Current exposure to phthalates and DINCH in European children and adolescents - Results from the HBM4EU Aligned Studies 2014 to 2021.

Phthalates are mainly used as plasticizers for polyvinyl chloride (PVC). Exposure to several phthalates is associated with different adverse effects most prominently on the development of reproductive functions. The HBM4EU Aligned Studies (2014-2021) have investigated current European exposure to ten phthalates (DEP, BBzP, DiBP, DnBP, DCHP, DnPeP, DEHP, DiNP, DiDP, DnOP) and the substitute DINCH to answer the open policy relevant questions which were defined by HBM4EU partner countries and EU institutions as the starting point of the programme. The exposure dataset includes ∼5,600 children (6-11 years) and adolescents (12-18 years) from up to 12 countries per age group and covering the North, East, South and West European regions. Study data from participating studies were harmonised with respect to sample size and selection of participants, selection of biomarkers, and quality and comparability of analytical results to provide a comparable perspective of European exposure. Phthalate and DINCH exposure were deduced from urinary excretions of metabolites, where concentrations were expressed as their key descriptor geometric mean (GM) and 95th percentile (P95). This study aims at reporting current exposure levels and differences in these between European studies and regions, as well as comparisons to human biomonitoring guidance values (HBM-GVs). GMs for children were highest for ∑DEHP metabolites (33.6 µg/L), MiBP (26.6 µg/L), and MEP (24.4 µg/L) and lowest for∑DiDP metabolites (1.91 µg/L) and ∑DINCH metabolites (3.57 µg/L). In adolescents highest GMs were found for MEP (43.3 µg/L), ∑DEHP metabolites (28.8 µg/L), and MiBP (25.6 µg/L) and lowest for ∑DiDP metabolites (= 2.02 µg/L) and ∑DINCH metabolites (2.51 µg/L). In addition, GMs and P95 stratified by European region, sex, household education level, and degree of urbanization are presented. Differences in average biomarker concentrations between sampling sites (data collections) ranged from factor 2 to 9. Compared to the European average, children in the sampling sites OCC (Denmark), InAirQ (Hungary), and SPECIMEn (The Netherlands) had the lowest concentrations across all metabolites and ESTEBAN (France), NAC II (Italy), and CROME (Greece) the highest. For adolescents, comparably higher metabolite concentrations were found in NEB II (Norway), PCB cohort (Slovakia), and ESTEBAN (France), and lower concentrations in POLAES (Poland), FLEHS IV (Belgium), and GerES V-sub (Germany). Multivariate analyses (Survey Generalized Linear Models) indicate compound-specific differences in average metabolite concentrations between the four European regions. Comparison of individual levels with HBM-GVs revealed highest rates of exceedances for DnBP and DiBP, with up to 3 and 5%, respectively, in children and adolescents. No exceedances were observed for DEP and DINCH. With our results we provide current, detailed, and comparable data on exposure to phthalates in children and - for the first time - in adolescents, and - for the first time - on DINCH in children and adolescents of all four regions of Europe which are particularly suited to inform exposure and risk assessment and answer open policy relevant questions.

From science to policy: How European HBM indicators help to answer policy questions related to phthalates and DINCH exposure.

Within the European Human Biomonitoring (HBM) Initiative HBM4EU we derived HBM indicators that were designed to help answering key policy questions and support chemical policies. The result indicators convey information on chemicals exposure of different age groups, sexes, geographical regions and time points by comparing median exposure values. If differences are observed for one group or the other, policy measures or risk management options can be implemented. Impact indicators support health risk assessment by comparing exposure values with health-based guidance values, such as human biomonitoring guidance values (HBM-GVs). In general, the indicators should be designed to translate complex scientific information into short and clear messages and make it accessible to policy makers but also to a broader audience such as stakeholders (e.g. NGO's), other scientists and the general public. Based on harmonized data from the HBM4EU Aligned Studies (2014-2021), the usefulness of our indicators was demonstrated for the age group children (6-11 years), using two case examples: one phthalate (Diisobutyl phthalate: DiBP) and one non-phthalate substitute (Di-isononyl cyclohexane-1,2- dicarboxylate: DINCH). For the comparison of age groups, these were compared to data for teenagers (12-18 years), and time periods were compared using data from the DEMOCOPHES project (2011-2012). Our result indicators proved to be suitable for demonstrating the effectiveness of policy measures for DiBP and the need of continuous monitoring for DINCH. They showed similar exposure for boys and girls, indicating that there is no need for gender focused interventions and/or no indication of sex-specific exposure patterns. They created a basis for a targeted approach by highlighting relevant geographical differences in internal exposure. An adequate data basis is essential for revealing differences for all indicators. This was particularly evident in our studies on the indicators on age differences. The impact indicator revealed that health risks based on exposure to DiBP cannot be excluded. This is an indication or flag for risk managers and policy makers that exposure to DiBP still is a relevant health issue. HBM indicators derived within HBM4EU are a valuable and important complement to existing indicator lists in the context of environment and health. Their applicability, current shortcomings and solution strategies are outlined.

