Global Urinary Volatolomics with (GC×)GC-TOF-MS.

Urinary volatolomics offers a noninvasive approach for disease detection and monitoring. Herein we present an improved methodology for global volatolomic profiling. Wide coverage was achieved by utilizing a multiphase sorbent for volatile organic compound (VOC) extraction. A single, midpolar column gas chromatography (GC) assay yielded substantially higher numbers of monitored VOCs compared to our previously reported single-sorbent method. Multidimensional GC (GC×GC) enhanced further biomarker discovery while data analysis was simplified by using a tile-based approach. At the same time, the required urine volume was reduced 5-fold from 2 to 0.4 mL. The applicability of the methodology was demonstrated in a pancreatic ductal adenocarcinoma cohort where previous findings were confirmed while a series of additional VOCs with diagnostic potential were discovered.

Cord blood metabolites and rapid postnatal growth as multiple mediators in the prenatal propensity to childhood overweight.

BACKGROUND: The mechanisms underlying childhood overweight and obesity are poorly known. Here, we investigated the direct and indirect effects of different prenatal exposures on offspring rapid postnatal growth and overweight in childhood, mediated through cord blood metabolites. Additionally, rapid postnatal growth was considered a potential mediator on childhood overweight, alone and sequentially to each metabolite. METHODS: Within four European birth-cohorts (N = 375 mother-child dyads), information on seven prenatal exposures (maternal education, pre-pregnancy BMI, weight gain and tobacco smoke during pregnancy, age at delivery, parity, and child gestational age), selected as obesogenic according to a-priori knowledge, was collected. Cord blood levels of 31 metabolites, associated with rapid postnatal growth and/or childhood overweight in a previous study, were measured via liquid-chromatography-quadrupole-time-of-flight-mass-spectrometry. Rapid growth at 12 months and childhood overweight (including obesity) between four and eight years were defined with reference to WHO growth charts. Single mediation analysis was performed using the imputation approach and multiple mediation analysis using the extended-imputation approach. RESULTS: Single mediation suggested that the effect of maternal education, pregnancy weight gain, parity, and gestational age on rapid postnatal growth but not on childhood overweight was partly mediated by seven metabolites, including cholestenone, decenoylcarnitine(C10:1), phosphatidylcholine(C34:3), progesterone and three unidentified metabolites; and the effect of gestational age on childhood overweight was mainly mediated by rapid postnatal growth. Multiple mediation suggested that the effect of gestational age on childhood overweight was mainly mediated by rapid postnatal growth and that the mediating role of the metabolites was marginal. CONCLUSION: Our findings provide evidence of the involvement of in utero metabolism in the propensity to rapid postnatal growth and of rapid postnatal growth in the propensity to childhood overweight. We did not find evidence supporting a mediating role of the studied metabolites alone between the studied prenatal exposures and the propensity to childhood overweight.

Cord blood metabolic signatures predictive of childhood overweight and rapid growth.

INTRODUCTION: Metabolomics may identify biological pathways predisposing children to the risk of overweight and obesity. In this study, we have investigated the cord blood metabolic signatures of rapid growth in infancy and overweight in early childhood in four European birth cohorts. METHODS: Untargeted liquid chromatography-mass spectrometry metabolomic profiles were measured in cord blood from 399 newborns from four European cohorts (ENVIRONAGE, Rhea, INMA and Piccolipiu). Rapid growth in the first year of life and overweight in childhood was defined with reference to WHO growth charts. Metabolome-wide association scans for rapid growth and overweight on over 4500 metabolic features were performed using multiple adjusted logistic mixed-effect models and controlling the false discovery rate (FDR) at 5%. In addition, we performed a look-up analysis of 43 pre-annotated metabolites, previously associated with birthweight or rapid growth. RESULTS: In the Metabolome-Wide Association Study analysis, we identified three and eight metabolites associated with rapid growth and overweight, respectively, after FDR correction. Higher levels of cholestenone, a cholesterol derivative produced by microbial catabolism, were predictive of rapid growth (p = 1.6 × 10-3). Lower levels of the branched-chain amino acid (BCAA) valine (p = 8.6 × 10-6) were predictive of overweight in childhood. The area under the receiver operator curve for multivariate prediction models including these metabolites and traditional risk factors was 0.77 for rapid growth and 0.82 for overweight, compared with 0.69 and 0.69, respectively, for models using traditional risk factors alone. Among the 43 pre-annotated metabolites, seven and five metabolites were nominally associated (P < 0.05) with rapid growth and overweight, respectively. The BCAA leucine, remained associated (1.6 × 10-3) with overweight after FDR correction. CONCLUSION: The metabolites identified here may assist in the identification of children at risk of developing obesity and improve understanding of mechanisms involved in postnatal growth. Cholestenone and BCAAs are suggestive of a role of the gut microbiome and nutrient signalling respectively in child growth trajectories.

