for children at school beginner age, 15microg/l for adult females and 25microg/l for adult males.

Polychlorinated biphenyls (PCBs) have been in widespread industrial use in the 1960s and 1970s. Despite a worldwide reduction, environmental exposure remains an issue especially in contaminated buildings. Due to the ubiquitous presence and poor degradation of PCBs, public health concerns continue to exist; however, evidence on the actual health effects of chronic low-dose exposure is scanty. The objective of the present study is an assessment of subjective complaints of exposed subjects in comparison to a non-exposed control group and their inter-relation to plasma levels of PCB congeners. The plasma concentrations of PCB congeners were measured in 583 subjects who had worked for an average of 14.7+/-9.6 years in a contaminated building in Germany, and 205 control subjects working in a non-contaminated building. Subjective complaints were assessed with the 24-item 'Giessen Subjective Complaints List' (GSCL-24). The subjects under chronic low-dose exposure scored significantly higher values on all the GSCL subscales except 'stomach complaints' in comparison to the non-exposed subjects and a 'normal' sample derived from the literature. However, thorough statistical analysis revealed no correlation of symptoms and PCB congener plasma concentration; the scores on the subscale 'exhaustion were even higher in subjects with low PCB concentration. Subjects working in a PCB-contaminated building report more subjective complaints in comparison to non-exposed subjects, but the complaints are not related to current PCB plasma concentrations.

The aim of this study was to evaluate exposure and health outcome of children living close to industrial sources. Exposure and health outcome was assessed in nearly 1000 children at school beginner age living in the vicinity of industrial sources of three different hot spots (Duisburg North, Duisburg South and Dortmund Horde) and in a rural area of North Rhine Westphalia (NRW), Germany. The cross-sectional study was undertaken between March and May 2000. Exposure assessment comprised modelling of ambient air quality data and human biomonitoring (HBM). Depending on the site-specific contaminants, HBM included the measurement of PAH (polycyclic aromatic hydrocarbons) and benzene metabolites in urine as well as heavy metals in blood and urine. Markers of early effects were DNA strand breaks as measured by the comet assay in lymphocytes and excretion of alpha-1-microglobuline and N-acetyl-beta-D-glucosamidase in urine. Health outcome was assessed by questionnaire, lung function test, dermatological examination as well as by RAST (radioallergosorbent test), patch tests and prick tests. The influence of exposure variables on biomarkers and health outcome was measured by means of multiple linear and logistic regression analysis. The most striking results were as follows. Children living close to a coke oven plant (Duisburg North) had increased levels of PAH metabolites in urine, and DNA exposure was increased. Children living at the Dortmund Horde hot spot (increased chromium and nickel ambient air levels from a steel mill) revealed a high prevalence of allergic sensitizations. Sensitization, especially against nickel, was associated with the current internal nickel exposure, and nickel in ambient air was positively associated with the frequency of allergic symptoms. Children from the hot spot areas had increased specific airway resistance and total lung capacity as compared to those of the reference area. In Duisburg North particularly, specific airway resistance and total lung capacity significantly increased with increasing TSP (total suspended particulate). The only positive associations between external and internal exposure were found between benzo[a]pyrene in ambient air and 1-hydroxypyrene in urine, and between lead in ambient air and in the blood of the children. It is concluded that despite improvements of the general air quality during the last decades, living in the vicinity of industrial sources results to some extent in increased internal contaminant exposure and in effects on health outcome. Still ongoing studies are aimed to find out whether the increased PAH and DNA exposure of children from Duisburg North had decreased after the coke oven plant had been shut down in 2003, and if the striking results on the high prevalence of allergic sensitization can be confirmed by introducing an expanded cross-sectional study at four hot spots with increased chromium and nickel ambient air levels.

In a retrospective human biomonitoring study we analyzed 24h urine samples taken from the German Environmental Specimen Bank for Human Tissues (ESBHum), which were collected from 634 subjects (predominantly students, age range 20-29 years, 326 females, 308 males) in 9 years between 1988 and 2003 (each n >or= 60), for the concentrations of primary and/or secondary metabolites of di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP), butylbenzyl phthalate (BBzP), di(2-ethylhexyl) phthalate (DEHP) and di-iso-nonyl phthalate (DiNP). Based on the urinary metabolite excretion we estimated daily intakes of the parent phthalates and investigated the chronological course of the phthalate exposure. In over 98% of the urine samples metabolites of all five phthalates were detectable indicating a ubiquitous exposure of the German population to all five phthalates throughout the last 20 years. The median daily intakes in the subsets between 1988 and 1993 were quite constant for DnBP (approx. 7 microg/kg bw/d) and DEHP (approx. 4 microg/kg bw/d). However, from 1996 the median levels of both phthalates decreased continuously until 2003 (DnBP 1.9 microg/kg bw/d; DEHP 2.4 microg/kg bw/d). By contrast, the daily intake values for DiBP were slightly increasing over the whole time frame investigated (median 1988: 1.1 microg/kg bw/d; median 2003: 1.4 microg/kg bw/d), approximating the levels for DnBP and DEHP. For BBzP we observed slightly decreasing values, even though the medians as of 1998 levelled off at around 0.2 microg/kg bw/d. Regarding daily DiNP exposure we found continuously increasing values, with the lowest median being 0.20 microg/kg bw/d for the subset of 1988 and the highest median for 2003 being twice as high. The trends observed in phthalate exposure may be associated with a change in production and usage pattern. Female subjects exhibited significantly higher daily intakes for the dibutyl phthalates (DnBP p=0.013; DiBP p=0.004). Compared to data from US National Health and Nutrition Examination Surveys (NHANES) exposure levels of the dibutyl phthalates were generally higher in our German study population, while levels of BBzP were somewhat lower. Overall, for a considerable 14% of the subjects we observed daily DnBP intakes above the tolerable daily intake (TDI) value deduced by the European Food Safety Authority (EFSA) (10 microg/kg bw/d). However, the frequency of exceedance decreased during the years and was beneath 2% in the 2003 subset. Even though transgressions of the exposure limit values of the EFSA and the US Environmental Protection Agency (US EPA) occurred only in a relatively small share of the subjects, one has to take into account the cumulative exposure to all phthalates investigated and possible dose-additive endocrine effects of these phthalates.