Reviewing the variability in urinary concentrations of non-persistent organic chemicals: evaluation across classes, sampling strategies and dilution corrections.

Various biomonitoring studies have been carried out to investigate the exposure of populations by measuring non-persistent organic chemicals in urine. To accurately assess the exposure, study designs should be carefully developed to maximise reproducibility and achieve good characterization of the temporal variability. To test these parameters, the intraclass correlation coefficients (ICCs) are calculated from repeated measurements and range from poor (<0.4) to excellent (≥0.75). Several studies have reported ICCs based on diverse study designs, but an overview, including recommendations for future studies, was lacking. Therefore, this review aimed to collect studies describing ICCs of non-persistent organic chemicals, discuss variations due to study design and formulate recommendations for future studies. More than 60 studies were selected, considering various chemical classes: bisphenols, pyrethroids, parabens, phthalates, alternative plasticizers and phosphate flame retardants. The variation in ICCs for an individual chemical was high (e.g. ICC of propyl paraben = 0.28-0.91), showing the large impact of the study design and of the specific exposure sources. The highest ICCs were reported for parabens (median = 0.52), while lowest ICCs were for 3-phenoxybenzoic acid (median = 0.08) and bisphenol A (median = 0.20). Overall, chemicals that had an exposure source with high variation, such as the diet, showed lower ICCs than those with more stable exposure sources, such as indoor materials. Urine correction by specific gravity had an overall positive effect on reducing the variability of ICCs. However, this effect was mostly seen in the adult population, while specific compounds showed less variation with creatinine correction. Single samples might not accurately capture the exposure to most non-persistent organic chemicals, especially when small populations are sampled. Future studies that examine compounds with low ICCs should take adequate measures to improve accuracy, such as correcting dilution with specific gravity or collecting multiple samples for one participant.

Development of an analytical method based on solid-phase extraction and LC-MS/MS for the monitoring of current-use pesticides and their metabolites in human urine.

Pyrethroids, organophosphorus pesticides and fipronil have been listed as priority chemicals in human biomonitoring studies because of their wide use and potential health effects in humans. The determination of 13 pesticides, including pyrethroids (deltamethrin, cypermethrin, permethrin, cyfluthrin, bifenthrin), organophosphorus (chlorpyrifos, chlorpyrifos-methyl, and malathion), fipronil, neonicotinoids (imidacloprid, acetamiprid and thiacloprid) and triazole (prothioconazole), together with 13 corresponding metabolites in human urine samples was achieved by solid-phase extraction and analysis by liquid chromatography coupled to tandem mass spectrometry. All targeted compounds, except malathion dicarboxylic acid, were measured with a mean within-accuracy (n = 5) of 71%-114% (RSD: 1%-14%) and between-run (n = 15) accuracy of 80%-118% (RSD: 2%-14%). Limits of quantitation of the targeted analytes ranged from 0.1 to 16 pg/mL. The detection result of urine samples from 25 volunteers indicated that the detection frequencies of 3,5,6-trichloro-2-pyridinol (median: 448 pg/mL), 6-chloropyridine-3-carboxylic acid (median: 193 pg/mL), 2-methyl-3-phenylbenzoic acid (median: 181 pg/mL), 3-phenoxybenzoic acid (median: 99 pg/mL), 2-isopropyl-6-methyl-4-pyrimidinol (median: 77 pg/mL), cyfluthrin (median: 59 pg/mL), cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cis-DCCA, median: 53 pg/mL), trans-DCCA (median: 25 pg/mL), prothioconazole (median: 21 pg/mL), imidacloprid (median: 7 pg/mL), and prothioconazole-desthio (median: 1 pg/mL) were > 50%. The obtained results show that the validated method is suitable for the human biomonitoring of these current-use pesticides and their metabolites.

Identification of chemicals of emerging concern in urine of Flemish adolescents using a new suspect screening workflow for LC-QTOF-MS.

