Genetic susceptibility to low-level lead exposure in men: Insights from ALAD polymorphisms.

The genetic susceptibility to low-level lead (Pb) exposure in general populations has been poorly investigated and is limited to the single nucleotide polymorphism (SNP) rs1800435 in the delta-aminolevulinic acid dehydratase gene (ALAD). This study explored associations between ten selected ALAD SNPs with Pb concentrations in blood (BPb) and urine (UPb) among 281 men aged 18-49 years from Slovenia, including 20 individuals residing in a Pb-contaminated area. The geometric mean (range) of BPb and UPb were 19.6 (3.86-84.7) µg/L and 0.69 (0.09-3.82) µg/L SG, respectively. The possible genetic influence was assessed by examining SNP haplotypes, individual SNPs, and the combination of two SNPs using multiple linear regression analyses. While no significant associations were found for haplotypes, the presence of variant alleles of rs1800435 and rs1805312 resulted in an 11% and 13% decrease in BPb, respectively, while the presence of variant allele of rs1139488 (homozygous only) exhibited significant 20% increase in BPb, respectively. Additionally, variant allele of rs1800435 resulted in lower UPb. Individual SNPs in the model explained only around 1 additional percentage point of BPb variability. In contrast, combination analyses identified six combinations of two SNPs, which significantly explained 3-22 additional percentage points of BPb variability, with the highest explanatory power observed for the rs1800435-rs1139488 and rs1139488-rs1805313 combinations. Moreover, excluding participants from the Pb-contaminated area indicated that exposure level influenced SNPs-Pb associations. Our results confirm the importance of the ALAD gene in Pb kinetics even at low exposure levels. Additionally, we demonstrated that identifying individuals with specific combinations of ALAD SNPs explained a larger part of Pb variability, suggesting that these combinations, pending confirmation in other populations and further evaluation through mechanistic studies, may serve as superior susceptibility biomarker in Pb exposure compared to individual SNPs.

Time Patterns in Internal Human Exposure Data to Bisphenols, Phthalates, DINCH, Organophosphate Flame Retardants, Cadmium and Polyaromatic Hydrocarbons in Europe.

Human biomonitoring (HBM) data in Europe are often fragmented and collected in different EU countries and sampling periods. Exposure levels for children and adult women in Europe were evaluated over time. For the period 2000-2010, literature and aggregated data were collected in a harmonized way across studies. Between 2011-2012, biobanked samples from the DEMOCOPHES project were used. For 2014-2021, HBM data were generated within the HBM4EU Aligned Studies. Time patterns on internal exposure were evaluated visually and statistically using the 50th and 90th percentiles (P50/P90) for phthalates/DINCH and organophosphorus flame retardants (OPFRs) in children (5-12 years), and cadmium, bisphenols and polycyclic aromatic hydrocarbons (PAHs) in women (24-52 years). Restricted phthalate metabolites show decreasing patterns for children. Phthalate substitute, DINCH, shows a non-significant increasing pattern. For OPFRs, no trends were statistically significant. For women, BPA shows a clear decreasing pattern, while substitutes BPF and BPS show an increasing pattern coinciding with the BPA restrictions introduced. No clear patterns are observed for PAHs or cadmium. Although the causal relations were not studied as such, exposure levels to chemicals restricted at EU level visually decreased, while the levels for some of their substitutes increased. The results support policy efficacy monitoring and the policy-supportive role played by HBM.

A regional comparison of children's blood cadmium, lead, and mercury in rural, urban and industrial areas of six European countries, and China, Ecuador, and Morocco.