The areas along the rivers Rhine, Ruhr and Wupper in North Rhine-Westphalia (NRW), Germany, represent the largest urban and industrial agglomeration in Europe with about 10 million inhabitants. Human biomonitoring (HBM) studies have been conducted in these areas since more than 30 years, mainly designed to evaluate internal exposure to air pollutants. Recent studies were focussed on residents living near industrial sources. The contaminants studied comprise heavy metals, metabolites of polycyclic aromatic hydrocarbons (PAH), persistent organic pollutants (POPs), volatile organic compounds (VOC), and markers of DNA exposure. Study groups were mainly children and elderly subjects. Human milk, blood, urine, teeth, hair and nails were investigated. Time trend analyses demonstrate a significant decline of exposure to many contaminants such as POPs and heavy metals. More recent studies suggest that there still is an increased internal exposure to metals, PAH and DNA damaging agents in children and women living very close to industrial sources.

In Germany, the Human Biomonitoring Commission of the German Federal Environment Agency was established in 1992 to develop scientifically based criteria for the application of human biomonitoring (HBM). The goal is to clarify fundamental and practical issues related to HBM. Following the assessment of pollutants in body fluids, the commission derives two different kinds of guideline values: reference values and HBM values (HBM I and HBM II values). This article gives a review of the current reference values, HBM values, and the work of the German Human Biomonitoring Commission.

Several aromatic amines (AA) could cause bladder cancer and are an occupational hygiene problem in the workplace. However, little is known about the percutaneous absorption of chemicals via impaired skin and about the efficacy of skin protection measures to reduce internal exposure.To determine the impact of skin status and of skin protection measures on the internal exposure to AA in workers manufacturing rubber products.51 workers occupationally exposed to aniline and o-toluidine were examined. The workplace conditions, risk factors for skin and the use of personal protective equipment were assessed by means of a self-administered questionnaire. The skin of hands and forearms was clinically examined. Exposure to aniline and o-toluidine was assessed by ambient air and biological monitoring (analyses of urine samples and of haemoglobin adducts).Haemoglobin-AA-adduct levels in workers with erythema (73%) were significantly higher (p0.04) than in workers with healthy skin (mean values: aniline 1150.4 ng/l vs 951.7 ng/l, o-toluidine 417.9 ng/l vs 118.3 ng/l). The multiple linear regression analysis showed that wearing gloves significantly reduced the internal exposure. A frequent use of skin barrier creams leads to a higher internal exposure of AA (p0.03). However, the use of skincare creams at the workplace was associated with a reduced internal exposure (p0.03). From these findings we assume that internal exposure of the workers resulted primarily from the percutaneous uptake.The study demonstrates a significantly higher internal exposure to AA in workers with impaired skin compared with workers with healthy skin. Daily wearing of gloves efficiently reduced internal exposure. However, an increased use of skin barrier creams enhances the percutaneous uptake of AA. Skincare creams seem to support skin regeneration and lead to reduced percutaneous uptake.

Background Polychlorinated biphenyls are toxic and slowly degrading substances that have been in widespread industrial use in the 1960s and 1970s. Despite a worldwide reduction, environmental exposure remains an issue; the assessment of an individual’s level of exposure at a given time in retrospect requires valid toxicokinetic modeling of the different PCB congeners. Objectives To develop and verify a toxicokinetic model of the in vivo-degradation of PCB 28 and 52 in adult humans. Methods The plasma concentrations of PCB congeners were measured in 583 persons working in a contaminated building for an average of 14.7 ± 9.6 years. The values were adapted to a simple pharmacokinetic model ( y t = y 0 × (1 − e − kt )); the steady state concentration y 0 was estimated based on the median values of persons working exposed more than 10 years. Results The steady state concentration of PCB 28 was 0.10 μg/l, that of PCB 52 0.02 μg/l. The half-life of the congeners was estimated based on a logarithmic regression analysis according to the model mentioned above; it was 2.18 (95% CI: 1.91–2.54) years for PCB 28 and 3.95 (95% CI: 3.55–4.45) years for PCB 52, respectively. Stepwise elimination of persons with very long employment duration did not reduce the estimated half-life. Conclusions The estimated half-life is higher than previously published data especially for PCB 52. Possible reasons and implications of this finding are discussed.

Acrylamide (AA) is a widely studied industrial chemical that is neurotoxic, mutagenic to somatic and germ cells, and carcinogenic in mice and rats. AA is also formed during cooking in many commonly consumed starchy foods. Our previous toxicokinetic investigations of AA and its genotoxic metabolite, glycidamide (GA), in rodents showed that AA is highly bioavailable from oral routes of administration, is widely distributed to tissues, and that the dietary route, in particular, favors metabolism to GA. Formation and accumulation of mutagenic GA–DNA adducts in many tissues support the hypothesis that AA is carcinogenic in rodent bioassays through metabolism to GA. The current investigation describes the quantification of 24 h urinary metabolites, including free AA and GA and their mercapturic acid conjugates (AAMA and GAMA, respectively), using LC/MS/MS in F344 rats and B6C3F1 mice following a dose of 0.1 mg/kg bw given by intravenous, gavage, and dietary routes of administration. Similar groups of rodents were used previously for serum/tissue toxicokinetic and adduct determinations (DNA and hemoglobin). The goal was to investigate relationships between urinary and circulating biomarkers of exposure, toxicokinetic parameters for AA and GA, and tissue GA–DNA adducts in rodents from single doses of AA. Significant linear correlations were observed between urinary levels of AA with AAMA and GA with GAMA in the current data sets for rats and mice. Concentrations of AA and AAMA correlated significantly with average AUC values determined previously for AA in groups of rats and mice similarly dosed with AA. Urinary GA and GAMA concentrations showed significant correlations with average AUC values for GA and liver GA–DNA adducts determined previously in rats and mice similarly dosed with AA. Despite statistical significance, considerable inter-animal variability was observed in all urinary measurements, which limited the degree of correlation with either average toxicokinetic or biomarker data collected from different groups of animals. These results suggest that urinary measurements of AA and its metabolites may be useful for prediction of internal exposures to AA and GA.

There are only few studies about the relationship of skin lesions and the percutaneous uptake of hazardous substances in exposed workers. Several aromatic amines are well known carcinogens for humans and/or animals. This case report emphasizes the relevance of dermal absorption of the aromatic amine ortho-toluidine considering four workers with different skin status (healthy, erythematous and burned skin as well as dishydrotic eczema) during the vulcanisation process of rubber products in a components supplier plant for automobile industry. The results of our case report show that dermal absorption of o-toluidine through damaged epidermal barrier is significantly higher than through healthy skin.