An essential step in human biomonitoring or molecular epidemiology programs is to estimate human exposure to environmental chemicals. Despite significant progress in the capabilities of analytical methods, the number of pollutants and their metabolites keeps increasing continuously. Some of these relatively unknown chemicals of emerging concern (CECs) may pose significant health risks to humans and biota, but remain virtually undetected in traditional HBM-studies. Non-target and suspect screening techniques based on high-resolution mass spectrometry (HRMS) perform the detection and identification of compounds without any a priori compound selection or chemical information and provide a more holistic overview of human exposure. In this study, 50 urine samples (25 female and 25 male) from a larger cohort of the Flemish Environment and Health Study (FLEHS IV, 2016-2020) have been submitted to suspect screening analysis, with the aim of detecting and identifying new CECs. For this purpose, an analytical method has been developed, optimised and evaluated in terms of analytical performance. Satisfactory results were obtained in terms of reproducibility, sensitivity and quality control. Data-mining was performed through the combination of two different workflows. The use of two complementary workflows enhanced the number of identified compounds. As a result, 45 CECs have been identified with a level of confidence ranged between 3 and 1. Most of the identified compounds were metabolisation products, many of which were currently not included in the targeted measurements of FLEHS IV. The identified chemicals and metabolites could be used as candidate biomarkers of exposure in future studies. Overall, the newly developed suspect screening workflow of this pilot study provided complementary and promising results for future HBM-programs.

Biomarkers of phthalates and alternative plasticizers in the Flemish Environment and Health Study (FLEHS IV): Time trends and exposure assessment.

Restrictions on the use of legacy phthalate esters (PEs) as plasticizer chemicals in several consumer products has led to the increased use of alternative plasticizers (APs), such as di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH) and di-(2-ethylhexyl) terephthalate (DEHTP). In the fourth cycle of the Flemish Environment and Health Study (FLEHS IV, 2016-2020), we monitored exposure to seven PEs (diethyl phthalate (DEP), di-(2-ethylhexyl) phthalate (DEHP), di-isobutyl phthalate (DiBP), di-n-butyl phthalate (DnBP), butylbenzyl phthalate (BBzP, di-isononyl phthalate (DINP), and di-isodecyl phthalate (DIDP))and three APs (DINCH, DEHTP, and di-(2-ethylhexyl) adipate (DEHA)) by measuring multiple biomarkers in urine of 416 adolescents from Flanders, Belgium (14-15 years old). The reference values show that exposure to PEs is still widespread, although levels of several PE metabolites (e.g., sum of DEHP metabolites, mono-normal-butyl phthalate (MnBP) and mono-benzyl phthalate (MBzP)) have decreased significantly compared to previous human biomonitoring cycles (2003-2018). On the other hand, metabolites of DINCH and DEHTP were detected in practically every participant. Concentrations of AP exposure biomarkers in urine were generally lower than PE metabolites, but calculations of estimated daily intakes (EDIs) showed that exposure to DINCH and DEHTP can be considerable. However, preliminary risk assessment showed that none of the EDI or urinary exposure levels of APs exceeded the available health-based guidance values, while a very low number of participants had levels of MiBP and MnBP exceeding the HBM value. Several significant determinants of exposure could be identified from multiple regression models: the presence of building materials containing PVC, ventilation habits, socio-economic status and season were all associated with PE and AP biomarker levels. Cumulatively, the results of FLEHS IV show that adolescents in Flanders, Belgium, are exposed to a wide range of plasticizer chemicals. Close monitoring over the last decade showed that the exposure levels of restricted PEs have decreased, while newer APs are now frequently detected in humans.

Exposure levels, determinants and risk assessment of organophosphate flame retardants and plasticizers in adolescents (14-15 years) from the Flemish Environment and Health Study.

