Mothers and children are related, even in exposure to chemicals present in common consumer products.

BACKGROUND: Phthalates, bisphenol A (BPA) and triclosan (TCS) are detectable in the vast majority of people. Most humans are continuously exposed to these chemicals due to their presence in food or in everyday consumer products. The measurement of these compounds in family members may help to explore the impact of major lifestyle factors on exposure. Mothers and (young) children are especially interesting to study, as they mostly share considerable parts of daily life together. MATERIALS AND METHODS: Phthalate metabolites, bisphenol A (BPA) and triclosan (TCS) were measured in first morning void urine, collected in mother-child pairs (n = 129) on the same day. The mothers (27-45y) and their children (6-11y) were recruited in the Brussels agglomeration and rural areas of Belgium in the context of the European COPHES-DEMOCOPHES human biomonitoring project. Face-to-face questionnaires gathered information on major exposure sources and lifestyle factors. Exposure determinants were assessed by multiple linear regression analysis. RESULTS: The investigated compounds were detectable in nearly all mothers (92.8-100%) and all children (95.2-100%). The range (P90 vs. P10) of differences in urinary concentrations within each age group was for most compounds around 10-20 fold, and was very high for TCS up to 35 and 350-fold in children and mothers respectively. Some participants exceeded the tolerable daily intake guidelines as far as they were available from the European Food Safety Authority (EFSA). Overall, for BPA, the urinary concentrations were similar among both age groups. Most urinary phthalate metabolites were higher in children compared to the mothers, except for monoethyl phthalate (MEP). TCS levels were generally higher in the mothers. Despite the difference in mothers' and children's urinary concentrations, the creatinine-corrected levels were correlated for all biomarkers (Spearman rank r = 0.32 to 0.66, p < 0.001). Furthermore, for phthalates, similar home and lifestyle factors were associated with the urinary concentrations in both age groups: home renovation during last two years or redecoration during the last year for di-ethyl phthalate (DEP); PVC in home for di-n-butyl phthalate (DnBP), di-iso-butyl phthalate (DiBP) and butyl benzyl phthalate (BBzP), and personal care products use for DiBP and DnBP. Based on questionnaire information on general food type consumption patterns, the exposure variability could not be explained. However, comparing the phthalate intake from the current study with earlier assessed Belgian food intake calculations for both ages, food in general was estimated to be the major intake source for di-ethyl hexyl phthalate (DEHP), with diminishing importance for BBzP, DiBP and DnBP. CONCLUSION: Our results confirm, that children and their mothers, sharing diets and home environments, also share exposure in common consumer products related chemicals. By collecting morning urine levels on the same day, and using basic questionnaires, suspected exposure routes could be unraveled.

Metabolism and elimination of methyl, iso- and n-butyl paraben in human urine after single oral dosage.

Parabens are used as preservatives in personal care and consumer products, food and pharmaceuticals. Their use is controversial because of possible endocrine disrupting properties. In this study, we investigated metabolism and urinary excretion of methyl paraben (MeP), iso-butyl paraben (iso-BuP) and n-butyl paraben (n-BuP) after oral dosage of deuterium-labeled analogs (10 mg). Each volunteer received one dosage per investigated paraben separately and at least 2 weeks apart. Consecutive urine samples were collected over 48 h. In addition to the parent parabens (free and conjugated) which are already used as biomarkers of internal exposure and the known but non-specific metabolites, p-hydroxybenzoic acid (PHBA) and p-hydroxyhippuric acid (PHHA), we identified new, oxidized metabolites with hydroxy groups on the alkyl side chain (3OH-n-BuP and 2OH-iso-BuP) and species with oxidative modifications on the aromatic ring. MeP represented 17.4 % of the dose excreted in urine, while iso-BuP represented only 6.8 % and n-BuP 5.6 %. Additionally, for iso-BuP, about 16 % was excreted as 2OH-iso-BuP and for n-BuP about 6 % as 3OH-n-BuP. Less than 1 % was excreted as ring-hydroxylated metabolites. In all cases, PHHA was identified as the major but non-specific metabolite (57.2-63.8 %). PHBA represented 3.0-7.2 %. For all parabens, the majority of the oral dose captured by the above metabolites was excreted in the first 24 h (80.5-85.3 %). Complementary to the parent parabens excreted in urine, alkyl-chain-oxidized metabolites of the butyl parabens are introduced as valuable and contamination-free biomarkers of exposure.

Case study: Possible differences in phthalates exposure among the Czech, Hungarian, and Slovak populations identified based on the DEMOCOPHES pilot study results.

