Bernd Rossbach

Internal exposure of the general population to DEHP and other phthalates—determination of secondary and primary phthalate monoester metabolites in urine☆

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:<0.5 (limit of quantification, LOQ) to 177 microg/L). A strong correlation was found between the excretion of 5OH-MEHP and 5oxo-MEHP with a correlation coefficient of r=0.991, indicating close metabolic proximity of those two parameters but also the absence of any contaminating interference. Median concentrations for the other monoester metabolites were for mono-n-butylphthalate (MnBuP) 181 microg/L, for monobenzylphthalate (MBzP) 21.0 microg/L, for monoethylphthalate (MEP) 90.2 microg/L and for mono-n-octylphthalate (MOP)<1.0 microg/L (LOQ). These results will help to perform health risk assessments for the phthalate exposure of the general population.

Determination of haemoglobin adducts of acrylamide and glycidamide in smoking and non-smoking persons of the general population

Acrylamide (AA) is a food-borne toxicant suspected to be carcinogenic to humans. It is formed in the heating process of starch-containing food. Currently, there is a great discussion about the possible human health risks connected with the dietary uptake of acrylamide. Haemoglobin adducts of acrylamide and its oxidative metabolite glycidamide are both markers of biochemical effect. However, because glycidamide has a higher carcinogenic potency than acrylamide itself, the glycidamide adduct might mirror the genotoxicity better than acrylamide adducts. In order to gain more information about the human metabolism of acrylamide, we investigated a small group of persons for the effective internal doses of acrylamide and glycidamide using haemoglobin adducts as parameters of biochemical effect. The collective was subdivided into non-smokers (n=13) and smokers (n=16) by determining the smoking-specific acrylonitrile haemoglobin adduct (N-cyanoethylvaline, CEV). The mean values for the adducts of acrylamide (N-2-carbamoylethylvaline, AAVal) and glycidamide (N-(R,S)-2-hydroxy-2-carbamoylethylvaline, GAVal) in nonsmokers was 19 pmol/g globin AAVal (range 7-31 pmol/g globin) and 17 pmol/g globin GAVal (range 9-23 pmol/g globin). For smokers mean levels of AAVal were 80 pmol/g globin (range: 25-199 pmol/g globin) and those of GAVal were 53 pmol/g globin (range: 22-119 pmol/g globin). Metabolism to glycidamide turned out to be significantly more effective in non-smokers than in the higher exposed smokers. Compared with studies in rats, the metabolic conversion of acrylamide to glycidamide as measured by haemoglobin adducts seems to occur to a similar extent in humans as in rats. Risk estimations on acrylamide based on experimental data obtained in rats obviously did not overestimate the cancer risk for the general population. Furthermore, our results might indicate that the dose-response curve for acrylamide is not linear. This would be in line with the results of animal experiments on rodents.

3-Hydroxybenzo[a]pyrene in the urine of workers with occupational exposure to polycyclic aromatic hydrocarbons in different industries.

Objectives: This study was conducted to assess external and internal exposure of workers to polycyclic aromatic hydrocarbons (PAHs). In this context, the analytical and diagnostical reliability of 3-hydroxybenzo[a]pyrene (3OH-BaP) as a biomarker of internal exposure to PAHs was established. Methods: Ambient and biological monitoring was carried out of 225 PAH-exposed employees of different industries. External exposure was determined by personal air sampling and analysis of 16 EPA-PAH. Internal exposure was examined by the urinary metabolites 3OH-BaP, 1-hydroxypyrene (OH-Pyr) and monohydroxylated phenanthrenes (OH-Phens). Results: Benzo[a]pyrene (BaP) was detected at all workplaces. Concentrations in the breathing zone of the workers ranged from below the limit of detection up to 44.3 μg/m3. In biological monitoring, urinary 3OH-BaP was found in median concentrations of 0.8 ng/g creatinine (crea) and the 95th percentile of 6.7 ng/g crea. The results ranged from the limit of detection up to 19.5 ng/g crea. Only 1% of the analysed samples showed concentrations below the limit of detection (0.05 ng/l). Regarding median concentrations, workers in coking plants showed lower 3OH-BaP concentrations (0.5 ng/g crea) than those employed in the production of fireproof material in refractories (1.1 ng/g crea), converter infeed (1.2 ng/g crea) and graphite electrode production (1.3 ng/g crea). Strong correlations of 3OH-BaP with OH-Pyr and the sum of OH-Phens were found for the workplaces converter infeed, coking plants and graphite electrode production (rPearson ranging from 0.618 to 0.867, p<0.001). The poor correlation of BaP in the air and 3OH-BaP in urine is most probably caused by routes of uptake other than via air—for example, dermal uptake. Conclusion: 3OH-BaP as a metabolite of the carcinogenic BaP could be shown to be a diagnostically specific and sensitive biomarker for determining the internal exposure of workers in different industries. Using this method, the estimation of health risks for workers can be fundamentally improved, because the 3OH-BaP represents the group of carcinogenic PAHs. The procedure for analysing 3OH-BaP is complex, but it is robust and produces reliable results.