The ubiquitous use of organophosphate flame retardants and plasticizers (PFRs) in a variety of consumer products has led to widespread human exposure. Since certain PFRs are developmental and carcinogenic toxicants, detailed exposure assessments are essential to investigate the risk associated with environmental exposure levels. However, such data are still lacking for European countries. In this study, concentrations of thirteen PFR metabolites were measured in urine samples from 600 adolescents from Flanders, Belgium. 1-Hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP), diphenyl phosphate (DPHP), bis(1,3-dichloro-isopropyl) phosphate (BDCIPP), 2-hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP), 2-ethylhexyl phenyl phosphate (EHPHP) and 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-HO-EHDPHP) were frequently detected (>83%) in all participants. Comparisons with study populations from outside the EU showed that urinary levels of DPHP, BDCIPP and BCIPHIPP were generally within the same range. Only exposure to 2-ethylhexyl diphenyl phosphate (EHDPHP) was presumably higher in Flemish adolescents. However, determinants analysis through multivariate regression analyses did not reveal significant predictors that may explain this finding. Significantly higher levels of BDCIPP were observed in participants with new decorations at home, while adolescents with highly educated parents had higher levels of BBOEHEP and BDCIPP. Furthermore, multiple PFR metabolite concentrations followed a seasonal pattern. Estimated daily intakes (EDIs) were calculated from the internal dose by including fractions of urinary excretion (FUE) estimated in in vitro metabolism studies. EDIs ranged from 6.3 ng/kg bw/day for TBOEP to 567.7 ng/kg bw/day for EHDPHP, which were well below the available oral reference doses for all investigated PFRs. This suggests that the associated risk is low at present. This is the first report on internal exposure to seven commonly used PFRs in a European population.

Short-term variability of bisphenols in spot, morning void and 24-hour urine samples.

Due to worldwide regulations on the application of the high production volume industrial chemical bisphenol A (BPA) in various consumer products, alternative bisphenols such as bisphenol F (BPF) and bisphenol S (BPS) are increasingly used. To assess human exposure to these chemicals, biomonitoring of urinary concentrations is frequently used. However, the short-term variability of alternative bisphenols has not been evaluated thoroughly yet, which is essential to achieve a correct estimation of exposure. In this study, we collected all spot urine samples from ten healthy adults for five consecutive days, and an additional 24 h pooled sample. We measured the concentrations of seven bisphenols (BPAF, BPF, BPA, BPB, BPZ, BPS and BPAP) in these samples using gas chromatography coupled to tandem mass spectrometry. BPA, BPF and BPS were frequently found in spot samples (>80%), while bisphenol AP (BPAP) was detected in 43% of spot samples. Calculations of intra-class correlation coefficients (ICCs) showed that reproducibility of these four bisphenols was relatively poor (<0.01-0.200) but improved when concentrations were corrected for urine dilution using creatinine levels (0.128-0.401). Of these four bisphenols, BPF showed the best reproducibility (ICC 0.200-0.439) and BPS the most variability (ICC <0.01-0.128). In general, the within-participant variability of bisphenol levels was the largest contributor to the total variance (47-100%). We compared repeated first morning voids to 24 h pooled urine and found no significantly different concentrations for BPA, BPF, BPS, or BPAP. Levels of BPA and BPF differed significantly depending on the sampling time throughout the day. The findings in this study suggest that collecting multiple samples per participant over a few days, in predefined time windows throughout the day, could result in a more reliable estimation of internal exposure to bisphenols.

Short-term temporal variability of urinary biomarkers of organophosphate flame retardants and plasticizers.

BACKGROUND: Exposure to organophosphate flame retardants and plasticizers (PFRs) is commonly estimated by measuring biomarker concentrations in spot urine samples. However, their concentrations in urine can vary greatly over time due to short biological half-lives and variable exposure, potentially leading to exposure misclassification. In this study, we examined the within- and between-individual and within- and between-day variability of PFR metabolites in spot and 24-hour pooled urine samples during five consecutive days. METHODS: We collected all spot urine samples from 10 healthy adults for 5 days. On one additional day, we collected 24-hour pooled urine samples. Samples were analyzed by solid-phase extraction coupled to high-performance liquid chromatography tandem mass spectrometry. We calculated intraclass correlation coefficients (ICCs) to assess the reproducibility of metabolite concentrations in morning void and spot samples. RESULTS: Fair-to-good reproducibility was observed for serial measurements of bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP), 2-hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP) and 2-ethyl-5-hydroxyhexyl diphenyl phosphate (5-HO-EHDPHP) (ICC: 0.396 - 0.599), whereas concentrations of diphenyl phosphate (DPHP) and 2-ethylhexyl phenyl phosphate (EHPHP) were more variable in time (ICC: 0.303 and 0.234). Reproducibility improved significantly when only morning void samples were considered and when concentrations were adjusted for urinary dilution. Collecting 24-hour pooled urine could be a reliable alternative for PFR biomarkers with poor short-term temporal variability. CONCLUSIONS: The between-day variability was minor compared to variability observed within the same day, which suggests that collecting multiple samples could reduce exposure missclassification. Differences in the observed between- and within-individual variance were compound specific and related to both the nature of the exposure (e.g., diet vs other exposure routes, multiple sources) and the individual toxicokinetic properties of the investigated PFRs.