OBJECTIVE: Phthalates and their metabolites are classified as endocrine modulators. They affect the hormonal balance in both children and adults. The aim of this publication was to compare the urinary levels of phthalate metabolites in selected populations of the Czech Republic (CZ), Slovakia (SK), and Hungary (HU) in relation to the sources of phthalate exposure identified by means of questionnaire (personal care products, floor and wall coverings, plastic toys, and some kinds of foods). METHODS: Data were obtained through the twin projects COPHES (COnsortium to Perform Human biomonitoring on a European Scale) and DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) from 2009 to 2012. The target groups were children aged 6-11 years old and their mothers up to 45 years of age. The metabolites of phthalates (monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobenzyl phthalate (MBzP), mono-cyclohexyl phthalate (MCHP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), and mono-(2-ethyl-5-oxohexyl) phthalate (5OXO-MEHP)) were analysed in first morning urine samples. After enzymatic glucuronide cleavage, the urine sample analyses were performed using ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) in one laboratory that qualified in the External Quality Assessment exercises organised by COPHES. RESULTS: Significant differences in phthalate exposure between countries were revealed for children only but not for mothers. The concentrations of 5-OH-MEHP (P<0.001), 5OXO-MEHP (P<0.001), and their sum (P<0.001) were the highest in SK compared to CZ and HU. The health based guidance values for the sum of DEHP metabolites 5-OH MEHP and 5OXO-MEHP established by the German Commission for biomonitoring of 300 µg/L and 500 µg/L for women adults and children, respectively, were only exceeded in one mother and three boys. A significant difference was also found for MEP (P=0.0149), with the highest concentrations detected in HU. In all countries, the increasing frequency of using personal care products significantly elevated the concentrations of MEP. CONCLUSION: Some differences were observed between countries in the concentrations of individual urinary phthalate metabolites in children. However, the questionnaire results give no direct explanation for the differences between the countries except the variation in using personal care products.

The Danish contribution to the European DEMOCOPHES project: A description of cadmium, cotinine and mercury levels in Danish mother-child pairs and the perspectives of supplementary sampling and measurements.

Human biomonitoring (HBM) is an important tool, increasingly used for measuring true levels of the body burdens of environmental chemicals in the general population. In Europe, a harmonized HBM program was needed to open the possibility to compare levels across borders. To explore the prospect of a harmonized European HBM project, DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) was completed in 17 European countries. The basic measurements performed in all implemented countries of DEMOCOPHES included cadmium, cotinine and phthalate metabolites in urine and mercury in hair. In the Danish participants, significant correlations between mothers and children for mercury in hair and cotinine in urine were found. Mercury in hair was further significantly associated with intake of fish and area of residence. Cadmium was positively associated with BMI in mothers and an association between cadmium and cotinine was also found. As expected high cotinine levels were found in smoking mothers. For both mercury and cadmium significantly higher concentrations were found in the mothers compared to their children. In Denmark, the DEMOCOPHES project was co-financed by the Danish ministries of health, environment and food safety. The co-financing ministries agreed to finance a number of supplementary measurements of substances of current toxicological, public and regulatory interest. This also included blood sampling from the participants. The collected urine and blood samples were analyzed for a range of other persistent and non-persistent environmental chemicals as well as two biomarkers of effect. The variety of supplementary measurements gives the researchers further information on the exposure status of the participants and creates a basis for valuable knowledge on the pattern of exposure to various chemicals.

Urinary BPA measurements in children and mothers from six European member states: Overall results and determinants of exposure.

For the first time in Europe, both European-wide and country-specific levels of urinary Bisphenol A (BPA) were obtained through a harmonized protocol for participant recruitment, sampling and quality controlled biomarker analysis in the frame of the twin projects COPHES and DEMOCOPHES. 674 child-mother pairs were recruited through schools or population registers from six European member states (Belgium, Denmark, Luxembourg, Slovenia, Spain and Sweden). Children (5-12 y) and mothers donated a urine sample. Information on socio-demographic characteristics, life style, dietary habits, and educational level of the parents was provided by mothers. After exclusion of urine samples with creatinine values below 300 mg/L or above 3000 mg/L, 653 children and 639 mothers remained for which BPA was measured. The geometric mean (with 95% confidence intervals) and 90th percentile were calculated for BPA separately in children and in mothers and were named "European reference values". After adjustment for confounders (age and creatinine), average exposure values in each country were compared with the mean of the "European reference values" by means of a weighted analysis of variance. Overall geometric means of all countries (95% CI) adjusted for urinary creatinine, age and gender were 2.04 (1.87-2.24) µg/L and 1.88 (1.71-2.07) µg/L for children (n=653) and mothers (n=639), respectively. Multiple regression analysis was used to identify significant environmental, geographical, personal or life style related determinants. Consumption of canned food and social class (represented by the highest educational level of the family) were the most important predictors for the urinary levels of BPA in mothers and children. The individual BPA levels in children were significantly correlated with the levels in their mothers (r=0.265, p<0.001), which may suggest a possible common environmental/dietary factor that influences the biomarker level in each pair. Exposure of the general European population was well below the current health-based guidance values and no participant had BPA values higher than the health-based guidance values.