Risk assessment of Bundeswehr (German Federal Armed Forces) permethrin-impregnated battle dress uniforms (BDU)

In an age when vector-borne diseases are emerging worldwide, personal protective measures are essential for shielding soldiers and other exposed persons from arthropod attack. The development of permethrin-impregnated clothing has been one recent advance in protecting persons at-risk. However, to date risk assessment has not been performed related to wearing permethrin-impregnated clothing over longer time periods. Therefore, this paper describes relevant toxicological aspects of permethrin and estimates the extent of dermal permethrin uptake by soldiers wearing impregnated uniforms by determining urine metabolites of permethrin. The exposure monitoring conducted in wearers of untreated uniforms did not show any signs of increased permethrin uptake and was similar to that of the general population in Germany. By contrast, studies involving the soldiers wearing permethrin-impregnated uniforms identified far higher internal exposure, the amounts of urine metabolites clearly above the reference value for the background exposure of the German population at large. Comparing the median excretion values, an approximately 200 times higher exposure can be assumed. The excretion levels of the subject with the maximum amount of metabolites correspond to an internal exposure of around 5-6microg/kg body weight and day thereby considering that biomonitoring could not take all urine metabolites and other elimination routes into account. Based on an oral absorption rate of 50%, the internal dose of 5-6microg/kg body weight and day would correspond to an oral uptake of permethrin which is around 20% of the ADI value of 50microg/kg body weight and day. In addition, based on these data and using a dermal absorption rate of 2% the permethrin dose reaching the skin was estimated to be 250microg/kg body weight and day. Considering a standard body weight and the area covered by the uniform, an exposure level of about 1.25microg permethrin/cm(2) skin and day can be calculated. Clinical subjective symptoms were recorded by means of a self-reporting questionnaire which has been developed and used for this specific purpose in environmental outpatient departments in both groups (wearers of impregnated versus non-impregnated uniforms). Only minor sensory impairments were identified in one of the studies (Kabul/Afghanistan) which may represent skin paraesthesiae. Based on these results, it can be assumed that the normal use of permethrin-treated uniforms does not affect human health to a clinical relevant extent. We recommend that the release rate of permethrin from the textile material should be strictly monitored by means of a quality assurance method. It should comply with standards to which the results of this study may contribute.

Uptake of permethrin from impregnated clothing

In order to examine exposure and health risks which can arise from permethrin-impregnated clothing, a controlled trial was conducted. In a study group consisting of 187 volunteers in total, a subgroup of 86 persons was equipped with permethrin-impregnated battle dress uniforms (BDU) for 28 days. One hundred and one persons served as a control group, wearing non-impregnated BDUs throughout the entire study period of 56 days. Internal exposure of all participants was assessed by determination of urinary permethrin metabolites (cis-DCCA, trans-DCCA and 3-PBA) on day 0, 14 and 28 of the wearing period and 28 days after termination of wearing. Exposure levels in the control group ranged within background exposure of the general German population at all four dates of sampling (medians Sigma DCCA+3-PBA were 0.09, 0.13, 0.23 and 0.10mug/l, respectively). For the group equipped with impregnated BDUs this applied to day 0 (0.31mug/l) only, while the following measurements revealed considerably higher metabolite concentrations (31.39, 22.01 and 1.44mug/l, respectively), especially while wearing impregnated clothing. Due to these results a substantial uptake of permethrin from impregnated BDUs has to be assumed. However, since calculations reveal a maximum permethrin uptake clearly below the acceptable daily intake (ADI), health impairments are rather unlikely.

Saliva as a matrix for human biomonitoring in occupational and environmental medicine

AbstractPurpose Human biomonitoring (HBM) implies the assessment of internal exposure to hazardous substances by measuring the substances, their metabolites or reaction products, as well as effect parameters in human body fluids. Along with blood, plasma and urine, saliva is of increasing interest as an alternative matrix for HBM.MethodsThis paper reviews studies that measure salivary background levels of hazardous substances, elevated levels after environmental or occupational exposure, as well as references which deal with physiological and toxicokinetic behaviour of saliva and salivary parameters, respectively.ResultsThe studies revealed that the determination of biomarkers in saliva is a promising approach for HBM, even if only few substances showed a satisfying correlation with exposure data or established biomonitoring matrices such as blood, plasma and urine. Saliva has been proven to be particularly suitable for substances of low molecular weight such as organic solvents, selected pesticides, cotinine, and for some specific trace elements. Besides several advantages, serious problems and limitations were identified. Above all, the complex interactions between substance properties, sampling procedure, sample preparation, measurement techniques or individual factors, and the salivary analyte level are discussed.ConclusionsA major conclusion of the review is that more scientific studies are needed in order to systematically collect data on parameters, influencing salivary analyte levels. Crucially required is a harmonisation of the sampling as well as the sample preparation techniques and procedures, which is indispensable to achieve an overall comparability and interpretability of salivary biomarker levels.

Application of headspace solid phase dynamic extraction gas chromatography/mass spectrometry (HS-SPDE-GC/MS) for biomonitoring of n-heptane and its metabolites in blood.