Determinants of exposure levels of bisphenols in flemish adolescents.

The broadly used industrial chemical bisphenol A (BPA), applied in numerous consumer products, has been under scrutiny in the past 20 years due to its widespread detection in humans and the environment and potential detrimental effects on human health. Following implemented restrictions and phase-out initiatives, BPA is replaced by alternative bisphenols, which have not received the same amount of research attention. As a part of the fourth cycle of the Flemish Environment and Health Study (FLEHS IV, 2016-2020), we monitored the internal exposure to six bisphenols in urine samples of 423 adolescents (14-15 years old) from Flanders, Belgium. All measured bisphenols were detected in the study population, with BPA and its alternatives bisphenol F (BPF) and bisphenol S (BPS) showing detection frequencies > 50%. The reference values show that exposure to these compounds is extensive. However, the urinary BPA level decreased significantly in Flemish adolescents compared to a previous cycle of the FLEHS (2008-2009). This suggests that the replacement of BPA with its analogues is ongoing. Concentrations of bisphenols measured in the Flemish adolescents were generally in the same order of magnitude compared to recent studies worldwide. Multiple regression models were used to identify determinants of exposure based on information on demographic and lifestyle characteristics of participants, acquired through questionnaires. Some significant determinants could be identified: sex, season, smoking behavior, educational level of the parents, recent consumption of certain foods and use of certain products were found to be significantly associated with levels of bisphenols. Preliminary risk assessment showed that none of the estimated daily intakes (EDIs) of BPA exceeded the tolerable daily intake, even in a high exposure scenario. For alternative bisphenols, no health-based guidance values are available, but in line with the measured urinary levels, their EDIs were lower than that of BPA. This study is, to the best of our knowledge, the first to determine internal exposure levels of other bisphenols than BPA in a European adolescent population.

Between- and within-individual variability of urinary phthalate and alternative plasticizer metabolites in spot, morning void and 24-h pooled urine samples.

Due to international regulations, commonly used phthalates such as di(2-ethylhexyl) phthalate (DEHP) are being replaced by other phthalates, such as di-isononyl phthalate (DINP), and di-isodecyl phthalate (DIDP) and by alternative plasticizers (APs) with similar chemical characteristics, like di(isononyl)cyclohexane-1,2-dicarboxylate (DINCH), di(2-ethylhexyl) terephthalate (DEHTP), or di-(2-ethylhexyl) adipate (DEHA). Urinary concentrations of metabolites are frequently used in the exposure assessment of non-persistent chemicals and for biomonitoring purposes, the intra- and inter-day variability of the metabolites should be known. However, the short-term variability of AP and several phthalate biomarkers has not been investigated yet. In this study, we collected all spot samples from 10 healthy adults for 5 consecutive days and 24h pooled urine on one additional day to investigate the short-term variability of 22 biomarkers of phthalates and APs. Metabolites of DEP, DEHP, DiBP, DnBP, DBzP, DINP and DIDP were found in high detection frequencies, while metabolites of most APs were found in approximately 50% of the samples. The short-term reproducibility of metabolites with diet as primary source (DEHP, DINP, DIDP) was poor (intraclass correlation coefficient - ICC < 0.4), whereas biomarkers of DEP, DnBP, DiBP and BBzP showed good consistency, most likely due to more continuous sources resulting in less between-day variance. ICC values of AP metabolites were similar to those of DEHP, but more studies are required to confirm these findings. Overall, reproducibility improved considerably when values were corrected for urinary dilution and when only morning voids samples were considered. Levels in morning voids samples were consistent for 5 days and comparable to 24-h pooled urine for all metabolites except for OH-MEHTP, sum DINP and sum DIDP, which supports the use of morning voids in human biomonitoring studies.

Biomonitoring and temporal trends of bisphenols exposure in Japanese school children.