OBJECTIVES: The authors aimed to evaluate whether blood cadmium (B-Cd), lead (B-Pb) and mercury (B-Hg) in children differ regionally in 9 countries, and to identify factors correlating with exposure. MATERIAL AND METHODS: The authors performed a cross-sectional study of children aged 7-14 years, living in 2007-2008 in urban, rural, or potentially polluted ("hot spot") areas (ca. 50 children from each area, in total 1363 children) in 6 European and 3 non-European countries. The authors analyzed Cd, Pb, and total Hg in blood and collected information on potential determinants of exposure through questionnaires. Regional differences in exposure levels were assessed within each country. RESULTS: Children living near industrial "hot-spots" had B-Cd 1.6 (95% CI: 1.4-1.9) times higher in the Czech Republic and 2.1 (95% CI:1.6-2.8) times higher in Poland, as compared to urban children in the same countries (geometric means [GM]: 0.13 µg/l and 0.15 µg/l, respectively). Correspondingly, B-Pb in the "hot spot" areas was 1.8 (95% CI: 1.6-2.1) times higher than in urban areas in Slovakia and 2.3 (95% CI: 1.9-2.7) times higher in Poland (urban GM: 19.4 µg/l and 16.3 µg/l, respectively). In China and Morocco, rural children had significantly lower B-Pb than urban ones (urban GM: 64 µg/l and 71 µg/l, respectively), suggesting urban exposure from leaded petrol, water pipes and/or coal-burning. Hg "hot spot" areas in China had B-Hg 3.1 (95% CI: 2.7-3.5) times higher, and Ecuador 1.5 (95% CI: 1.2-1.9) times higher, as compared to urban areas (urban GM: 2.45 µg/l and 3.23 µg/l, respectively). Besides industrial exposure, traffic correlated with B-Cd; male sex, environmental tobacco smoke, and offal consumption with B-Pb; and fish consumption and amalgam fillings with B-Hg. However, these correlations could only marginally explain regional differences. CONCLUSIONS: These mainly European results indicate that some children experience about doubled exposures to toxic elements just because of where they live. These exposures are unsafe, identifiable, and preventable and therefore call for preventive actions. Int J Occup Med Environ Health. 2023;36(3):349-64.

HBM4EU-MOM: Prenatal methylmercury-exposure control in five countries through suitable dietary advice for pregnancy - Study design and characteristics of participants.

BACKGROUND: Seafood is a major source of vital nutrients for optimal fetal growth, but at the same time is the main source of exposure to methylmercury (MeHg), an established neurodevelopmental toxicant. Pregnant women must be provided with dietary advice so as to include safely fish in their diet for nutrition and mercury control. The aim of this work is to present the design of a multicentre randomized control trial (RCT), which combines human biomonitoring (HBM) with dietary interventions using seafood consumption advice to pregnant women for MeHg control, and to collect information about other possible sources of exposure to mercury. It also presents the materials developed for the implementation of the study and the characteristics of the study participants, which were self-reported in the first trimester of pregnancy. METHODS: The "HBM4EU-MOM" RCT was performed in the frame of the European Human Biomonitoring Initiative (HBM4EU) in five coastal, high fish-consuming European countries (Cyprus, Greece, Spain, Portugal and Iceland). According to the study design, pregnant women (≥120/country, ≤20 weeks gestational age) provided a hair sample for total mercury assessment (THg) and personal information relevant to the study (e.g., lifestyle, pregnancy status, diet before and during the pregnancy, information on seafood and factors related to possible non-dietary exposures to mercury) during the first trimester of pregnancy. After sampling, participants were randomly assigned to "control" (habitual practices) or "intervention" (received the harmonized HBM4EU-MOM dietary advice for fish consumption during the pregnancy and were encouraged to follow it). Around child delivery, participants provided a second hair sample and completed another tailored questionnaire. RESULTS: A total of 654 women aged 18-45 years were recruited in 2021 in the five countries, primarily through their health-care providers. The pre-pregnancy BMI of the participants ranged from underweight to obese, but was on average within the healthy range. For 73% of the women, the pregnancy was planned. 26% of the women were active smokers before the pregnancy and 8% continued to smoke during the pregnancy, while 33% were passive smokers before pregnancy and 23% remained passively exposed during the pregnancy. 53% of the women self-reported making dietary changes for their pregnancy, with 74% of these women reporting making the changes upon learning of their pregnancy. Of the 43% who did not change their diet for the pregnancy, 74% reported that their diet was already balanced, 6% found it difficult to make changes and 2% were unsure of what changes to make. Seafood consumption did not change significantly before and during the first trimester of pregnancy (overall average ∼8 times per month), with the highest frequency reported in Portugal (≥15 times per month), followed by Spain (≥7 times per month). During the first-trimester of pregnancy, 89% of the Portuguese women, 85% of the Spanish women and <50% of Greek, Cypriot and Icelandic women reported that they had consumed big oily fish. Relevant to non-dietary exposure sources, most participants (>90%) were unaware of safe procedures for handling spillage from broken thermometers and energy-saving lamps, though >22% experienced such an incident (>1 year ago). 26% of the women had dental amalgams. ∼1% had amalgams placed and ∼2% had amalgams removed during peri-pregnancy. 28% had their hair dyed in the past 3 months and 40% had body tattoos. 8% engaged with gardening involving fertilizers/pesticides and 19% with hobbies involving paints/pigments/dyes. CONCLUSIONS: The study design materials were fit for the purposes of harmonization and quality-assurance. The harmonized information collected from pregnant women suggests that it is important to raise the awareness of women of reproductive age and pregnant women about how to safely include fish in their diet and to empower them to make proper decisions for nutrition and control of MeHg, as well as other chemical exposures.