Di-iso-nonylphthalate (DINP) is the major plasticizer for polyvinylchloride (PVC) polymers. Two DINP products are currently produced: DINP 1 and DINP 2. We analyzed the isononyl alcohol mixtures (INA) used for the synthesis of these two DINP plasticizer products and thus identified 4-methyloctanol-1 as one of the major constituents of the alkyl side chains of DINP 1 (8.7%) and DINP 2 (20.7%). Based on this isomer, we postulated the major DINP metabolites renally excreted by humans: mono-(4-methyl-7-hydroxy-octyl)phthalate (7OH-MMeOP), mono-(4-methyl-7-oxo-octyl)phthalate (7oxo-MMeOP) and mono-(4-methyl-7-carboxy-heptyl)phthalate (7carboxy-MMeHP). We present a fast and reliable on-line clean-up HPLC method for the simultaneous determination of these three DINP metabolites in human urine. We used ESI-tandem mass spectrometry for detection and isotope dilution for quantification (limit of quantification 0.5microg/l). Via these three oxidised DINP isomer standards, we quantified the excretion of all oxidised DINP isomers with hydroxy (OH-MINP), oxo (oxo-MINP) and carboxy (carboxy-MINP) functional groups. With this approach, we can for the first time reliably quantify the internal burden of the general population to DINP. Mean urinary metabolite concentrations in random samples from the general German population (n=25) were 14.9microg/l OH-MINP, 8.9microg/l oxo-MINP and 16.4microg/l carboxy-MINP. Metabolites strongly correlated with each other over all samples analyzed (R>0.99, p

We conducted a cross-shift study with 66 bitumen-exposed mastic asphalt workers and 49 construction workers without exposure to bitumen. Exposure was assessed using personal monitoring of airborne bitumen exposure, urinary 1-hydroxypyrene (1-OHP), and the sum of 1-, 2 + 9–,3-,4-hydroxyphenanthrene (OHPH). Genotoxic effects in WBC were determined with nonspecific DNA adduct levels of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and the formation of DNA strand breaks and alkali-labile sites. Concentration of fumes and aerosols of bitumen correlated significantly with the concentrations of 1-OHP and OHPH after shift (rs = 0.27; P = 0.03 and rs = 0.55; P < 0.0001, respectively). Bitumen-exposed workers had more DNA strand breaks than the reference group (P < 0.0001) at both time points and a significant correlation with 1-OHP and OHPH in the postshift urines (rs = 0.32; P = 0.001 and rs = 0.27; P = 0.004, respectively). Paradoxically, we measured higher levels of DNA strand breaks, although not significant, in both study groups before shift. 8-OxodGuo adduct levels did not correlate with DNA strand breaks. Further, 8-oxodGuo levels were associated neither with personal exposure to bitumen nor with urinary metabolite concentrations. Significantly more DNA adducts were observed after shift not only in bitumen-exposed workers but also in the reference group. Only low-exposed workers had significantly elevated 8-oxodGuo adduct levels before as well as after shift (P = 0.0002 and P = 0.02, respectively). Our results show that exposure to fumes and aerosols of bitumen may contribute to an increased DNA damage assessed with strand breaks. (Cancer Epidemiol Biomarkers Prev 2006;15(4):645–51)

Possible health hazards of fumes and aerosols of bitumen are in discussion, and data on their adverse effects on human airways under current exposure conditions are limited. To assess the irritative effects of exposure to fumes and aerosols of bitumen on the airways, a cross-sectional cross-shift study was conducted including external and internal exposure measurements, spirometry and especially non-invasive methods like nasal lavage collection and induction of sputum in order to identify and evaluate more precisely inflammatory process in the upper and lower airways. The cross-shift study comprised 74 mastic asphalt workers who were exposed to fumes and aerosols of bitumen and 49 construction workers without this exposure as reference group. Questionnaire, spirometry, ambient monitoring and urinary analysis were performed. Humoral and cellular parameters were measured in nasal lavage fluid (NALF) and induced sputum. For data analysis, a mixed linear model was performed on the different outcomes with exposure group, time of measurement (pre-, post-shift), current smoking, German nationality and age as fixed factors and subjects as random factor. Based on personal exposure measurements during shift, mastic asphalt workers were classified into a low (or =10 mg/m(3); n = 46) and a high (10 mg/m(3); n = 28) exposure group. High exposure was accompanied by significant higher urinary post-shift concentrations of 1-hydroxypyrene and the sum of hydroxyphenanthrenes. Acute respiratory symptoms were reported more frequently in the high exposure group after shift. Significant cross-shift declines in lung function parameters (forced expiratory volume in 1 s [FEV(1) (% predicted)] and forced vital capacity [FVC (% predicted)]) were measured in mastic asphalt workers. Pre-shift FEV(1) (% predicted) and FVC (% predicted) were higher in the low exposure group. In pre- and post-shift NALF samples, interleukin (IL)-1beta-, IL-8- and total protein concentrations were lower in the low exposure group compared to the reference and the high exposure group. Pre- and post-shift neutrophil percentages in both nasal and sputum samples were also lower in the low exposure group. Significantly higher pre- and post-shift sputum concentrations of IL-8, IL-6, nitrogen oxide (NO) derivatives and total protein were detected especially in highly exposed workers. Irritative effects of exposure to fumes and aerosols of bitumen on the upper and lower airways were apparent, especially in mastic asphalt workers with exposure above 10 mg/m(3).

Methylglyoxal (MG) is a sugar degradation product, which is endogenously formed by fragmentation of triose phosphates during glycolysis, ketone body metabolism of acetone, and catabolism of threonine. Food, beverages, and medical products are important exogenous sources with concentrations of up to 100 microM MG. MG is a reactive dicarbonyl compound, which easily modifies amino groups of proteins (glycation reaction) and thereby induces proinflammatory responses. Moreover, increased mutation frequencies in mammalian cells after treatment with MG have been reported, which are caused by stable modifications of DNA bases. Thus far, two types of adducts have been identified, which are formed during the reaction of free guanine or 2'-deoxyguanosine with high MG concentrations. In this study, we investigated the prolonged exposure of DNA to physiological MG concentrations. DNA was incubated with MG, enzymatically hydrolyzed to release the free nucleosides, and then analyzed by LC-MS/MS. We detected four products, which were derived from the reaction of 2'-deoxyguanosine and 2'-deoxyadenosine with 1 and 2 equiv of MG each. The adducts with 1 equiv of MG were identified as N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) and N6-(1-carboxyethyl)-2'-deoxyadenosine. LC-MS/MS was optimized for these compounds, and incubation of DNA was repeated using physiological concentrations of 10 microM MG. Thereby, CEdG proved to be the most sensitive and suitable marker for the reaction of DNA with MG (negative MRM mode, three mass transitions [M - 1](-) 338-->178, 338-->106, and 338-->149).