The widely used chemical bisphenol A (BPA), applied in various consumer products, has been under scrutiny in the past 20 years due to its widespread detection in humans and potential detrimental effects on human health. Following the implementation of restrictions and phase-out initiatives, BPA has been replaced by other structurally similar bisphenols, which have not yet received the same level of research attention. In this study, we aimed to 1) investigated the internal exposure to seven bisphenols in morning void urine samples (n = 396) from 7-year-old children from Hokkaido, Japan and 2) assess possible time trends in the concentrations of bisphenols between 2012 and 2017. Information on demographic, indoor environment and dietary characteristics of participants were acquired through a self-administered questionnaire. All bisphenols were detected in the study population, with BPA, BPF and BPS showing detection frequencies >50%. Concentrations of bisphenols measured in the Japanese children in our study were generally lower compared to studies worldwide. We found that BPA concentrations decreased significantly over the study time period (average 6.5% per year), whereas BPS rose with 2.8% per year. Levels of BPA and BPF were higher in autumn compared to winter. Higher urinary BPF levels were significantly associated with higher concentrations of the oxidative stress biomarker, 8-hydroxy-2'-deoxyguanosine (8-OHdG). BPA and BPF levels were higher in children from families with lower household income. Bisphenol concentrations were significantly influenced by some other personal (e.g. household income), food intake (e.g. vegetables and cow milk) and indoor housing characteristics (e.g. flooring). This is the first study to report longitudinal time trends of bisphenols in Japan. The presented findings imply that further research on bisphenols is warranted in the future to monitor whether these time trends continue.

Exposure to organophosphate esters, phthalates, and alternative plasticizers in association with uterine fibroids.

Exposure to endocrine disrupting chemicals is suggested to be responsible for the development or progression of uterine fibroids. However, little is known about risks related to emerging chemicals, such as organophosphate esters (OPEs) and alternative plasticizers (APs). A case-control study was conducted to investigate whether exposures to OPEs, APs, and phthalates, were associated with uterine fibroids in women of reproductive age. For this purpose, the cases (n = 32) and the matching controls (n = 79) were chosen based on the results of gynecologic ultrasonography among premenopausal adult women in Korea and measured for metabolites of several OPEs, APs, and major phthalates. Logistic regression models were employed to assess the associations between chemical exposure and disease status. Factor analysis was conducted for multiple chemical exposure assessments as a secondary analysis. Among OPE metabolites, diphenyl phosphate (DPHP), 2-ethylhexyl phenyl phosphate (EHPHP), and 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP) were detected in >80% of the subjects. Among APs, metabolites of di-isononyl phthalate (DINP) and di(2-propylheptyl) phthalate (DPrHpP) were detected in >75% of the urine samples. The odds ratios (ORs) of uterine fibroids were significantly higher among the women with higher exposures to tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and tris(2-butoxyethyl) phosphate (TBOEP), di(2-ethylhexyl) terephthalate (DEHTP), DPrHpP, and di-(iso-nonyl)-cyclohexane-1,2-dicarboxylate (DINCH). In addition, urinary concentrations of mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), a sum of five di(2-ethylhexyl) phthalate metabolites (∑5DEHP), and mono(4-methyl-7-hydroxyoctyl) phthalate (OH-MINP) were significantly higher in the cases. In factor analysis, a factor heavily loaded with DPrHpP and DEHP was significantly associated with uterine fibroids, supporting the observation from the single chemical regression model. We found for the first time that several metabolites of OPEs and APs are associated with increased risks of uterine fibroids among pre-menopausal women. Further epidemiological and mechanistic studies are warranted to validate the associations observed in the present study.

Temporal trends and determinants of PFR exposure in the Hokkaido Study.

The phase-out of polybrominated diphenyl ethers (PBDE) flame retardants has led to the rapid increase of alternatives such as phosphate flame retardants and plasticizers (PFRs) in many consumer products. Exposure to these additive chemicals is widespread and potentially harmful to humans and the environment. In the present study, we assessed the exposure to PFRs through the analysis of metabolites in urine collected from 7-year old children from Hokkaido, Japan between 2012 and 2017. This allowed us to investigate temporal and seasonal trends for PFR metabolite concentrations and to study determinants of exposure. Thirteen metabolites of seven PFRs were measured in morning spot urine samples (n = 400). Multiple regression models were used to quantify the yearly increase in metabolite concentrations per sampling year. Information on the demographics, indoor environment and dietary habits of the participants were derived from self-administered questionnaires. PFR metabolite concentrations were comparable to our previous study of school children (7-12 years old). Eight PFR metabolites were detected in >50% of the samples. During the study time period, concentrations of three metabolites increased significantly: bis(1,3-dichloro-2-propyl) phosphate (BDCIPP; 13.3% per year), 1-hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP; 12.9% per year), and 2-ethylhexyl phenyl phosphate (EHPHP; 6.7% per year). We also found seasonality as a determinant for several PFR metabolites, with 2-fold higher levels in summer for BCIPHIPP and BDCIPP. Concentrations were also significantly impacted by ventilation habits. More frequent window opening or use of mechanical ventilation was consistently associated with higher levels of PFR metabolites in children's urine. This is the first study to show that human exposure to PFRs has increased in recent years in Japan, which indicates that further research into this class of chemicals is warranted.