Mercury analysis in hair: Comparability and quality assessment within the transnational COPHES/DEMOCOPHES project.

Human biomonitoring (HBM) is an effective tool for assessing actual exposure to chemicals that takes into account all routes of intake. Although hair analysis is considered to be an optimal biomarker for assessing mercury exposure, the lack of harmonization as regards sampling and analytical procedures has often limited the comparison of data at national and international level. The European-funded projects COPHES and DEMOCOPHES developed and tested a harmonized European approach to Human Biomonitoring in response to the European Environment and Health Action Plan. Herein we describe the quality assurance program (QAP) for assessing mercury levels in hair samples from more than 1800 mother-child pairs recruited in 17 European countries. To ensure the comparability of the results, standard operating procedures (SOPs) for sampling and for mercury analysis were drafted and distributed to participating laboratories. Training sessions were organized for field workers and four external quality-assessment exercises (ICI/EQUAS), followed by the corresponding web conferences, were organized between March 2011 and February 2012. ICI/EQUAS used native hair samples at two mercury concentration ranges (0.20-0.71 and 0.80-1.63) per exercise. The results revealed relative standard deviations of 7.87-13.55% and 4.04-11.31% for the low and high mercury concentration ranges, respectively. A total of 16 out of 18 participating laboratories the QAP requirements and were allowed to analyze samples from the DEMOCOPHES pilot study. Web conferences after each ICI/EQUAS revealed this to be a new and effective tool for improving analytical performance and increasing capacity building. The procedure developed and tested in COPHES/DEMOCOPHES would be optimal for application on a global scale as regards implementation of the Minamata Convention on Mercury.

Exposure determinants of cadmium in European mothers and their children.

The metal cadmium (Cd) is a widespread environmental pollutant with documented adverse effects on the kidneys and bones from long-term environmental exposure, but with insufficiently elucidated public health consequences such as risk of cardiovascular disease, hormone-related cancer in adults and developmental effects in children. This study is the first pan-European human biomonitoring project that succeeded in performing harmonized measurements of Cd in urine in a comparable way in mother-child couples from 16 European countries. The aim of the study was to evaluate the overall Cd exposure and significant determinants of Cd exposure. A study population of 1632 women (24-52 years of age), and 1689 children (5-12 years of age), from 32 rural and urban areas, was examined within a core period of 6 months in 2011-2012. Women were stratified as smokers and non-smokers. As expected, smoking mothers had higher geometric mean (gm) urinary cadmium (UCd; 0.24 µg/g crea; n=360) than non-smoking mothers (gm 0.18 µg/g crea; n=1272; p<0.0001), and children had lower UCd (gm 0.065 µg/g crea; n=1689) than their mothers at the country level. Non-smoking women exposed to environmental tobacco smoke (ETS) at home had 14% (95% CI 1-28%) higher UCd than those who were not exposed to ETS at home (p=0.04). No influence of ETS at home or other places on UCd levels was detected in children. Smoking women with primary education as the highest educational level of the household had 48% (95% CI 18-86%) higher UCd than those with tertiary education (p=0.0008). The same observation was seen in non-smoking women and in children; however they were not statistically significant. In children, living in a rural area was associated with 7% (95% CI 1-13%) higher UCd (p=0.03) compared to living in an urban area. Children, 9-12 years had 7% (95% CI 1-13%) higher UCd (p=0.04) than children 5-8 years. About 1% of the mothers, and 0.06% of the children, exceeded the tolerable weekly intake (TWI) appointed by EFSA, corresponding to 1.0 µg Cd/g crea in urine. Poland had the highest UCd in comparison between the 16 countries, while Denmark had the lowest. Whether the differences between countries are related to differences in the degree of environmental Cd contamination or to differences in lifestyle, socioeconomic status or dietary patterns is not clear.

Lessons learnt on recruitment and fieldwork from a pilot European human biomonitoring survey.