High amounts of acrylamide in some foods result in an estimated daily mean intake of 50 μg for a western style diet. Animal studies have shown the carcinogenicity of acrylamide upon oral exposure. However, only sparse human toxicokinetic data is available for acrylamide, which is needed for the extrapolation of human cancer risk from animal data. We evaluated the toxicokinetics of acrylamide in six young healthy volunteers after the consumption of a meal containing 0.94 mg of acrylamide. Urine was collected up to 72 hours thereafter. Unchanged acrylamide, its mercapturic acid metabolite N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), its epoxy derivative glycidamide, and the respective metabolite of glycidamide, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA), were quantified in the urine by liquid chromatography-mass spectrometry. Toxicokinetic variables were obtained by noncompartmental methods. Overall, 60.3 ± 11.2% of the dose was recovered in the urine. Although no glycidamide was found, unchanged acrylamide, AAMA, and GAMA accounted for urinary excretion of (mean ± SD) 4.4 ± 1.5%, 50.0 ± 9.4%, and 5.9 ± 1.2% of the dose, respectively. Apparent terminal elimination half-lives for the substances were 2.4 ± 0.4, 17.4 ± 3.9, and 25.1 ± 6.4 hours. The ratio of GAMA/AAMA amounts excreted was 0.12 ± 0.02. In conclusion, most of the acrylamide ingested with food is absorbed in humans. Conjugation with glutathione exceeds the formation of the reactive metabolite glycidamide. The data suggests an at least 2-fold and 4-fold lower relative internal exposure for glycidamide from dietary acrylamide in humans compared with rats or mice, respectively. This should be considered for quantitative cancer risk assessment. (Cancer Epidemiol Biomarkers Prev 2006;15(2):266–71)

The aim of this study was to investigate whether it is possible to detect high-resolution computed tomography (HRCT) findings in aluminium powder workers, which are consistent with early stages of aluminosis. 62 male workers from 8 departments of two plants producing aluminium (Al) powder were investigated using a standardized questionnaire, physical examination, lung function analysis, biological monitoring of Al in plasma and urine, chest X-ray, HRCT and immunological tests. Chronic bronchitis was observed in 15 (24.2%) of the workers, and four workers (6.5%) reported shortness of breath during exercise. HRCT findings in 15 workers (24.2%) were characterized by ill-defined centrilobular nodular opacities. Workers with ill-defined centrilobular nodular opacities had a lower vital capacity than workers who had no such HRCT-findings (90.9 % pred. vs. 101.8 % pred., p = 0.01). Biological monitoring in plasma and urine revealed higher internal exposure to Al in affected workers (33.5 μg/l plasma to 15.4 μg/l plasma, p = 0.01) and (340.5 μg/g creat. to 135.1 μg/g creat., p = 0.007). Years of exposure and concentration of aluminum in urine and plasma appear to be the best predictors for HRCT findings. Age and decreased vital capacity show borderline significance. We conclude that aluminosis is still relevant in occupational medicine. With HRCT it is possible to detect early stages of aluminosis and biological monitoring can be used to define workers at high risk.

The European Council Directive 98/24 on the protection of the health and safety of workers exposed to chemical agents sets out provisions for environmental and biological monitoring, making specific reference to binding limit values and health surveillance measures for those with exposure to leadTo compare how the Directive has been implemented at a national level in EU countries and to determine whether workers receive equivalent protection.Information on selected key issues was collected from 14 EU countries by means of a structured questionnaire.National occupational exposure limit values generally reflect that set by the Directive (0.15 mg/m(3)), but in five cases lower limits are set. National binding biological limit values range from 20 microg/100 ml blood in one country up to 80 microg/100 ml blood in others. The risk to the unborn child is generally recognised with specific measures for women of child-bearing potential or those that are pregnant or breast feeding. In only three countries are special arrangements included for young workers. Limits at which medical surveillance is put into effect are more consistent at 40 microg/100 ml in most countries. The Directive also refers to guidelines for health surveillance but none have been issued with respect to lead. Thus monitoring strategies and requirements for analytical performance vary considerably.The results of this survey suggest that protection of workers against the risk of exposure to lead at work is far from uniform across the European Union. Such disparity may also have implications on the requirements set at national level for laboratories measuring lead in blood and/or air. In the interest of harmonisation within the EU, further consideration should be given to the Annex II of the EC Directive 98/24, taking into account the suggestions for lower binding limit values for lead; this should include full guidelines for medical surveillance and requirements for laboratories should be issued.

We investigated the human metabolism of AA to the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-L-cysteine (AAMA) and N-(R/S)-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-L: -cysteine (GAMA) which are derived from AA itself and from its oxidative genotoxic metabolite glycidamide (GA), respectively. A healthy male volunteer received a single dose of about 1 mg deuterium-labelled acrylamide (d(3)-AA), representing 13 microg/kg body weight, in drinking water. Urine samples before dosing and within 46 h after the dose were analysed for d(3)-AAMA and d(3)-GAMA by LC-ESI-MS/MS. A first phase of increase in urinary concentration was found to last 18 h with a broad plateau between 8 and 18 h for AAMA, and 22 h for GAMA. Elimination half-lives of both AAMA and GAMA were estimated to be approximately 3.5 h for the first phase and more than 10 h up to few days for the second phase. Total recovery in urine after 24 h was about 51% as the sum of AAMA and GAMA and hereby well in accordance with former studies in rats. After 2 days AAMA, accounting for altogether 52% of the total AA dose, was the major metabolite of AA in humans. GAMA, accounting for 5%, appeared as a minor metabolite of AA. In humans we found a urinary ratio of 0.1 for GAMA/AAMA compared to previously reported values of 0.2 for rats and 0.5 for mice. Therefore, the metabolic fate of AA in humans was more similar to that in rats than in mice as already demonstrated in terms of the haemoglobin adducts. Consequently a genotoxic potency of AA mediated by GA could be supposed to be comparable in rats and humans.