How to use human biomonitoring in chemical risk assessment: Methodological aspects, recommendations, and lessons learned from HBM4EU.

One of the aims of the European Human Biomonitoring Initiative, HBM4EU, was to provide examples of and good practices for the effective use of human biomonitoring (HBM) data in human health risk assessment (RA). The need for such information is pressing, as previous research has indicated that regulatory risk assessors generally lack knowledge and experience of the use of HBM data in RA. By recognising this gap in expertise, as well as the added value of incorporating HBM data into RA, this paper aims to support the integration of HBM into regulatory RA. Based on the work of the HBM4EU, we provide examples of different approaches to including HBM in RA and in estimations of the environmental burden of disease (EBoD), the benefits and pitfalls involved, information on the important methodological aspects to consider, and recommendations on how to overcome obstacles. The examples are derived from RAs or EBoD estimations made under the HBM4EU for the following HBM4EU priority substances: acrylamide, o-toluidine of the aniline family, aprotic solvents, arsenic, bisphenols, cadmium, diisocyanates, flame retardants, hexavalent chromium [Cr(VI)], lead, mercury, mixture of per-/poly-fluorinated compounds, mixture of pesticides, mixture of phthalates, mycotoxins, polycyclic aromatic hydrocarbons (PAHs), and the UV-filter benzophenone-3. Although the RA and EBoD work presented here is not intended to have direct regulatory implications, the results can be useful for raising awareness of possibly needed policy actions, as newly generated HBM data from HBM4EU on the current exposure of the EU population has been used in many RAs and EBoD estimations.

Exposure to Phthalates in European Children, Adolescents and Adults since 2005: A Harmonized Approach Based on Existing HBM Data in the HBM4EU Initiative.