Within the European Environment and Health Action Plan an initiative to establish a coherent human biomonitoring approach in Europe was started. The project COPHES (COnsortium to Perform Human biomonitoring on a European Scale ) developed recommendations for a harmonized conduct of a human biomonitoring (HBM) survey which came into action as the pilot study DEMOCOPHES (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale). Seventeen European countries conducted a survey with harmonized instruments for, inter alia, recruitment, fieldwork and sampling, in autumn/winter 2011/2012. Based on the countries' experiences of conducting the pilot study, following lessons learnt were compiled: the harmonized fieldwork instruments (basic questionnaire, urine and hair sampling) turned out to be very valuable for future HBM surveys on the European scale. A school approach was favoured by most of the countries to recruit school-aged children according to the established guidelines and country specific experiences. To avoid a low participation rate, intensive communication with the involved institutions and possible participants proved to be necessary. The communication material should also include information on exclusion criteria and offered incentives. Telephone contact to the participants the day before fieldwork during the survey can prevent the forgetting of appointments and first morning urine samples. To achieve comparable results on the European scale, training of interviewers in all issues of recruitment, fieldwork and sampling through information material and training sessions is crucial. A survey involving many European countries needs time for preparation and conduct. Materials for quality control prepared for all steps of recruitment, fieldwork and sampling proved to be important to warrant reliable results.

Policy recommendations and cost implications for a more sustainable framework for European human biomonitoring surveys.

The potential of Human Biomonitoring (HBM) in exposure characterisation and risk assessment is well established in the scientific HBM community and regulatory arena by many publications. The European Environment and Health Strategy as well as the Environment and Health Action Plan 2004-2010 of the European Commission recognised the value of HBM and the relevance and importance of coordination of HBM programmes in Europe. Based on existing and planned HBM projects and programmes of work and capabilities in Europe the Seventh Framework Programme (FP 7) funded COPHES (COnsortium to Perform Human Biomonitoring on a European Scale) to advance and improve comparability of HBM data across Europe. The pilot study protocol was tested in 17 European countries in the DEMOCOPHES feasibility study (DEMOnstration of a study to COordinate and Perform Human biomonitoring on a European Scale) cofunded (50%) under the LIFE+ programme of the European Commission. The potential of HBM in supporting and evaluating policy making (including e.g. REACH) and in awareness raising on environmental health, should significantly advance the process towards a fully operational, continuous, sustainable and scientifically based EU HBM programme. From a number of stakeholder activities during the past 10 years and the national engagement, a framework for sustainable HBM structure in Europe is recommended involving national institutions within environment, health and food as well as European institutions such as ECHA, EEA, and EFSA. An economic frame with shared cost implications for national and European institutions is suggested benefitting from the capacity building set up by COPHES/DEMOCOPHES.

Communication in a Human biomonitoring study: Focus group work, public engagement and lessons learnt in 17 European countries.

A communication strategy was developed by The Consortium to Perform Human Biomonitoring on a European Scale (COPHES), as part of its objectives to develop a framework and protocols to enable the collection of comparable human biomonitoring data throughout Europe. The framework and protocols were tested in the pilot study DEMOCOPHES (Demonstration of a study to Coordinate and Perform Human biomonitoring on a European Scale). The aims of the communication strategy were to raise awareness of human biomonitoring, encourage participation in the study and to communicate the study results and their public health significance. It identified the audiences and key messages, documented the procedure for dissemination of results and was updated as the project progressed. A communication plan listed the tools and materials such as press releases, flyers, recruitment letters and information leaflets required for each audience with a time frame for releasing them. Public insight research was used to evaluate the recruitment material, and the feedback was used to improve the documents. Dissemination of results was coordinated in a step by step approach by the participating countries within DEMOCOPHES, taking into account specific national messages according to the needs of each country. Participants received individual results, unless they refused to be informed, along with guidance on what the results meant. The aggregate results and policy recommendations were then communicated to the general public and stakeholders, followed by dissemination at European level. Several lessons were learnt that may assist other future human biomonitoring studies. Recruitment took longer than anticipated and so social scientists, to help with community engagement, should be part of the research team from the start. As a European study, involving multiple countries, additional considerations were needed for the numerous organisations, different languages, cultures, policies and priorities. Therefore, communication documents should be seen as templates with essential information clearly indicated and the option for each country to tailor the material to reflect these differences. Future studies should consider setting up multidisciplinary networks of medical professionals and communication experts, and holding training workshops to discuss the interpretation of results and risk communication. Publicity and wide dissemination of the results helped to raise awareness of human biomonitoring to the general public, policy makers and other key stakeholders. Effective and timely communication, at all stages of a study, is essential if the potential of human biomonitoring research to improve public health is to be realised.