Neurodevelopmental exposome: The effect of in utero co-exposure to heavy metals and phthalates on child neurodevelopment.

In this study, the exposome paradigm has been applied on a mother-child cohort adopting an optimised untargeted metabolomics approach for human urine followed by advanced bioinformatics analysis. Exposome-wide association algorithms were used to draw links between in utero co-exposure to metals and phthalates, metabolic pathways deregulation, and clinically observed phenotypes of neurodevelopmental disorders such as problems in linguistic, motor development and cognitive capacity. Children (n = 148) were tested at the first and second year of their life using the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley-III). Their mothers had been exposed to metals and phthalates during the pregnancy, according to human biomonitoring results from previously performed studies. Untargeted metabolomics analysis of biobanked urine samples from the mothers was performed using a combination of the high throughput analytical methods liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR). Most perturbed metabolic pathways from co-exposure heavy metals and phthalates were pathways related to the tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation, indicating the possibility of disruption of mitochondrial respiration. Overproduction of reactive oxygen species (ROS); the presence of glutathione peroxidase 3 (GPx3) during pregnancy and presence of glutathione peroxidase 1 (GPx1) in the umbilical cord were linked to verbal development problems. Another finding of the study is that in real life, adverse outcomes occur as a combination of environmental and social factors, all of them acting synergistically towards the deployment of an observed phenotype. Finally, the two-steps association process (exposure to pathways and pathways to adverse outcomes) was able to (a) provide associations that are not evident by directly associating exposure to outcomes and (b) provides additional insides on the mechanisms of environmental disease.

Development of a generic lifelong physiologically based biokinetic model for exposome studies.

The current study within the frame of the HEALS project aims at the development of a lifelong physiologically based biokinetic (PBBK) model for exposome studies. The aim was to deliver a comprehensive modelling framework for addressing a large chemical space. Towards this aim, the delivered model can easily adapt parameters from existing ad-hoc models or complete the missing compound specific parameters using advanced quantitative structure activity relationship (QSAR). All major human organs are included, as well as arterial, venous, and portal blood compartments. Xenobiotics and their metabolites are linked through the metabolizing tissues. This is mainly the liver, but also other sites of metabolism might be considered (intestine, brain, skin, placenta) based on the presence or not of the enzymes involved in the metabolism of the compound of interest. Each tissue is described by three mass balance equations for (a) red blood cells, (b) plasma and interstitial tissue and (c) cells respectively. The anthropometric parameters of the models are time dependent, so as to provide a lifetime internal dose assessment, as well as to describe the continuously changing physiology of the mother and the developing fetus. An additional component of flexibility is that the biokinetic processes that relate to metabolism are related with either Michaelis-Menten kinetics, as well as intrinsic clearance kinetics. The capability of the model is demonstrated in the assessment of internal exposure and the prediction of expected biomonitored levels in urine for three major compounds within the HEALS project, namely bisphenol A (BPA), Bis(2-ethylhexyl) phthalate (DEHP) and cadmium (Cd). The results indicated that the predicted urinary levels fit very well with the ones from human biomonitoring (HBM) studies; internal exposure to plasticizers is very low (in the range of ng/L), while internal exposure to Cd is in the range of µg/L.

Human biomonitoring data analysis for metals in an Italian adolescents cohort: An exposome approach.