Phthalates are mainly used as plasticizers and are associated inter alia with adverse effects on reproductive functions. While more and more national programs in Europe have started monitoring internal exposure to phthalates and its substitute 1,2-Cyclohexanedicarboxylic acid (DINCH), the comparability of results from such existing human biomonitoring (HBM) studies across Europe is challenging. They differ widely in time periods, study samples, degree of geographical coverage, design, analytical methodology, biomarker selection, and analytical quality assurance level. The HBM4EU initiative has gathered existing HBM data of 29 studies from participating countries, covering all European regions and Israel. The data were prepared and aggregated by a harmonized procedure with the aim to describe-as comparably as possible-the EU-wide general population's internal exposure to phthalates from the years 2005 to 2019. Most data were available from Northern (up to 6 studies and up to 13 time points), Western (11; 19), and Eastern Europe (9; 12), e.g., allowing for the investigation of time patterns. While the bandwidth of exposure was generally similar, we still observed regional differences for Butyl benzyl phthalate (BBzP), Di(2-ethylhexyl) phthalate (DEHP), Di-isononyl phthalate (DiNP), and Di-isobutyl phthalate (DiBP) with pronounced decreases over time in Northern and Western Europe, and to a lesser degree in Eastern Europe. Differences between age groups were visible for Di-n-butyl phthalate (DnBP), where children (3 to 5-year olds and 6 to 11-year olds) had lower urinary concentrations than adolescents (12 to 19-year-olds), who in turn had lower urinary concentrations than adults (20 to 39-year-olds). This study is a step towards making internal exposures to phthalates comparable across countries, although standardized data were not available, targeting European data sets harmonized with respect to data formatting and calculation of aggregated data (such as developed within HBM4EU), and highlights further suggestions for improved harmonization in future studies.

Exposure of children and adolescents from Northeastern Slovenia to per- and polyfluoroalkyl substances.

Per- and polyfluoroalkyl substances (PFAS) are of high concern for the environment, wildlife, and human health due to their persistence and potential to cause adverse health effects. Despite political measures to restrict the production and distribution of PFAS and to limit the exposure of populations, PFAS can be measured at commonly high detection frequencies in human samples. Thus, this pilot study aimed to determine the serum concentrations of PFPA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFHxS, PFHpS, PFOS, PFHxA, PFDoDA, and PFBS in 113 girls and 112 boys (age 7-10 and 12-15) from Northeastern Slovenia - a rural area characterized by agricultural activities - and to identify potential sources of exposure using questionnaire data. PFAS were analysed by liquid chromatography coupled to mass spectrometry after phospholipid removal. 9 out of 12 analytes were detected at detection frequencies above 30%, with the highest geometric means (GM) being observed for PFOS (GM 1.9 ng/mL) > PFOA (GM 1.0 ng/mL) > PFHxS (GM 0.3 ng/mL) = PFNA (GM 0.3 ng/mL). We identified the participants' socio-economic status, age, sex, sampling region, public water supply, and the consumption of fish and seafood, cereals, and locally produced fruits, vegetables, and mushrooms as the predominant determinants of exposure. Furthermore, we compared our results with the serum and plasma concentrations reported for similar age groups in other studies and concluded that PFAS exposure in this highly agricultural area in Slovenia is notably low. This is the first study systematic HBM study of PFAS exposure in Slovenia, although it was conducted on a limited number of participants representative of rural and agricultural areas, it represents a good basis for upgrading the approach to a nationwide HBM study.

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.

ALAD and APOE polymorphisms are associated with lead and mercury levels in Italian pregnant women and their newborns with adequate nutritional status of zinc and selenium.

The impacts of single-nucleotide polymorphisms (SNPs) in ALAD and VDR genes on Pb health effects and/or kinetics are inconclusive at low exposure levels, while studies including APOE SNPs are rare. In this study, we examined the associations of ALAD, VDR and APOE SNPs with exposure biomarkers of Pb and other trace elements (TEs) in Italian pregnant women (N = 873, aged 18-44 years) and their newborns (N = 619) with low-level mixed-element exposure through diet, the environment or endogenously. DNA from maternal peripheral venous blood (mB), sampled during the second and third trimesters, was genotyped for ALAD (rs1800435, rs1805313, rs1139488, rs818708), VDR (rs2228570, rs1544410, rs7975232, rs731236) and APOE (rs429358, rs7421) using TaqMan SNP assays. Personal and lifestyle data and TE levels (mB, maternal plasma, hair and mixed umbilical cord blood [CB]) from the PHIME project were used. Multiple linear regression models, controlling for confounding variables, were performed to test the associations between SNPs and TEs. The geometric means of mB-Pb, mB-Hg, mB-As and mB-Cd (11.0 ng/g, 2.16 ng/g, 1.38 ng/g and 0.31 ng/g, respectively) indicated low exposure levels, whereas maternal plasma Zn and Se (0.72 µg/mL and 78.6 ng/g, respectively) indicated adequate micronutritional status. Variant alleles of ALAD rs1800435 and rs1805313 were negatively associated with mB-Pb levels, whereas a positive association was observed for rs1139488. None of the VDR SNPs or their haplotypes had any association with Pb levels. Regarding APOE, the ϵ4 allele was associated with lower mB-Hg and CB-Hg, while a positive association was found with the ϵ2 allele and CB-Pb when the model included only newborn girls. The observed associations indicate possible modification effects of ALAD and APOE SNPs on Pb or Hg kinetics in women and their newborns with low exposure to non-essential TEs, as well as an adequate nutritional status of Zn and Se.