Fish consumption patterns and hair mercury levels in children and their mothers in 17 EU countries.

The toxicity of methylmercury (MeHg) in humans is well established and the main source of exposure is via the consumption of large marine fish and mammals. Of particular concern are the potential neurodevelopmental effects of early life exposure to low-levels of MeHg. Therefore, it is important that pregnant women, children and women of childbearing age are, as far as possible, protected from MeHg exposure. Within the European project DEMOCOPHES, we have analyzed mercury (Hg) in hair in 1799 mother-child pairs from 17 European countries using a strictly harmonized protocol for mercury analysis. Parallel, harmonized questionnaires on dietary habits provided information on consumption patterns of fish and marine products. After hierarchical cluster analysis of consumption habits of the mother-child pairs, the DEMOCOPHES cohort can be classified into two branches of approximately similar size: one with high fish consumption (H) and another with low consumption (L). All countries have representatives in both branches, but Belgium, Denmark, Spain, Portugal and Sweden have twice as many or more mother-child pairs in H than in L. For Switzerland, Czech Republic, Hungary, Poland, Romania, Slovenia and Slovakia the situation is the opposite, with more representatives in L than H. There is a strong correlation (r=0.72) in hair mercury concentration between the mother and child in the same family, which indicates that they have a similar exposure situation. The clustering of mother-child pairs on basis of their fish consumption revealed some interesting patterns. One is that for the same sea fish consumption, other food items of marine origin, like seafood products or shellfish, contribute significantly to the mercury levels in hair. We conclude that additional studies are needed to assess and quantify exposure to mercury from seafood products, in particular. The cluster analysis also showed that 95% of mothers who consume once per week fish only, and no other marine products, have mercury levels 0.55 µg/g. Thus, the 95th percentile of the distribution in this group is only around half the US-EPA recommended threshold of 1 µg/g mercury in hair. Consumption of freshwater fish played a minor role in contributing to mercury exposure in the studied cohort. The DEMOCOPHES data shows that there are significant differences in MeHg exposure across the EU and that exposure is highly correlated with consumption of fish and marine products. Fish and marine products are key components of a healthy human diet and are important both traditionally and culturally in many parts of Europe. Therefore, the communication of the potential risks of mercury exposure needs to be carefully balanced to take into account traditional and cultural values as well as the potential health benefits from fish consumption. European harmonized human biomonitoring programs provide an additional dimension to national HMB programs and can assist national authorities to tailor mitigation and adaptation strategies (dietary advice, risk communication, etc.) to their country's specific requirements.

Metabolism of the plasticizer and phthalate substitute diisononyl-cyclohexane-1,2-dicarboxylate (DINCH(®)) in humans after single oral doses.

Hexamoll(®) DINCH(®) (diisononyl-cyclohexane-1,2-dicarboxylate) is a new high-molecular-weight plasticizer and a phthalate substitute. In this study, the metabolism of DINCH(®) was investigated by oral dosage of three male volunteers with approximately 50 mg Hexamoll(®) DINCH(®) (resulting in individual doses between 0.552 and 0.606 mg/kg body weight). Their urine samples were consecutively collected over 48 h. In analogy to di-iso-nonylphthalate (DINP) metabolism, we quantified the simple monoester mono-isononyl-cyclohexane-1,2-dicarboxylate (MINCH) and its secondary oxidized metabolites with HPLC-MS/MS via isotope dilution analysis. Additionally, we quantified the unspecific full breakdown product, cyclohexane-1,2-dicarboxylic acid (CHDA), via standard addition. All postulated metabolites were present in all samples analyzed. The unspecific CHDA was identified as the major urinary metabolite representing 23.7 % of the dose as the mean of the three volunteers (range 20.0-26.5 %). 14.8 % (11.3-16.7 %) of the dose was excreted as monoesters with oxidative modifications, in particular OH-MINCH 10.7 % (7.7-12.9 %), oxo-MINCH 2.0 % (1.5-2.6 %) and carboxy-MINCH 2.0 % (1.8-2.3 %). Less than 1 % was excreted as the simple monoester MINCH. In sum, 39.2 % (35.9-42.4 %) of the DINCH(®) dose was excreted as these metabolites in urine within 48 h. Over 90 % of the metabolites investigated were excreted within 24 h after application. The secondary oxidized metabolites, with elimination half-times between 10 and 18 h, proved to be apt and specific biomarkers to determine DINCH(®) exposure. With this study, we provide reliable urinary excretion factors to calculate DINCH(®) intakes based on these metabolites in environmental and occupational studies.