The first Italian human biomonitoring survey (PROBE - PROgramme for Biomonitoring general population Exposure) considered a reference population of adolescents, aged 13-15 years, living in urban and rural areas and investigated their exposure to metals. The study was expanded up to 453 adolescents living in the same areas of Latium Region (Italy) and blood samples were analyzed for 19 metals (As, Be, Cd, Co, Cr, Hg, Ir, Mn, Mo, Ni, Pb, Pd, Pt, Rh, Sb, Sn, Tl, V, and W) by sector field inductively coupled plasma mass spectrometry. The exposure assessment was contextualized following an exposome approach that considered several determinants related to the subjects, available environmental parameters and geo-coding of residence address. To assess the influence of exposure determinants and modifiers on children biomarkers levels we used two independent methodologies. The first makes use of the so-called Environment-Wide Association Study (EWAS) methodology while the second was based on the application of a Generalized Liner Model (GLM) capturing co-exposures to pairs of key determinants. Based on our analysis, Hg and As were positively associated with dietary pathways (primarily linked to fish and to a lesser extent to milk consumption) while Cr showed a more complex interaction between co-exposure to different dietary pathways (milk and fish) coupled to proximity of residence to industrial activities. In addition to diet, socio-economic status of the mother revealed robust statistical associations with Cd, Ni and W biomonitoring levels in the respective children.

The contribution to policies of an exposome-based approach to childhood obesity.

Childhood obesity is an increasingly severe public health problem, with a prospective impact on health. We propose an exposome approach to identify actionable risk factors for this condition. Our assumption is that relationships between external exposures and outcomes such as rapid growth, overweight, or obesity in children can be better understood through a "meet-in-the-middle" model. This is based on a combination of external and internal exposome-based approaches, that is, the study of multiple exposures (in our case, dietary patterns) and molecular pathways (metabolomics and epigenetics). This may strengthen causal reasoning by identifying intermediate markers that are associated with both exposures and outcomes. Our biomarker-based studies in the STOP consortium suggest (in several ways, including mediation analysis) that branched-chain amino acids (BCAAs) could be mediators of the effect of dietary risk factors on childhood overweight/obesity. This is consistent with intervention and animal studies showing that higher intake of BCAAs has a positive impact on body composition, glycemia, and satiety. Concerning food, of particular concern is the trend of increasing intake of ultra-processed food (UPF), including among children. Several mechanisms have been proposed to explain the impact of UPF on obesity and overweight, including nutrient intake (particularly proteins), changes in appetite, or the role of additives. Research from the Avon Longitudinal Study of Parents and Children cohort has shown a relationship between UPF intake and trajectories in childhood adiposity, while UPF was related to lower blood levels of BCAAs. We suggest that an exposome-based approach can help strengthening causal reasoning and support policies. Intake of UPF in children should be restricted to prevent obesity.

Perspectives and challenges of epigenetic determinants of childhood obesity: A systematic review.

The tremendous increase in childhood obesity prevalence over the last few decades cannot merely be explained by genetics and evolutionary changes in the genome, implying that gene-environment interactions, such as epigenetic modifications, likely play a major role. This systematic review aims to summarize the evidence of the association between epigenetics and childhood obesity. A literature search was performed via PubMed and Scopus engines using a combination of terms related to epigenetics and pediatric obesity. Articles studying the association between epigenetic mechanisms (including DNA methylation and hydroxymethylation, non-coding RNAs, and chromatin and histones modification) and obesity and/or overweight (or any related anthropometric parameters) in children (0-18 years) were included. The risk of bias was assessed with a modified Newcastle-Ottawa scale for non-randomized studies. One hundred twenty-one studies explored epigenetic changes related to childhood obesity. DNA methylation was the most widely investigated mechanism (N = 101 studies), followed by non-coding RNAs (N = 19 studies) with evidence suggestive of an association with childhood obesity for DNA methylation of specific genes and microRNAs (miRNAs). One study, focusing on histones modification, was identified. Heterogeneity of findings may have hindered more insights into the epigenetic changes related to childhood obesity. Gaps and challenges that future research should face are herein described.

The built environment as determinant of childhood obesity: A systematic literature review.