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.

Exposure to flame retardants in European children - Results from the HBM4EU aligned studies.

Many legacy and emerging flame retardants (FRs) have adverse human and environmental health effects. This study reports legacy and emerging FRs in children from nine European countries from the HBM4EU aligned studies. Studies from Belgium, Czech Republic, Germany, Denmark, France, Greece, Slovenia, Slovakia, and Norway conducted between 2014 and 2021 provided data on FRs in blood and urine from 2136 children. All samples were collected and analyzed in alignment with the HBM4EU protocols. Ten halogenated FRs were quantified in blood, and four organophosphate flame retardants (OPFR) metabolites quantified in urine. Hexabromocyclododecane (HBCDD) and decabromodiphenyl ethane (DBDPE) were infrequently detected (<16% of samples). BDE-47 was quantified in blood from Greece, France, and Norway, with France (0.36 ng/g lipid) having the highest concentrations. BDE-153 and -209 were detected in <40% of samples. Dechlorane Plus (DP) was quantified in blood from four countries, with notably high median concentrations of 16 ng/g lipid in Slovenian children. OPFR metabolites had a higher detection frequency than other halogenated FRs. Diphenyl phosphate (DPHP) was quantified in 99% of samples across 8 countries at levels ∼5 times higher than other OPFR metabolites (highest median in Slovenia of 2.43 ng/g lipid). FR concentrations were associated with lifestyle factors such as cleaning frequency, employment status of the father of the household, and renovation status of the house, among others. The concentrations of BDE-47 in children from this study were similar to or lower than FRs found in adult matrices in previous studies, suggesting lower recent exposure and effectiveness of PBDE restrictions.

Association of Zn and Cu Levels in Cord Blood and Maternal Milk with Pregnancy Outcomes among the Slovenian Population.

Trace elements, including zinc (Zn) and copper (Cu), are known to play important roles in human health. The present study aimed to assess the levels of Zn and Cu in cord blood and maternal milk and to analyze their association with maternal and infant characteristics and pregnancy outcomes in a Slovenian study population of mothers and their neonates recruited within the PHIME prospective cohort study. The study included 324 mothers, but the data on Zn and Cu levels in both cord blood and maternal milk was available for 243 mothers. Questionnaires were used to assess the socio-demographic and health status of the mothers, their lifestyle habits (including detailed nutritional habits), and their residential and occupational histories. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) was used to measure Zn and Cu levels in cord blood and maternal milk. Low Zn levels in cord blood were associated with lower gestational age and birth weight and were correlated with an increased probability of the birth of small for gestational age (SGA) infants. Maternal smoking influenced the Cu levels in both cord blood and maternal milk. Cord blood Cu levels were higher and Cu levels in maternal milk were lower in smoking compared to non-smoking mothers. Most importantly, a decreased Zn/Cu ratio in cord blood was associated with lower gestational age and lower birth weight. This indicates the overall positive effects of Zn and negative effects of Cu on pregnancy outcomes.

Cadmium exposure in adults across Europe: Results from the HBM4EU Aligned Studies survey 2014-2020.