Determination of 2,3-dihydroxypropionamide, an oxidative metabolite of acrylamide, in human urine by gas chromatography coupled with mass spectrometry.

The general population is exposed to acrylamide (AA) mainly through food and tobacco smoke. AA is classified as probably carcinogenic to humans. Glycidamide (GA), as the primary oxidative metabolite, was identified to be the ultimate genotoxic agent. This warrants full investigation of the oxidative pathway in AA metabolism and the share of the oxidative compared to the reductive pathway. 2,3-Dihydroxy-propionamide (OH-PA) as the direct hydrolysis product of GA has been shown to be a major urinary oxidative metabolite in human AA metabolism. We developed an analytical method to reliably quantify OH-PA in urine by GC-MS after a multistep procedure including "stripping" on a solid phase material, lyophilization, silylation and re-extraction. With a detection limit of 1 µg/L, our method is sensitive enough to quantify OH-PA in all urine samples of the general population. Within and between series precisions were between 1.9% and 8.2% and mean recoveries between 97% and 101%. We applied this method to 30 urine samples from the general population. In all the samples, OH-PA was present in concentrations between 6.8 and 109.4 µg/L (median, 49.7 µg/L) with no difference between smokers and non-smokers. OH-PA concentrations were approximately ten times higher than expected from the metabolism of AA via GA. Currently, we cannot confirm OH-PA to be a specific biomarker of the oxidative pathway of AA metabolism. Other sources than AA respectively GA might need to be considered for the formation of OH-PA.

Excretion of 2,3-dihydroxy-propionamide (OH-PA), the hydrolysis product of glycidamide, in human urine after single oral dose of deuterium-labeled acrylamide.

A dose of 0.99 mg d(3)-acrylamide (d(3)-AA) (13.2 µg/kg body weight) was ingested by a healthy male volunteer. Urine samples were collected over a period of 46 h after the intake and analyzed for the hydrolysis product of glycidamide (GA), 2,3-dihydroxy-propionamide (OH-PA), a metabolite of the toxicologically relevant oxidative AA metabolism pathway; 5.4% of the administered d(3)-AA dose was eliminated as OH-PA within 46 h after ingestion. Therefore, OH-PA represents a major metabolite of the oxidative metabolism pathway. Elimination kinetics of OH-PA is similar to the oxidative metabolites N-acetyl-S-(2-carbamoyl-2-hydroxyethyl)-cysteine (GAMA) and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)-cysteine (iso-GAMA). The major excretion of d(3)-OH-PA took place between 8 and 22 h with the highest urinary d(3)-OH-PA concentration (c (max)) of 69.3 µg/L urine, 18 h (t (max)) postdose. OH-PA (5.4%), together with the other known urinary metabolites of the oxidative pathway GAMA (4.6%) and iso-GAMA (0.8%), represents 10.8% of the total AA dose. The share of the oxidative pathway metabolites is much smaller than the share of the reductive pathway metabolite N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) that represents 51.7% of the ingested d(3)-AA dose. However, this new quantitative human data on OH-PA together with the previous data on the other oxidative pathway metabolites are of special importance when evaluating the carcinogenic potential of AA and when comparing human data with data from animal studies.

Assessment of micronuclei in lymphocytes from workers exposed to vapours and aerosols of bitumen.

We investigated the micronucleus frequencies in peripheral blood lymphocytes of 225 mastic asphalt workers (age 17-62 years) and 69 non-bitumen-exposed road construction workers (age 18-64 years) in Germany before and after the working shift. Median shift exposure to vapours and aerosols of bitumen of exposed workers was 3.0 mg/m³. Micronuclei (MN) were determined with a standard method using cytochalasin B. Median MN frequency was 6.0 (interquartile range (IQR) 4.0-8.5) MN/1,000 binucleated lymphocytes (MN/1,000 BNC) in exposed workers and 6.0 (IQR 4.0-8.3) MN/1,000 BNC in non-exposed workers before shift. After shift, we observed 6.5 (IQR 4.4-9.3) MN/1,000 BNC in exposed workers and 6.5 (IQR 4.0-9.0) MN/1,000 BNC in non-exposed workers. Regression models were applied with the log-transformed MN frequency as the dependent variable in order to estimate the effects of exposure to vapours and aerosols of bitumen and of potential confounders. Age was the strongest predictor of MN formation in both exposed workers and referents. Our data suggest that MN formation was not associated with concentration of vapours and aerosols of bitumen during shift at the individual level. Although similar MN frequencies were observed in both groups, the modelling of factors potentially influencing MN frequency revealed a weak group difference in the post-shift model. We conclude that this small difference cannot be judged to be a relevant mutagenic effect of exposure to vapours and aerosols of bitumen, also with regard to the lack of adjustment for multiple testing and the lack of a group effect in the original data.