We evaluated the epidemiological evidence on the built environment and its link to childhood obesity, focusing on environmental factors such as traffic noise and air pollution, as well as physical factors potentially driving obesity-related behaviors, such as neighborhood walkability and availability and accessibility of parks and playgrounds. Eligible studies were (i) conducted on human children below the age of 18 years, (ii) focused on body size measurements in childhood, (iii) examined at least one built environment characteristic, (iv) reported effect sizes and associated confidence intervals, and (v) were published in English language. A z test, as alternative to the meta-analysis, was used to quantify associations due to heterogeneity in exposure and outcome definition. We found strong evidence for an association of traffic-related air pollution (nitrogen dioxide and nitrogen oxides exposure, p < 0.001) and built environment characteristics supportive of walking (street intersection density, p < 0.01 and access to parks, p < 0.001) with childhood obesity. We identified a lack of studies that account for interactions between different built environment exposures or verify the role and mechanism of important effect modifiers such as age.

A systematic review of metabolomic studies of childhood obesity: State of the evidence for metabolic determinants and consequences.

Childhood obesity has become a global epidemic and carries significant long-term consequences to physical and mental health. Metabolomics, the global profiling of small molecules or metabolites, may reveal the mechanisms of development of childhood obesity and clarify links between obesity and metabolic disease. A systematic review of metabolomic studies of childhood obesity was conducted, following Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines, searching across Scopus, Ovid, Web of Science and PubMed databases for articles published from January 1, 2005 to July 8, 2020, retrieving 1271 different records and retaining 41 articles for qualitative synthesis. Study quality was assessed using a modified Newcastle-Ottawa Scale. Thirty-three studies were conducted on blood, six on urine, three on umbilical cord blood, and one on saliva. Thirty studies were primarily cross-sectional, five studies were primarily longitudinal, and seven studies examined effects of weight-loss following a life-style intervention. A consistent metabolic profile of childhood obesity was observed including amino acids (particularly branched chain and aromatic), carnitines, lipids, and steroids. Although the use of metabolomics in childhood obesity research is still developing, the identified metabolites have provided additional insight into the pathogenesis of many obesity-related diseases. Further longitudinal research is needed into the role of metabolic profiles and child obesity risk.

Molecular mediators of the association between child obesity and mental health.

Biological mechanisms underlying the association between obesity and depression remain unclear. We investigated the role of metabolites and DNA methylation as mediators of the relationship between childhood obesity and subsequent poor mental health in the English Avon Longitudinal Study of Parents and Children. Obesity was defined according to United Kingdom Growth charts at age 7 years and mental health through the Short Mood and Feelings Questionnaire (SMFQ) completed at age 11 years. Metabolites and DNA methylation were measured by nuclear magnetic resonance spectroscopy and Illumina array in blood at the age of 7 years. The associations between obesity and SMFQ score, as continuous count data or using cut-offs to define depressive symptoms (SMFQ >7) or depression (SMFQ >11), were tested using adjusted Poisson and logistic regression. Candidate metabolite mediators were identified through metabolome-wide association scans for obesity and SMFQ score, correcting for false-discovery rate. Candidate DNA methylation mediators were identified through testing the association of putative BMI-associated CpG sites with SMFQ scores, correcting for look-up false-discovery rate. Mediation by candidate molecular markers was tested. Two-sample Mendelian randomization (MR) analyses were additionally applied to test causal associations of metabolites with depression in independent adult samples. 4,018 and 768 children were included for metabolomics and epigenetics analyses, respectively. Obesity at 7 years was associated with a 14% increase in SMFQ score (95% CI: 1.04, 1.25) and greater odds of depression (OR: 1.46 (95% CI: 0.78, 2.38) at 11 years. Natural indirect effects (mediating pathways) between obesity and depression for tyrosine, leucine and conjugated linoleic acid were 1.06 (95% CI: 1.00, 1.13, proportion mediated (PM): 15%), 1.04 (95% CI: 0.99, 1.10, PM: 9.6%) and 1.06 (95% CI: 1.00, 1.12, PM: 13.9%) respectively. In MR analysis, one unit increase in tyrosine was associated with 0.13 higher log odds of depression (p = 0.1). Methylation at cg17128312, located in the FBXW9 gene, had a natural indirect effect of 1.05 (95% CI: 1.01,1.13, PM: 27%) as a mediator of obesity and SMFQ score. Potential biologically plausible mechanisms involving these identified molecular features include neurotransmitter regulation, inflammation, and gut microbiome modulation. These results require replication in further observational and mechanistic studies.

Metabolic profiles of ultra-processed food consumption and their role in obesity risk in British children.