The objectives of the study were to estimate the current exposure to cadmium (Cd) in Europe, potential differences between the countries and geographic regions, determinants of exposure and to derive European exposure levels. The basis for this work was provided by the European Human Biomonitoring Initiative (HBM4EU) which established a framework for alignment of national or regional HBM studies. For the purpose of Cd exposure assessment, studies from 9 European countries (Iceland, Denmark, Poland, Czech Republic, Croatia, Portugal, Germany, France, Luxembourg) were included and urine of 20-39 years old adults sampled in the years 2014-2021 (n = 2510). The measurements in urine were quality assured by the HBM4EU quality assurance/quality control scheme, study participants' questionnaire data were post-harmonized. Spatially resolved external data, namely Cd concentrations in soil, agricultural areas, phosphate fertilizer application, traffic density and point source Cd release were collected for the respective statistical territorial unit (NUTS). There were no distinct geographic patterns observed in Cd levels in urine, although the data revealed some differences between the specific study sites. The levels of exposure were otherwise similar between two time periods within the last decade (DEMOCOPHES - 2011-2012 vs. HBM4EU Aligned Studies, 2014-2020). The age-dependent alert values for Cd in urine were exceeded by 16% of the study participants. Exceedances in the different studies and locations ranged from 1.4% up to 42%. The studies with largest extent of exceedance were from France and Poland. Association analysis with individual food consumption data available from participants' questionnaires showed an important contribution of vegetarian diet to the overall exposure, with 35% higher levels in vegetarians as opposed to non-vegetarians. For comparison, increase in Cd levels due to smoking was 25%. Using NUTS2-level external data, positive associations between HBM data and percentage of cropland and consumption of Cd-containing mineral phosphate fertilizer were revealed, which indicates a significant contribution of mineral phosphate fertilizers to human Cd exposure through diet. In addition to diet, traffic and point source release were identified as significant sources of exposure in the study population. The findings of the study support the recommendation by EFSA to reduce Cd exposure as also the estimated mean dietary exposure of adults in the EU is close or slightly exceeding the tolerable weekly intake. It also indicates that regulations are not protecting the population sufficiently.

Suspect and non-targeted screening-based human biomonitoring identified 74 biomarkers of exposure in urine of Slovenian children.

Human exposure to organic contaminants is widespread. Many of these contaminants show adverse health effects on human population. Human biomonitoring (HBM) follows the levels and the distribution of biomarkers of exposure (BoE), but it is usually done in a targeted manner. Suspect and non-targeted screening (SS/NTS) tend to find BoE in an agnostic way, without preselection of compounds, and include finding evidence of exposure to predicted, unpredicted known and unknown chemicals. This study describes the application of high-resolution mass spectrometry (HRMS)-based SS/NTS workflow for revealing organic contaminants in urine of a cohort of 200 children from Slovenia, aged 6-9 years. The children originated from two regions, urban and rural, and the latter were sampled in two time periods, summer and winter. We tentatively identified 74 BoE at the confidence levels of 2 and 3. These BoE belong to several classes of pharmaceuticals, personal care products, plasticizers and plastic related products, volatile organic compounds, nicotine, caffeine and pesticides. The risk of three pesticides, atrazine, amitraz and diazinon is of particular concern since their use was limited in the EU. Among BoE we tentatively identified compounds that have not yet been monitored in HBM schemes and demonstrate limited exposure data, such as bisphenol G, polyethylene glycols and their ethers. Furthermore, 7 compounds with unknown use and sources of exposure were tentatively identified, either indicating the entry of new chemicals into the market, or their metabolites and transformation products. Interestingly, several BoE showed location and time dependency. Globally, this study presents high-throughput approach to SS/NTS for HBM. The results shed a light on the exposure of Slovenian children and raise questions on potential adverse health effects of such mixtures on this vulnerable population.

A Tiered Approach for Assessing Individual and Combined Risk of Pyrethroids Using Human Biomonitoring Data.