Solid phase dynamic extraction (SPDE) is an innovative sample preparation and enrichment technique in connection with gas chromatography (GC). Using SPDE, we developed a method for simultaneous determination of n-heptane and its mono-oxygenated metabolites heptane-4-one, 3-one, 2-one, 4-ol, 3-ol, 2-ol, and 1-ol in blood. After adjustment of various extraction and desorption parameters, method validation resulted in limits of detection (LOD) between 0.006 (heptane-4-one) and 0.021mg/L (heptane-1-ol). Intra-assay coefficients of variation ranged between 4.8% and 20.8% while relative recovery ranged between 100% and 117% (spiked concentration 0.128mg/L, n=8). The method was applied to blood samples, which have been collected from 20 volunteers after controlled inhalative exposure to 167, 333, and 500ppm n-heptane. After 3h of exposure, n-heptane and heptane-2-one were detectable in all samples in concentrations ranging up to 2.903 and 0.495mg/L, while the concentrations of the remaining analytes were closer to the respective LOD or even below. A significant linear relationship with ambient exposure (R(2)=0.701, p<0.001, n=55) was found for n-heptane in blood, which could be helpful for evaluation of biological exposure limits in future. Due to its high abundance in blood, 2-heptanone could be an interesting candidate as a biomarker also in alternative matrices such as urine or saliva.

Biomonitoring in wearers of permethrin impregnated battle dress uniforms in Afghanistan and Germany

Objectives To analyse differences in uptake of the insecticide permethrin in wearers of non-impregnated and permethrin impregnated battle dress uniforms (BDU) in Afghanistan and Germany. Methods In two separate studies, in April 2003–January 2004 (study I, n=549) and in February–April 2005 (study II, n=195), healthy female and male members of the German Federal Armed Forces were equipped with permethrin impregnated BDU (two sub-cohorts in Germany and one in Afghanistan) while members equipped with non-impregnated uniforms served as a control group. Human biomonitoring was conducted before, during and after wearing the uniforms by measuring permethrin metabolites in urine samples via GC–MS. Results Subjects of the Afghan and German control groups had permethrin levels in the range of the German general population. In contrast, subjects wearing impregnated BDU had about 200-fold higher exposure levels. Within this group, subjects located in Afghanistan and smokers had significantly higher exposure levels. Internal exposure decreased with increasing duration of use of impregnated BDU. Conclusions There is no evidence for a higher background permethrin contamination in military bases located in Afghanistan compared to Germany. Daily use of permethrin impregnated BDU is associated with significantly higher permethrin uptake compared to the general population. Hand–mouth contact by smoking can increase uptake which also seems to be influenced by the duration of use of impregnated BDU.

Uptake and elimination of permethrin related to the use of permethrin treated clothing for forestry workers

Wearing of permethrin treated clothing usually implicates an uptake of permethrin by the user. Aim of our study was to examine the kinetics of internal permethrin exposure in volunteers during and after a single 8h-use of treated clothing as well as factors potentially influencing permethrin uptake. 28 male volunteers (age: 20-34 years) were equipped with permethrin treated jackets and pants from two different suppliers. The clothing was worn for 8h, simulating differing external conditions, including comfort conditions as well as conditions of increased temperature and humidity without and with additional physical workload. Internal permethrin exposure was monitored by determination of permethrin metabolites (DCCA and 3-PBA) in a set of 12 urine samples, covering a period of 504 h from the beginning of the wearing interval. Time-concentration curves showed an increase of internal exposure associated with wearing of the clothing (individual maximum: 109.5 μg/L) followed by a first-order like decay (mean half-life: 38.5 h). Metabolite excretion was affected by the make of clothing, which could be explained by differing permethrin contents of the garment. Furthermore, internal exposure increased with increasing temperature/humidity and additional physical workload. Assuming dermal uptake of permethrin, this may be ascribed to an alteration of the barrier function of the skin.

Biomonitoring and evaluation of permethrin uptake in forestry workers using permethrin-treated tick-proof pants.

We conducted a randomized case–control trial to analyze uptake of the insecticide/arcaricide permethrin in wearers of permethrin-impregnated and non-impregnated pants in German forestry. Eighty-two male workers were each equipped for a 16-week period with permethrin-treated (test group) or with non-treated work pants (control group). Pants with or without lining to protect against cuts, obtained from two different distributors, were worn in each group. Urinary permethrin metabolite levels were measured by GC-MS/MS before, during and after wearing of the pants. Permethrin uptake was calculated using additional questionnaire data. In the control group, metabolite levels in the range of environmental background exposure (median: ~0.5 μg/l) were measured. Subjects wearing impregnated pants showed consistently significantly higher exposure levels even before the first use of the pants with a maximum after 1 week of wearing the pants (median: ~12.5 μg/l). Significant differences in internal exposure were found depending on which of the distributors the pants came from. Metabolite levels decreased probably due to permethrin losses associated with laundering the pants. Calculated permethrin uptake is below the value corresponding to the WHO-proposed acceptable daily intake. Based on our data, a marginally increased cancer risk compared with the general population cannot be excluded when wearing impregnated pants over a working-lifetime period.