Combined exposure to phthalate esters and phosphate flame retardants and plasticizers and their associations with wheeze and allergy symptoms among school children.

BACKGROUND: Phthalate esters and phosphate flame retardants and plasticizers (PFRs) are both used as plasticizers and are commonly detected in indoor environments. Although both phthalates and PFRs are known to be associated with children's wheeze and allergic symptoms, there have been no previous studies examining the effects of mixtures of these exposures. OBJECTIVES: To investigate the association between exposure to mixtures of phthalate esters and PFRs, and wheeze and allergic symptoms among school-aged children. METHODS: A total of 128 elementary school-aged children were enrolled. Metabolites of 3 phthalate esters and 7 PFRs were measured in urine samples. Parent-reported symptoms of wheeze, rhinoconjunctivitis, and eczema were evaluated using the International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire. In the primary model, we created a phthalate ester and PFR mixture exposure index, and estimated odds ratios (ORs) using weighted quantile sum (WQS) regression and quantile g (qg)-computation. The two highest chemicals according to qg-computation weight %s were combined to create a combination high × high exposure estimate, with ORs calculated using the "low × low" exposure group as the reference category. Concentrations of each metabolite were corrected by multiplying this value by the sex- and body size-Standardised creatinine concentration and dividing by the observed creatinine value. All models were adjusted for sex, grade, dampness index and annual house income. RESULTS: The odds ratio of rhinoconjunctivitis for the association between exposure to chemical mixtures according to the WQS index positive models was; OR = 2.60 (95% confidence interval [CI]: 1.38-5.14). However, wheeze and eczema of the WQS index positive model, none of the WQS index negative models or qg-computation result yielded statistically significant results. Combined exposure to the two highest WQS weight %s of "high-high" ΣTCIPP and ΣTPHP was associated with an increased prevalence of rhino-conjunctivitis, OR = 5.78 (1.81-18.43) to the "low × low" group. CONCLUSIONS: Significant associations of mixed exposures to phthalates and PFRs and increased prevalence of rhinoconjunctivitis was found among elementary school-aged children in the WQS positive model. Mixed exposures were not associated with any of allergic symptoms in the WQS negative model or qg-computation approach. However, the combined effects of exposure to two PFRs suggested an additive and/or multiplicative interaction, potentially increasing the prevalence of rhinoconjunctivitis. A further study with a larger sample size is needed to confirm these results.

Organophosphate ester flame retardants and plasticizers in a Chinese population: Significance of hydroxylated metabolites and implication for human exposure.

Organophosphate esters (OPEs) are widely used as flame retardants, plasticizers and defoamers and their exposure are likely associated with a number of adverse effects in humans. In this study, tris(chloroethyl) phosphate and thirteen OPE metabolites including six hydroxylated OPEs (HO-OPEs) were analyzed in 46 urine samples, collected from 8 provinces located across different regions in China. 1-Hydroxy-2-propyl bis(1-chloro-2-propyl) phosphate (BCIPHIPP) and 2-hydroxyethyl bis(2-butoxyethyl) phosphate (BBOEHEP) were major metabolites of their parent compounds with detection frequencies of 54.3%-89.1%, which were all higher than their corresponding OPE diesters (2.2%-6.5%). The urine-based estimated daily intake (EDI) of OPEs ranged from 0.06 ng/kg·bw for tris(2-butoxyethyl) phosphate (TBOEP) to 273 ng/kg·bw for 2-ethylhexyl phenyl phosphate. Analyzed with concentrations in paired dust samples, dust exposure to OPEs and their diesters may explain 0.28%-23.8% of the urine-based EDI of OPEs and the contribution of dust TBOEP was the highest. Although direct exposure to OPE diesters in dust showed a minor contribution, their intake via food and drinking water may account for a larger portion of urinary OPE metabolites. Overall, the hazard quotients of four OPEs indicated no immediate exposure risk for the investigated Chinese residents but the cumulative and long-term chronic effects involving exposure to other OPEs and OPE diesters are worth further concerns.

Towards establishing indicative values for metabolites of organophosphate ester contaminants in human urine.