Pyrethroids are a major insecticide class, suitable for biomonitoring in humans. Due to similarities in structure and metabolic pathways, urinary metabolites are common to various active substances. A tiered approach is proposed for risk assessment. Tier I was a conservative screening for overall pyrethroid exposure, based on phenoxybenzoic acid metabolites. Subsequently, probabilistic approaches and more specific metabolites were used for refining the risk estimates. Exposure was based on 95th percentiles from HBM4EU aligned studies (2014-2021) covering children in Belgium, Cyprus, France, Israel, Slovenia, and The Netherlands and adults in France, Germany, Israel, and Switzerland. In all children populations, the 95th percentiles for 3-phenoxybenzoic acid (3-PBA) exceeded the screening value. The probabilistic refinement quantified the risk level of the most exposed population (Belgium) at 2% or between 1-0.1% depending on the assumptions. In the substance specific assessments, the 95th percentiles of urinary concentrations in the aligned studies were well below the respective human biomonitoring guidance values (HBM-GVs). Both information sets were combined for refining the combined risk. Overall, the HBM data suggest a low health concern, at population level, related to pyrethroid exposure for the populations covered by the studies, even though a potential risk for highly exposed children cannot be completely excluded. The proposed tiered approach, including a screening step and several refinement options, seems to be a promising tool of scientific and regulatory value in future.

Glyphosate and AMPA in Human Urine of HBM4EU Aligned Studies: Part A Children.

Few data are available on the exposure of children to glyphosate (Gly) in Europe. Within HBM4EU, new HBM exposure data were collected from aligned studies at five sampling sites distributed over Europe (studies: SLO CRP (SI); ORGANIKO (CY); GerES V-sub (DE); 3XG (BE); ESTEBAN (FR)). Median Gly concentrations in urine were below or around the detection limit (0.1 µg/L). The 95th percentiles ranged between 0.18 and 1.03 µg Gly/L. The ratio of AMPA (aminomethylphosphonic acid; main metabolite of Gly) to Gly at molar basis was on average 2.2 and the ratio decreased with higher Gly concentrations, suggesting that other sources of AMPA, independent of metabolism of Gly to AMPA in the monitored participants, may concurrently operate. Using reverse dosimetry and HBM exposure data from five European countries (east, west and south Europe) combined with the proposed ADI (acceptable daily intake) of EFSA for Gly of 0.1 mg/kg bw/day (based on histopathological findings in the salivary gland of rats) indicated no human health risks for Gly in the studied populations at the moment. However, the absence of a group ADI for Gly+AMPA and ongoing discussions on e.g., endocrine disrupting effects cast some uncertainty in relation to the current single substance ADI for Gly. The carcinogenic effects of Gly are still debated in the scientific community. These outcomes would influence the risk conclusions presented here. Finally, regression analyses did not find clear associations between urinary exposure biomarkers and analyzed potential exposure determinants. More information from questionnaires targeting exposure-related behavior just before the sampling is needed.

Harmonization of Human Biomonitoring Studies in Europe: Characteristics of the HBM4EU-Aligned Studies Participants.