Each year thousands of healthy volunteers undergo apheresis procedures to donate blood components and safe lives. However, many disposables used in apheresis contain di(2-ethylhexyl)phthalate (DEHP). This way, donors are exposed to DEHP, which is a reproductive and developmental toxicant in animals and a suspected endocrine modulator in humans. We quantified the DEHP exposure of six plasma donors, six discontinuous-flow platelet donors and six continuous-flow platelet donors by determining three specific metabolites in urine (5OH-MEHP: mono(2-ethyl-5-hydroxyhexyl)phthalate; 5oxo-MEHP: mono(2-ethyl-5-oxo-hexyl)phthalate and MEHP: mono(2-ethylhexyl)phthalate). We found maximum concentrations in urine samples after the discontinuous-flow plateletpheresis procedure with 826 microg/l for 5OH-MEHP, 774 microg/l for 5oxo-MEHP and 266 microg/l for MEHP (mean of the six volunteers). Metabolite excretions were found to be significantly (p

A cross-sectional study was conducted in 170 German workers exposed to polycyclic aromatic hydrocarbons (PAH) to investigate the role of 11 polymorphisms of CYP1A1, CYP1A2, CYP1B1, CYP3A4, EPHX1, GSTM1, GSTT1, and GSTP1 in the association between occupational exposure to PAH and urinary PAH metabolites. Polymorphisms were genotyped with real-time PCR. Exposure to 16 PAH was measured by personal air sampling. Urinary concentrations of 1-hydroxypyrene (1-OHP) and the sum of 1-, 2+9-, 3-, and 4-hydroxyphenanthrenes (OHPhe) were determined post-shift. Urinary 1-OHP and OHPhe correlated significantly with exogenous pyrene (Spearman r=0.52, p

The metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans was studied after three doses of 0.35 mg (4.7 microg/kg), 2.15 mg (28.7 microg/kg) and 48.5 mg (650 microg/kg) of D4-ring-labelled DEHP were administered orally to a male volunteer. Two new metabolites, mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) and mono[2-(carboxymethyl)hexyl]phthalate (2cx-MMHP) were monitored for 44 h in urine and for 8 h in serum for the high-dose case, in addition to the three metabolites previously analysed: mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP). For the medium- and low-dose cases, 24 h urine samples were analysed. Up to 12 h after the dose, 5OH-MEHP was the major urinary metabolite, after 12 h it was 5cx-MEPP, and after 24 h it was 2cx-MMHP. The elimination half-lives of 5cx-MEHP and 2cx-MMHP were between 15 and 24 h. After 24 h 67.0% (range: 65.8-70.5%) of the DEHP dose was excreted in urine, comprising 5OH-MEHP (23.3%), 5cx-MEPP (18.5%), 5oxo-MEHP (15.0%), MEHP (5.9%) and 2cx-MMHP (4.2%). An additional 3.8% of the DEHP dose was excreted on the second day, comprising 2cx-MMHP (1.6%), 5cx-MEPP (1.2%), 5OH-MEHP (0.6%) and 5oxo-MEHP (0.4%). In total about 75% of the administered DEHP dose was excreted in urine after two days. Therefore, in contrast to previous studies, most of the orally administered DEHP is systemically absorbed and excreted in urine. No dose dependency in metabolism and excretion was observed. The secondary metabolites of DEHP are superior biomonitoring markers compared to any other parameters, such as MEHP in urine or blood. 5OH-MEHP and 5oxo-MEHP in urine reflect short-term and 5cx-MEHP and 2cx-MMHP long-term exposure. All secondary metabolites are unsusceptible to contamination. Furthermore, there are strong hints that the secondary oxidised DEHP metabolites-not DEHP or MEHP-are the ultimate developmental toxicants.

Aim: This pilot study attempts to assess how far the standardized questionnaires are a valid tool to detect the food-related burden of acrylamide. Acrylamide is a toxic substance classified by the International Agency of Research on Cancer, as well as the Deutsche Forschungsgemeinschaft, as a probable human carcinogen. Methods: A venous blood sample was taken in order to determine the smoking-specific acrylnitrile adduct N-cyanoethylvaline and the acrylamide adduct N-2-carbamoylethylvaline in a female study population expecting delivery soon. A standardized questionnaire was used to determine the consumption of acrylamide-contaminated food. The results of our questionnaire were transferred to a linear evaluation system. Finally, anamnestic data of the questionnaire were correlated to objective parameters such as blood levels of hemoglobin adducts of acrylamide and acrylonitrile. Results: A positive correlation between the acrylamide intake and the levels of hemoglobin adducts in our study population was not proven. Conclusions: Evaluation of food-related exposure to acrylamide is difficult due to several reasons. Firstly, the validity of anamnestic data strongly depends on the patient’s ability to remember precisely all consumed food (quality as well as quantity) over a 3-month period. In addition, the contamination of acrylamide in food varies from one product to another; even the contamination of the same product is variable. Therefore, the missing correlation between the questionnaire and hemoglobin adduct rates is rather due to restricted validity of anamnestic data.

Objectives: Some phthalates, among them di-n-butylphthalate (DnBP) and butylbenzylphthalate (BBzP), are known reproductive and developmental toxicants in animals and suspected endocrine disruptors in humans. Children are probably the most susceptible to these effects. To obtain an estimate of internal exposure to DnBP and BBzP we compared the excretion of their metabolites in the urine of nursery school children with that of their teachers and parents. Methods: We measured the urinary mono-ester metabolites of DnBP, mono-n-butylphthalate (MnBP), and BBzP, monobenzylphthalate (MBzP), in first-morning voids of 36 children (median age 4.7 years) and 19 adults (37.4 years). Results: In all samples both metabolites were detected. Urinary MnBP concentrations (in microgrammes per litre) of the children and adults were 139 and 91.8 (median), respectively. MBzP concentrations were 22.1 μg/l and 12.7 μg/l (median), respectively. Concentrations in microgrammes per gramme creatinine for MnBP were 161 for the children and 91.8 for the adults (median). The maximum concentration found for children (2249 μg/g) was approximately 15-times higher than that for adults (149 μg/g). This maximum value for children was attributed to medication that contained DnBP. If this child was excluded, the maximum concentration was 517 μg/g. MBzP concentrations for children and adults were 37.0 μg/g and 9.8 μg/g (median), respectively. The maximum concentration found for children (193 μg/g) was approximately seven-times higher than that for adults (26.7 μg/g). Creatinine-adjusted concentrations were significantly higher for children for both MBzP and MnBP (P

We developed a new 3-D HPLC method for on-line clean-up and simultaneous quantification of two important naphthalene metabolites, 1-naphthol and 2-naphthol, in human urine. Except an enzymatic hydrolysis no further sample pre-treatment is necessary. The metabolites are stripped from urinary matrix by on-line extraction on a restricted access material pre-column (RAM RP-8), transferred in backflush mode onto a silica-based CN-(cyano)phase column for further purification from interfering substances. By another successive column switching step both analytes are transferred with a minimum of overlapping interferences onto a C12 bonded reversed phase column with trimethylsilyl endcapping where the final separation is carried out. The entire arrangement is software controlled. Eluting analytes are quantified by fluorescence detection (227/430 nm) after an external calibration. Within a total run time of 40 min we can selectively quantify both naphthols with detection limits in the lower ppb range (1.5 and 0.5 microg/l for 1- and 2-naphthol, respectively) with excellent reliability (ensured by precision, accuracy, matrix-independency and FIOH quality assurance program participation). First results on a collective of 53 occupationally non exposed subjects showed mean levels of 11.0 microg/l (1-naphthol) and 12.9 microg/l (2-naphthol). Among smokers (n=21) a significantly elevated mean level of urinary naphthols was determined (1-naphthol: 19.2 microg/l and 2-naphthol: 23.7 microg/l) in comparison to non smokers (n=32; 1-naphthol: 5.6 microg/l, 2-naphthol: 5.6 microg/l).