BACKGROUND & AIMS: Higher consumption of ultra-processed foods (UPF) has been associated with childhood obesity, but underlying mechanisms remain unclear. We investigated plasma nuclear magnetic resonance metabolic profiles of higher UPF consumption and their role in obesity risk in the British ALSPAC cohort. METHODS: We performed cross-sectional and prospective metabolome wide association analyses of UPF, calculated from food diaries using the NOVA classification. In cross-sectional analysis, we tested the association between UPF consumption and metabolic profile at 7 years (N = 4528), and in the prospective analysis we tested the association between UPF consumption at 13 years and metabolic profile at 17 years (N = 3086). Effects of UPF-associated metabolites at 7 years on subsequent fat mass accumulation were assessed using growth curve models. RESULTS: At 7 years, UPF was associated with 115 metabolic traits including lower levels of branched-chain and aromatic amino acids and higher levels of citrate, glutamine, and monounsaturated fatty acids, which were also associated with greater fat mass accumulation. Reported intake of nutrients mediated associations with most metabolites, except for citrate. CONCLUSIONS: UPF consumption among British children is associated with perturbation of multiple metabolic traits, many of which contribute to child obesity risk.

Integration of proteomic and metabolomic analyses: New insights for mapping informal workers exposed to potentially toxic elements.

Occupational exposure to potentially toxic elements (PTEs) is a concerning reality of informal workers engaged in the jewelry production chain that can lead to adverse health effects. In this study, untargeted proteomic and metabolomic analyses were employed to assess the impact of these exposures on informal workers' exposome in Limeira city, São Paulo state, Brazil. PTE levels (Cr, Mn, Ni, Cu, Zn, As, Cd, Sn, Sb, Hg, and Pb) were determined in blood, proteomic analyses were performed for saliva samples (n = 26), and metabolomic analyses in plasma (n = 145) using ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight (Q-TOF) mass spectrometry. Blood PTE levels of workers, controls, and their family members were determined by inductively coupled plasma-mass spectrometry (ICP-MS). High concentration levels of Sn and Cu were detected in welders' blood (p < 0.001). Statistical analyses were performed using MetaboAnalyst 4.0. The results showed that 26 proteins were upregulated, and 14 proteins downregulated on the welder group, and thirty of these proteins were also correlated with blood Pb, Cu, Sb, and Sn blood levels in the welder group (p < 0.05). Using gene ontology analysis of these 40 proteins revealed the biological processes related to the upregulated proteins were translational initiation, SRP-dependent co-translational protein targeting to membrane, and viral transcription. A Metabolome-Wide Association Study (MWAS) was performed to search for associations between blood metabolites and exposure groups. A pathway enrichment analysis of significant features from the MWAS was then conducted with Mummichog. A total of 73 metabolomic compounds and 40 proteins up or down-regulated in welders were used to perform a multi-omics analysis, disclosing seven metabolic pathways potentially disturbed by the informal work: valine leucine and isoleucine biosynthesis, valine leucine and isoleucine degradation, arginine and proline metabolism, ABC transporters, central carbon metabolism in cancer, arachidonic acid metabolism and cysteine and methionine metabolism. The majority of the proteins found to be statistically up or downregulated in welders also correlated with at least one blood PTE level, providing insights into the biological responses to PTE exposures in the informal work exposure scenario. These findings shed new light on the effects of occupational activity on workers' exposome, underscoring the harmful effects of PTE.

Determinants of accelerated metabolomic and epigenetic aging in a UK cohort.

Markers of biological aging have potential utility in primary care and public health. We developed a model of age based on untargeted metabolic profiling across multiple platforms, including nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry in urine and serum, within a large sample (N = 2,239) from the UK Airwave cohort. We validated a subset of model predictors in a Finnish cohort including repeat measurements from 2,144 individuals. We investigated the determinants of accelerated aging, including lifestyle and psychological risk factors for premature mortality. The metabolomic age model was well correlated with chronological age (mean r = .86 across independent test sets). Increased metabolomic age acceleration (mAA) was associated after false discovery rate (FDR) correction with overweight/obesity, diabetes, heavy alcohol use and depression. DNA methylation age acceleration measures were uncorrelated with mAA. Increased DNA methylation phenotypic age acceleration (N = 1,110) was associated after FDR correction with heavy alcohol use, hypertension and low income. In conclusion, metabolomics is a promising approach for the assessment of biological age and appears complementary to established epigenetic clocks.