Human biomonitoring has become a pivotal tool for supporting chemicals' policies. It provides information on real-life human exposures and is increasingly used to prioritize chemicals of health concern and to evaluate the success of chemical policies. Europe has launched the ambitious REACH program in 2007 to improve the protection of human health and the environment. In October 2020 the EU commission published its new chemicals strategy for sustainability towards a toxic-free environment. The European Parliament called upon the commission to collect human biomonitoring data to support chemical's risk assessment and risk management. This manuscript describes the organization of the first HBM4EU-aligned studies that obtain comparable human biomonitoring (HBM) data of European citizens to monitor their internal exposure to environmental chemicals. The HBM4EU-aligned studies build on existing HBM capacity in Europe by aligning national or regional HBM studies. The HBM4EU-aligned studies focus on three age groups: children, teenagers, and adults. The participants are recruited between 2014 and 2021 in 11 to 12 primary sampling units that are geographically distributed across Europe. Urine samples are collected in all age groups, and blood samples are collected in children and teenagers. Auxiliary information on socio-demographics, lifestyle, health status, environment, and diet is collected using questionnaires. In total, biological samples from 3137 children aged 6-12 years are collected for the analysis of biomarkers for phthalates, HEXAMOLL® DINCH, and flame retardants. Samples from 2950 teenagers aged 12-18 years are collected for the analysis of biomarkers for phthalates, Hexamoll® DINCH, and per- and polyfluoroalkyl substances (PFASs), and samples from 3522 adults aged 20-39 years are collected for the analysis of cadmium, bisphenols, and metabolites of polyaromatic hydrocarbons (PAHs). The children's group consists of 50.4% boys and 49.5% girls, of which 44.1% live in cities, 29.0% live in towns/suburbs, and 26.8% live in rural areas. The teenagers' group includes 50.6% girls and 49.4% boys, with 37.7% of residents in cities, 31.2% in towns/suburbs, and 30.2% in rural areas. The adult group consists of 52.6% women and 47.4% men, 71.9% live in cities, 14.2% in towns/suburbs, and only 13.4% live in rural areas. The study population approaches the characteristics of the general European population based on age-matched EUROSTAT EU-28, 2017 data; however, individuals who obtained no to lower educational level (ISCED 0-2) are underrepresented. The data on internal human exposure to priority chemicals from this unique cohort will provide a baseline for Europe's strategy towards a non-toxic environment and challenges and recommendations to improve the sampling frame for future EU-wide HBM surveys are discussed.

Risk Assessment of Dietary Exposure to Organophosphorus Flame Retardants in Children by Using HBM-Data.

Due to their extensive usage, organophosphorus flame retardants (OPFRs) have been detected in humans and in the environment. Human are exposed to OPFRs via inhalation of indoor air, dust uptake or dietary uptake through contaminated food and drinking water. Only recently, few studies addressing dietary exposure to OPFRs were published. In this study, we used human biomonitoring (HBM) data of OPFRs to estimate how much the dietary intake may contribute to the total exposure. We estimated by reverse dosimetry, the daily intake of tris (2-chloroethyl) phosphate (TCEP), tris (1-chloro-2-propyl) phosphate (TCIPP), tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) for children using HBM data from studies with sampling sites in Belgium, Denmark, France, Germany, Slovenia and Slovakia. For estimating the dietary exposure, a deterministic approach was chosen. The occurrence data of selected food categories were used from a published Belgium food basket study. Since the occurrence data were left-censored, the Lower bound (LB)-Upper bound (UB) approach was used. The estimated daily intake (EDI) calculated on the basis of urine metabolite concentrations ranged from 0.03 to 0.18 µg/kg bw/d for TDCIPP, from 0.05 to 0.17 µg/kg bw/d for TCIPP and from 0.02 to 0.2 µg/kg bw/d for TCEP. Based on national food consumption data and occurrence data, the estimated dietary intake for TDCIPP ranged from 0.005 to 0.09 µg/kg bw/d, for TCIPP ranged from 0.037 to 0.2 µg/kg bw/d and for TCEP ranged from 0.007 to 0.018 µg/kg bw/d (summarized for all countries). The estimated dietary intake of TDCIPP contributes 11-173% to the EDI, depending on country and LB-UB scenario. The estimated dietary uptake of TCIPP was in all calculations, except in Belgium and France, above 100%. In the case of TCEP, it is assumed that the dietary intake ranges from 6 to 57%. The EDI and the estimated dietary intake contribute less than 3% to the reference dose (RfD). Therefore, the estimated exposure to OPFRs indicates a minimal health risk based on the current knowledge of available exposure, kinetic and toxicity data. We were able to show that the dietary exposure can have an impact on the general exposure based on our underlying exposure scenarios.