To assess the suitability of different methods for biological monitoring of internal dose to N,N-dimethylformamide (DMF) in occupational settings.The determination of urinary metabolites of DMF, N-hydroxymethyl- N-methylformamide (HMMF), N-methylformamide (NMF) and N-acetyl- S-(N-methylcarbamoyl) cysteine (AMCC) was carried out by four selected analytical procedures. Two methods solely measured total NMF (HMMF and NMF). The other two methods measured both total NMF and AMCC in one analytical run. All four methods were tested on 34 urine samples from workers exposed to DMF.Comparison of the four methods for determination of total NMF in urine showed that results were similar for three methods, while the remaining one provided NMF levels significantly lower (by 22%) than the other methods. Thus, all but one of the tested methods for the determination of total NMF can be considered to be suitable for biological monitoring of internal dose to DMF. The two tested methods for the determination of AMCC afforded results that showed high correlation but differed significantly (by 10%).The choice of the biomonitoring method depends mainly on the purpose for which the measurement is conducted. For evaluation of acute exposures or to assess safety measures in the working area, an updated version of the traditional method of Kimmerle and Eben (1975a, b) for the determination of total NMF in urine is sufficient. For risk assessment after exposure to DMF, the determination of AMCC should be carried out, since AMCC, but not total NMF, is supposed to be related to the toxicity of DMF. However, there is still a need to develop an easier, more sensitive and more selective method for the determination of AMCC in urine until AMCC can be considered for regulatory purposes in occupational settings.

Objective: Workers in various industries can be exposed to polycyclic aromatic hydrocarbons (PAHs). The relationship between biomarkers of genotoxic risk, PAH compounds in air (ambient monitoring) and PAH metabolites in urine (internal exposure) were studied in 17 workers exposed to PAHs in a fireproof-material producing plant before and 3 months after the PAH profile was altered in the binding pitch. Methods: Two biomarkers of exposure, specific DNA adducts of (±)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) and non-specific DNA adduct of 8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxodGuo) were determined in white blood cells (WBCs). In addition, DNA strand breaks were analysed in lymphocytes by single-cell gel electrophoresis in a genotoxic risk assessment. Sixteen PAH compounds in air were determined by personal air sampling, and hydroxylated metabolites of phenanthrene, pyrene and naphthalene were determined in urine. Results: After substitution of the binding pitch the concentrations of benzo[a]pyrene in air decreased (P

A number of phthalates and their metabolites are suspected of having teratogenic and endocrine disrupting effects. Especially the developmental and reproductive effects of di(2-ethylhexyl)phthalate (DEHP) are under scrutiny. In this study we determined the concentrations of the secondary, chain oxidized monoester metabolites of DEHP, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) in urine samples from the general population. The utilization of the secondary metabolites minimized any risk of contamination by the ubiquitously present phthalate parent compounds. Included in the method were also the simple monoester metabolites of DEHP, dioctylphthalate (DOP), di-n-butylphthalate (DnBuP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Automated sample preparation was performed applying a column switching liquid chromatography system enabling online extraction of the urine on a restricted access material (RAM) and separation on a reversed phase analytical column. Detection was performed by negative ESI-tandem mass spectrometry in multiple reaction monitoring mode and quantification by isotope dilution. The excretion of DEHP and the other phthalates was studied by analyzing first morning urine samples from 53 women and 32 men aged 7-64 years (median: 34.2 years) living in northern Bavaria (Germany) who were not occupationally exposed to phthalates. Phthalate metabolites, secondary and primary ones, were detected in all specimens. Concentrations were found to vary strongly from phthalate to phthalate and subject to subject with differences spanning more than three orders of magnitude. Median concentrations for excretion of DEHP metabolites were 46.8 microg/L for 5OH-MEHP (range 0.5-818 microg/L), 36.5 microg/L for 5oxo-MEHP (range 0.5-544 microg/L), and 10.3 microg/L for MEHP (range

Human metabolism of di(2-ethylhexyl)phthalate (DEHP) was studied after a single oral dose of 48.1 mg to a male volunteer. To avoid interference by background exposure the D4-ring-labelled DEHP analogue was dosed. Excretion of three metabolites, mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP), mono(2-ethyl-5-oxohexyl)phthalate (5oxo-MEHP) and mono(2-ethylhexyl)phthalate (MEHP), was monitored for 44 h in urine and for 8 h in serum. Peak concentrations of all metabolites were found in serum after 2 h and in urine after 2 h (MEHP) and after 4 h (5OH-MEHP and 5oxo-MEHP). While the major metabolite in serum was MEHP, the major metabolite in urine was 5OH-MEHP, followed by 5oxo-MEHP and MEHP. Excretion in urine followed a multi-phase elimination model. After an absorption and distribution phase of 4 to 8 h, half-life times of excretion in the first elimination phase were approximately 2 h with slightly higher half-life times for 5OH- and 5oxo-MEHP. Half-life times in the second phase-beginning 14 to 18 h post dose-were 5 h for MEHP and 10 h for 5OH-MEHP and 5oxo-MEHP. In the time window 36 to 44 h, no decrease in excreted concentrations of 5OH- and 5oxo-MEHP was observed. In the first elimination phase (8 to 14 h post dose), mean excretion ratios of MEHP to 5oxo-MEHP and MEHP to 5OH-MEHP were 1 to 1.8 and 1 to 3.1. In the second elimination phase up to 24 h post dose mean excretion ratios of MEHP to 5oxo-MEHP to 5OH-MEHP were 1 to 5.0 to 9.3. The excretion ratio of 5OH-MEHP to 5oxo-MEHP remained constant through time at 1.7 in the mean. After 44 h, 47% of the DEHP dose was excreted in urine, comprising MEHP (7.3%), 5OH-MEHP (24.7%) and 5oxo-MEHP (14.9%).