Modulation of urinary polycyclic aromatic hydrocarbon metabolites by enzyme polymorphisms in workers of the German Human Bitumen Study.

Data concerning the influence of sequence variants of metabolizing enzymes on the effect modulation of current exposure to vapors and aerosols of bitumen in humans are limited. To assess the influence of 18 single-nucleotide polymorphisms (SNP) in genes coding for enzymes involved in polycyclic aromatic hydrocarbon (PAH) and amine metabolism regarding their impact on urinary markers 1-hydroxpyrene (1-OHP) and the sum of 1-, 2+9-, 3-, 4-hydroxyphenanthrene (OHPHE). Based on personal ambient monitoring data for bitumen emissions, 218 German workers exposed to vapors and aerosols of bitumen during a shift and 96 German roadside construction workers without exposure to bitumen but with similar working tasks were studied. SNP determination based on DNA aliquots isolated from blood samples by real-time PCR or direct sequencing. The impact of sequence variants on the urinary levels of 1-OHP and sum of OHPHE was estimated with mixed linear models, adjusted for age, creatinine, exposure, smoking, SNP, and time of measurement. In the mixed linear model, an increasing metabolite level of OHPHE was only slightly modulated by the CC variant of the cytochrome P450 SNP CYP1A1 3801T>C (rs4646903; P = 0.051). In contrast, GSTM1 carriers showed a significant (P= 0.046) and double-mutated variants of three NAT2-specific SNP (NAT2*341CC, P = 0.06; NAT2*481TT, P = 0.06; NAT2*803GG, P = 0.042) displayed a decreasing influence on OHPHE levels. None of the SNP studied showed a significant effect on 1-OHP. The modulating SNP effects on OHPHE in the adjusted model were less pronounced when compared with the effects observed in a recent study with 170 workers occupationally exposed to PAH in German industries. This may be due to the much lower PAH exposure in the Human Bitumen Study.

Urinary metabolites of polycyclic aromatic hydrocarbons in workers exposed to vapours and aerosols of bitumen.

Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons (PAH) were investigated as potential biomarkers of bitumen exposure in a cross-shift study in 317 exposed and 117 non-exposed workers. Personal measurements of the airborne concentration of vapours and aerosols of bitumen during a working shift were weakly associated with post-shift concentrations of 1-hydroxypyrene (1-OHP) and 1-, 2+9-, 3- and 4-hydroxyphenanthrenes (further referred to their sum as OHPHE), but not 1- and 2-hydroxynaphthalene (OHNA). Smoking showed a strong influence on the metabolite concentrations, in particular on OHNA. Pre-shift concentrations of 1-OHP and OHPHE did not differ between the study groups (P = 0.16 and P = 0.89, respectively). During shift, PAH metabolite concentrations increased in exposed workers and non-exposed smokers. Statistical modelling of post-shift concentrations revealed a small increase in 1-OHP by a factor of 1.02 per 1 mg/m(3) bitumen (P = 0.02) and 1.04 for OHPHE (P < 0.001). A group difference was observed that was diminished in non-smokers. Exposed non-smokers had a median post-shift 1-OHP concentration of 0.42 µg/l, and non-smoking referents 0.13 µg/l. Although post-shift concentrations of 1-OHP and OHPHE were slightly higher than those in the general population, they were much lower than in coke-oven workers. The small content of PAHs in vapours and aerosols of bitumen, the increasing use of additives to asphalt mixtures, the strong impact of smoking and their weak association with airborne bitumen limit the use of PAH metabolites as specific biomarkers of bitumen exposure.

Bitumen workers handling mastic versus rolled asphalt in a tunnel: assessment of exposure and biomarkers of irritation and genotoxicity.