In 2015, nine laboratories from Belgium, USA, Canada, China, and Australia participated in an interlaboratory exercise to quantify metabolites of organophosphate ester (OPE) contaminants in pooled human urine. Pooled human urine available as SRM 3673 (Organic contaminants in non-smokers' urine) was obtained from the U.S. National Institute of Standards and Technology and was analyzed for its content of OPE metabolites. Each participating laboratory received 10 mL sample and used its own validated method and standards to report the concentrations of the OPE metabolites of its choice. Four OPE metabolites were consistently measured by most laboratories and they were the following diesters: bis(1,3-dichloro-2-propyl) phosphate (BDCIPP), diphenyl phosphate (DPHP), bis(2-chloroethyl) phosphate (BCEP), and bis(1-chloro-2-propyl) phosphate (BCIPP). Concentrations of other OPE metabolites in SRM 3673 were also reported but are only considered as informative values since they were measured by three laboratories at most. All laboratories used liquid chromatography with tandem mass spectrometry (LC-MS/MS) with or without solid-phase extraction (SPE). This is the first study to report indicative values for OPE metabolites in a human urine Standard Reference Material. It is expected that these indicative values obtained for these four metabolites will be used as quality control to ensure compatibility of results in biomonitoring studies and by other researchers who validate their own methods for the quantification of OPE metabolites in human urine.

Multiple exposures to organophosphate flame retardants alter urinary oxidative stress biomarkers among children: The Hokkaido Study.

Organophosphate flame retardants (PFRs) are used as additives in plastics and other applications such as curtains and carpets as a replacement for brominated flame retardants. As such, exposure to PFR mixtures is widespread, with children being more vulnerable than adults to associated health risks such as allergies and inflammation. Oxidative stress is thought to be able to modulate the development of childhood airway inflammation and atopic dermatitis. To evaluate these associations, the present study investigated the relationship between urinary PFR metabolites, their mixtures and urinary oxidative stress biomarkers in children as part of the Hokkaido Study on Environment and Children's Health. The levels of the oxidative stress biomarkers, such as 8-hydroxy-2'-deoxyguanosine (8-OHdG), hexanoyl-lysine (HEL), and 4-hydroxynonenal (HNE), and of 14 PFR metabolites were measured in morning spot urine samples of 7-year-old children (n = 400). Associations between PFR metabolites or PFR metabolite mixtures and oxidative stress biomarkers were examined by multiple regression analysis and weighted quantile sum regression analysis, respectively. We found that the non-chlorinated PFR metabolites, 2-ethylhexyl phenyl phosphate (EHPHP), bis(2-butoxyethyl) phosphate (BBOEP), and diphenyl phosphate (DPHP) were associated with increased levels of oxidative stress biomarkers. Furthermore, the PFR metabolite mixture was associated with increased levels of HEL and HNE, but not 8-OHdG. The combination of elevated top 2 PFR metabolites was not associated with higher urinary oxidative stress marker levels. This is the first study to report associations between urinary PFR metabolites and oxidative stress biomarkers among children.

Metabolites of phosphate flame retardants and alternative plasticizers in urine from intensive care patients.

Several regulatory offices called for the phase-out of di (2-ethylhexyl) phthalate (DEHP) in medical devices if safer alternatives are available. In medical devices, the occurrence of alternative plasticizers (APs) is widely variable among types of devices. However, plasticizer use is constantly evolving, as there is no reference to guide manufacturers in the choice and amount to be integrated into their products. As intensive care unit (ICU) patients need numerous indwelling plastic devices during their treatment, we hypothesized that these patients are exposed to APs and phosphate flame retardants and plasticizers (PFRs). Urinary metabolites of APs and PFRs were analyzed in the urine of adult ICU patients (n = 24) over a time period of four days. Our results show that adult ICU patients are exposed to PFRs as well as to APs concentrations were much lower compared to the levels of DEHP metabolites in the same samples. However, significantly higher than in controls (n = 15) this exposure resulted in detectable urinary levels in almost every patient and at every studied time point. Increasing temporal trends were observed for several metabolites from admission until day 3 at ICU. The use of specific medical devices, such as continuous venovenous hemofiltration (CVVH) and extracorporeal membrane oxygenation (ECMO), was associated with an increase in urinary concentrations for several PFR metabolites, despite the lack of information on the presence of these plasticizer chemicals in such medical devices. Further research into the possibly toxic effects of these chemicals released from medical devices is urgently needed.