We analyzed 85 urine samples of the general German population for human specific metabolites of phthalates. By that we avoided contamination with the parent phthalates being omnipresent in the environment and for the first time could deduce each individual's internal exposure to phthalates without contamination. Determined were the secondary metabolites mono(2-ethyl-5-hydroxyhexyl)phthalate (5OH-MEHP) and mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) of di(2-ethylhexyl)phthalate (DEHP) and the primary monoester metabolites of DEHP, di-noctylphthalate (DnOP), di-n-butylphthalate (DnBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Based on these internal exposure values we calculated the daily intake of the parent phthalates using urinary metabolite excretion factors. For DEHP we determined a median intake of 13.8 micrograms/kg body weight/day and an intake at the 95th percentile of 52.1 micrograms/kg body weight/day. The tolerable daily intake (TDI) value settled by the EU Scientific Committee for Toxicity, Ecotoxicity and the Environment (CSTEE) is 37 micrograms/kg body weight/day. Twelve percent of the subjects (10 out of 85 samples) within our collective of the general population are exceeding this value. Thirty-one percent of the subjects (26 out of 85 samples) had values higher than the reference dose (RfD) of 20 micrograms/kg body weight/day of the U.S. Environmental Protection Agency (EPA). For DnBP, BBzP, DEP and DnOP intake values at the 95th percentile were 16.2, 2.5, 22.1 and 0.42 micrograms/kg body weight/day respectively. Our results unequivocally prove that the general German population is exposed to DEHP to a much higher extent than previously believed. This is of greatest importance for public health since DEHP is not only the most important phthalate with respect to its production, use, occurrence and omnipresence but also the phthalate with the greatest endocrine disrupting potency. DEHP is strongly suspected to be a developmental and reproductive toxicant. We are not aware of any other environmental contaminant for which the TDI and RfD are exceeded to such an extent within the general population. The transgressions of the TDI and RfD for DEHP are accompanied by considerable ubiquitous exposures to DnBP and BbzP, two phthalates under scrutiny for similar toxicological mechanisms.

We developed a new and fast multidimensional on-line HPLC-method for the quantitative determination of the secondary, chain oxidized monoester metabolites of diethylhexylphthalate (DEHP), 5-hydroxy-mono-(2-ethylhexyl)-phthalate (5OH-MEHP) and 5-oxo-mono-(2-ethylhexyl)-phthalate (5oxo-MEHP) in urine samples from the general population. Also included in the method were the simple monoester metabolites of DEHP, dioctylphthalate (DOP), dibutylphthalate (DBP), butylbenzylphthalate (BBzP) and diethylphthalate (DEP). Except for enzymatic hydrolysis for deconjugation of the metabolites no further sample pre-treatment step is necessary. The phthalate metabolites are stripped from urinary matrix by on-line extraction on a restricted access material (LiChrospher((R)) ADS-8) precolumn, transferred in backflush-mode and chromatographically resolved by reversed-phase HPLC. Eluting metabolites are detected by ESI-tandem mass spectrometry in negative ionization mode and quantified by isotope dilution. Within a total run time of 25 min we can selectively and sensitively quantify seven urinary metabolites of six commonly occurring phthalate diesters including the controversial di(2-ethylhexyl)phthalate (DEHP). The detection limits for all analytes are in the low ppb range (0.5-2.0 microgram/l urine). First results on a small non-exposed group (n=8) ranged for 5OH-MEHP from 0.59 to 124 microgram/l, for 5oxo-MEHP fromLOQ to 73.0 microgram/l, and for MEHP fromLOQ to 41.1 microgram/l. The other short chain monoester metabolites were detectable in every sample with mean concentrations for MnBuP of 36.5 microgram/l, for MBzP of 7.19 microgram/l and MEP of 1.0 mg/l. With this rapid and economic method we can determine the internal exposure of the general population to DEHP and other phthalates as well as the body burden of occupationally and medically exposed subjects. The results can help to rank the risks of phthalates in the areas of carcinogenesis, peroxisome proliferation and endocrine disruption. Since secondary, functionalized metabolites of DEHP are included in the method an enduring problem of the past is excluded: sample contamination in the pre-analytical and analytical phase by both di- and monoesters.

A newly developed method permits the simultaneous quantitative determination of various aromatic amines (or metabolites of aromatic nitro compounds, respectively) in human urine in one analytical run. Applying this method it is possible to determine aniline, toluidines, 4-isopropylaniline, o-anisidine, 3- and 4-chloroaniline, 4-bromoaniline, aminonitrotoluenes, aminodinitrotoluenes, 3,5- and 3,4-dichloroaniline, alpha- and beta-naphtylamine and 4-aminodiphenyl. After separation from the urinary matrix by a simple liquid-liquid extraction at pH 6.2-6.4 the analytes are converted into their pentafluoropropionic acid amides. Separation and quantitative analysis is carried out by capillary gas chromatography and mass-selective detection in the single ion monitoring mode. The limits of detection were within the range from 0.05 microg/l (4-aminobiphenyl, o-anisidine, 3,5-dichloroaniline) to 2 microg/l urine (4-amino-2,6-dinitrotoluene). The relative standard deviation of the within-series imprecision (determined at spiked concentrations of 2.0 microg/l and 10 microg/l) was between 2.9 and 13.6% depending on analyte and concentration. The relative recovery rates were in the range of 70-121%. The analytes that do not contain a nitro function showed better performance regarding the analytical reliability criteria. In order to determine the suitability of this new method for biological monitoring we analysed 20 12-h urine samples of persons without known exposure to aromatic amines, nitroaromatics or precursors in a pilot study. In these samples various aromatic amines could be clearly identified. The general population renally excretes aniline (median: 3.5 microg/l; 95th percentile: 7.9 microg/l), o- (0.12 microg/l; 2.7 microg/l), m- (0.17 microg/l; 2.2 microg/l) and p-toluidine (0.11 microg/l; 0.43 microg/l), and o-anisidine (0.22 microg/l; 0.68 microg/l). Additionally, we found that the persons investigated also excrete 3- (0.05 microg/l; 0.55 microg/l) and 4-chloroaniline (0.11 microg/l; 0.57 microg/l) as well as 3,5-dichloroaniline (0.18 microg/l; 1.5 microg/l). 3,4-Dichloroaniline was found in some specimens (20%) in concentrations near the limit of detection (0.05 microg/l; 0.12 microg/l). We did not detect alpha- or beta-naphtylamine, 4-aminobiphenyl or metabolites of explosives in the samples.