Emission levels of vapours and aerosols of bitumen are different when processing rolled asphalt compared to mastic asphalt, with working temperatures up to 180 and 250°C, respectively. During the Human Bitumen Study, we examined six workers handling rolled asphalt and mastic asphalt in two consecutive weeks at the same construction site in a tunnel. In addition to the determination of exposure to bitumen and polycyclic aromatic hydrocarbons (PAH) during shift, we examined urinary PAH metabolites, irritative and genotoxic effects before and after shift. Median personal shift concentration of vapours and aerosols of bitumen was 1.8 (range 0.9-2.4) mg/m(3) during the application of rolled asphalt and 7.9 (range 4.9-11.9) mg/m(3) when mastic asphalt was applied. Area measurement of vapours and aerosols of bitumen revealed higher concentrations than the personal measurements for mastic asphalt (mastic asphalt: 34.9 mg/m(3); rolled asphalt: 1.8 mg/m(3)). Processing mastic asphalt was associated also with higher PAH concentrations. Urinary 1-hydroxypyrene and the sum of 1-, 2+ 9-, 3- and 4-hydroxyphenanthrene increased slightly during shift without clear difference between mastic and rolled asphalt application. However, the post-shift urinary PAH-metabolite concentrations did not reflect the different PAH exposure during mastic and rolled asphalt application. Individual workers could be identified by their spirometry results indicating that these data reflect more chronic than acute effects. In most cases, an increase of 8-oxodGuo adducts was observed during shift that was independent of the asphalt application. 8-oxodGuo and (+)-anti-BPDE-DNA adducts were higher than in exposed workers of the Human Bitumen Study independent of the asphalt application. The DNA-strand breaks were considerably higher pre-shift and decreased during shift. In this study, mastic asphalt application led to significantly higher exposure to vapours and aerosols of bitumen, as well as to airborne PAH, compared to rolled asphalt application. Nevertheless, no differences in the excretion of urinary PAH metabolites, lung function impairment and genotoxic markers were detected. However, higher levels of genotoxicity markers on both examination days compared with the results of the Human Bitumen Study may indicate a possible influence of the specific tunnel setting.

DNA adducts and strand breaks in workers exposed to vapours and aerosols of bitumen: associations between exposure and effect.

To study the associations between exposure to vapours and aerosols of bitumen and genotoxic effects, a cross-sectional and cross-shift study was conducted in 320 exposed workers and 118 non-exposed construction workers. Ambient air measurements were carried out to assess external exposure to vapours and aerosols of bitumen. Hydroxylated metabolites of naphthalene, phenanthrene and pyrene were measured in urine, whereas (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide ((+)-anti-BPDE), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxodGuo) and DNA strand breaks were determined in blood. Significantly higher levels of 8-oxodGuo adducts and DNA strand breaks were found in both pre- and post-shift blood samples of exposed workers compared to those of the referents. No differences between exposed workers and referents were observed for (+)-anti-BPDE. Moreover, no positive associations between DNA damage and magnitude of airborne exposure to vapours and aerosols of bitumen could be observed in our study. Additionally, no relevant association between the urinary metabolites of PAH and the DNA damage in blood was observed. Overall, our results indicate increased oxidative DNA damage in workers exposed to vapours and aerosols of bitumen compared to non-exposed referents at the group level. However, increased DNA strand breaks in bitumen workers were still within the range of those found in non-exposed and healthy persons as reported earlier. Due to the lack of an association between oxidative DNA damage and exposure levels at the workplaces under study, the observed increase in genotoxic effects in bitumen workers cannot be attributed to vapours and aerosols of bitumen.

Air sampling and determination of vapours and aerosols of bitumen and polycyclic aromatic hydrocarbons in the Human Bitumen Study.

The chemical complexity of emissions from bitumen applications is a challenge in the assessment of exposure. Personal sampling of vapours and aerosols of bitumen was organized in 320 bitumen-exposed workers and 69 non-exposed construction workers during 2001-2008. Area sampling was conducted at 44 construction sites. Area and personal sampling of vapours and aerosols of bitumen showed similar concentrations between 5 and 10 mg/m(3), while area sampling yielded higher concentrations above the former occupational exposure limit (OEL) of 10 mg/m(3). The median concentration of personal bitumen exposure was 3.46 mg/m(3) (inter-quartile range 1.80-5.90 mg/m(3)). Only few workers were exposed above the former OEL. The specificity of the method measuring C-H stretch vibration is limited. This accounts for a median background level of 0.20 mg/m³ in non-exposed workers which is likely due to ubiquitous aliphatic hydrocarbons. Further, area measurements of polycyclic aromatic hydrocarbons (PAHs) were taken at 25 construction sites. U.S. EPA PAHs were determined with GC/MS, with the result of a median concentration of 2.47 µg/m(3) at 15 mastic asphalt worksites associated with vapours and aerosols of bitumen, with a Spearman correlation coefficient of 0.45 (95% CI -0.13 to 0.78). PAH exposure at mastic-asphalt works was higher than at reference worksites (median 0.21 µg/m(3)), but about one order of magnitude lower compared to coke-oven works. For a comparison of concentrations of vapours and aerosols of bitumen and PAHs in asphalt works, differences in sampling and analytical methods must to be taken into account.