Biological monitoring of chemical exposure in the workplace has become increasingly important in the assessment of health risk as an integral part of the overall occupational health and safety strategy. In environmental medicine biological monitoring plays also an important role in the assessment of excessive, acute or chronic exposure to chemical agents. To guarantee that the results obtained in biological monitoring are comparable with threshold limit values and results from other laboratories, the analysis must be carried out with tested and reliable analytical methods and accompanied by a quality assurance scheme. Confounding influences and interferences during the pre-analytical phase can be minimised by recommendations from experienced laboratories. For internal quality control commercially available control samples with an assigned concentration are used. External quality control programs for biological monitoring are offered by several institutions. The external quality control program of the German Society of Occupational and Environmental Medicine has been organised since 1982. In the meantime the 27th program has been carried out offering 96 analytes in urine, blood and plasma for 47 substances. This program covers most of the parameters relevant to occupational and environmental medicine. About 350 laboratories take part in these intercomparison programs. At present, ten German and 14 international laboratories are commissioned to determine the assigned values. The data evaluated from the results of the intercomparison programs give a good overview of the current quality of the determination of analytes assessed in occupational and environmental toxicological laboratories. For the analysis of inorganic substances in blood and urine the tolerable variation ranges from 7.5 to 43.5%. For organic substances in urine the tolerable variation ranges from 12 to 48%. The highest variations (36–60%) were found for the analysis of organochlorine compounds in plasma. The tolerable variations for the determination of solvents in blood by head space gas chromatography range from 26 to 57%. If the recommendations for the pre-analytical phase, the selection of reliable analytical methods by the laboratory and the carrying out of adequate quality control are observed, the pre-requisites for reliable findings during biological monitoring are fulfilled

We have developed and validated a new, reliable and very sensitive method for the determination of the urinary metabolites of the most common pyrethroids in one analytical run. After acidic hydrolysis for the cleavage of conjugates, the analytes cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-Cl(2)CA), trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (trans-Cl(2)CA), cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (Br(2)CA), 4-fluoro-3-phenoxybenzoic acid (F-PBA) and 3-phenoxybenzoic acid (3-PBA) were extracted from the matrix with a liquid-liquid extraction procedure using n-hexane under acidic conditions. For further clean-up, NaOH was added to the organic phase and the carboxylic acids were re-extracted into the aqueous phase. After acidification and extraction into n-hexane again, the metabolites were then derivatised to volatile esters using N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamid (MTBSTFA). Separation and detection were carried out using capillary gas chromatography with mass-selective detection (GC-MS). 2-Phenoxybenzoic acid (2-PBA) served as internal standard for the quantification of the pyrethroid metabolites. The limit of detection for all analytes was 0.05 microg/l urine. The RSD of the within-series imprecision was between 2.0 and 5.4% at a spiked concentration of 0.4 microg/l and the relative recovery was between 79.3 and 93.4%, depending on the analyte. This method was used for the analysis of urine samples of 46 persons from the general population without known exposure to pyrethroids. The metabolites cis-Cl(2)CA, trans-Cl(2)CA and 3-PBA could be found in 52, 72 and 70% of all samples with median values of 0.06, 0.11 and 0.16 microg/l, respectively. Br(2)CA and F-PBA could also be detected in 13 and 4% of the urine samples.

In a chemical plant, ethylene oxide (EO) and propylene oxide (PO) were used for the production of surfactants for the textile industry. Within health supervision, we investigated the internal exposure of the workers using hemoglobin adducts as parameters of biochemical effects. The 95th percentile for N-2-hydroxyethylvaline (HEV) was 1280 pmol/g globin (=29.4 microg/l blood) in blood from exposed workers compared with 100 pmol/g globin (or 2.3 microg/l) in controls. N-(R,S)-2-hydroxypropylvaline (HPV) both in workers and controls was below the detection limit (80 pmol/g globin or 2 microg/l). The levels of the adducts of acrylonitrile (ACN) and acrylamide (AA) were also determined, though they were mainly accounted for by smoking and diet. Median values of N-2-cyanoethylvaline (CEV) were below 4 pmol/g globin (or 0.1 microg/l) in non-smokers (n=24) and 131 pmol/g globin (or 3.3 microg/l) in smokers (n=38). Median values of N-2-carbamoylethylvaline (AAV) were 22 pmol/g globin (or 0.6 microg/l) in non-smokers compared with 89 pmol/g globin (or 2.4 microg/l) in smokers. Correlations were found between smoking habits and adduct levels of CEV and AAV.

Inhabitants (1177) of a residential area in Frankfurt/Main have been investigated with respect to internal exposure to pyrethroids. Biological monitoring revealed a body burden of pyrethroids. The 95th per thousand for the urinary metabolites of pyrethroids, such as permethrin and cypermethrin, cis and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (cis-DCCA and trans-DCCA), was determined to be 0.5 and 1.4 microg/l, respectively. 95th per thousand for cis-3-(2,2-dibromovinyl)-2,2-dimethylcyclopropane-1-carboxylic acid (DBCA), a specific metabolite of deltamethrin, and 4-fluoro-3-phenoxybenzoic acid (F-PBA), a metabolite of cyfluthrin, were 0.3 and 0.27 microg/l, respectively. The metabolic pattern found for these samples points out that pyrethroids are probably ingested orally with daily diet.

It was the aim of this study to ascertain whether xylenes form phenylmercapturic acids via aromatic epoxides in the human metabolism. Aromatic epoxides are suspected to exert mutagenic properties. Therefore we developed an LC/MS/MS procedure for the determination of these mercapturic acids. Using this method we were able to detect dimethylphenylmercapturic acid (DPMA) in urine samples of persons occupationally exposed to xylenes. The unequivocal LC/MS/MS detection of phenylmercapturic acid metabolites of xylene thanks to authentic standards was verified with an independent GC/MS method. Xylene concentrations in the air of the workplaces ranged between 0.7 and 58.1 ppm (median 12.6 ppm). The excretion of methylhippuric acid in the urine samples of the workers (n=27) ranges from 19.8 to 2332.5 mg/l (median 450.9 mg/l). DPMA was detected in only 9 samples of 27 exposed persons. According to a rough calculation DPMA is only formed in a ratio of 0.0003% respective to the xylene main metabolite MHA. That means that even under occupationally relevant xylene exposure potential mutagenic potencies should be negligible. DPMA in urine is not sensitive enough for general biomonitoring purposes due to the low ratio of excretion. MHA should therefore be used for biomonitoring from now on. Furthermore, our results show that irrespective of the structure of xylene isomers there is no preferences in metabolism.