Home-based and informal work exposes the families to high levels of potentially toxic elements.

The city of Limeira presents a relevant productive chain of jewelry and fashion jewelry, including a scenario of outsourcing informal home practices. It is highly complex to understand the potentially toxic elements (PTE: Cr, Mn, Ni, Cu, Zn, As, Cd, Sn, Sb, Hg, and Pb) exposures of the workers because this productive chain encompasses households. This study aimed to investigate the associations between blood PTE levels and informal work in the home environment. Fifty-two families divided into Exposed group (n = 112) and Control group (n = 53) were included. Families' blood (n = 165) and welder's breathing zone air samples (n = 9) were collected and PTEs concentrations were determined by ICP-MS. Questionnaires were applied to collect sociodemographic information and workplace details. Principal component analysis, Mann-Whitney test, cluster and a logistic regression analysis based on environment-wide association studies (EWAS) were carried out. Ni, Cu, Zn, Cd and Pb concentrations in the air samples were higher than occupational guidelines. Eighty percent of the workers were female, and 43.5% of those females then worked as welder. A significant difference was found for Pb concentration between the exposed and control group (p < 0.0001) and between sexes (p = 0.0046). For Cu (p < 0.0001) and Sb (p = 0.0434), differences were found between the sexes. The receiver operating characteristic of the EWAS was 0.80, providing evidence of a potential model to associate exposure levels and occupational factors. PTEs concentrations in the air samples raised concerns, particularly for children, who were in the same exposure scenario. Inadequate work conditions were observed in the houses, revealing the need of public actions to protect these families.

PhenoMeNal: processing and analysis of metabolomics data in the cloud.

BACKGROUND: Metabolomics is the comprehensive study of a multitude of small molecules to gain insight into an organism's metabolism. The research field is dynamic and expanding with applications across biomedical, biotechnological, and many other applied biological domains. Its computationally intensive nature has driven requirements for open data formats, data repositories, and data analysis tools. However, the rapid progress has resulted in a mosaic of independent, and sometimes incompatible, analysis methods that are difficult to connect into a useful and complete data analysis solution. FINDINGS: PhenoMeNal (Phenome and Metabolome aNalysis) is an advanced and complete solution to set up Infrastructure-as-a-Service (IaaS) that brings workflow-oriented, interoperable metabolomics data analysis platforms into the cloud. PhenoMeNal seamlessly integrates a wide array of existing open-source tools that are tested and packaged as Docker containers through the project's continuous integration process and deployed based on a kubernetes orchestration framework. It also provides a number of standardized, automated, and published analysis workflows in the user interfaces Galaxy, Jupyter, Luigi, and Pachyderm. CONCLUSIONS: PhenoMeNal constitutes a keystone solution in cloud e-infrastructures available for metabolomics. PhenoMeNal is a unique and complete solution for setting up cloud e-infrastructures through easy-to-use web interfaces that can be scaled to any custom public and private cloud environment. By harmonizing and automating software installation and configuration and through ready-to-use scientific workflow user interfaces, PhenoMeNal has succeeded in providing scientists with workflow-driven, reproducible, and shareable metabolomics data analysis platforms that are interfaced through standard data formats, representative datasets, versioned, and have been tested for reproducibility and interoperability. The elastic implementation of PhenoMeNal further allows easy adaptation of the infrastructure to other application areas and 'omics research domains.

Integrated exposure and risk characterization of bisphenol-A in Europe.

The current study aims at a comprehensive risk characterization of bisphenol A (BPA) supported by an integrated exposure modelling framework that comprises far field and near field exposure modelling coupled to a dynamic lifetime PBTK model. Exposure analysis was done on European data of BPA food residues and human biomonitoring (HBM). The latter were further assimilated through an advanced exposure reconstruction modelling framework to estimate the corresponding external and internal systemic dose of BPA and its metabolites. Special attention was paid on the assessment of exposure to BPA during critical developmental stages such as gestation by modelling the mother-fetus toxicokinetic interaction. Our findings showed that current exposure levels in Europe are below the temporary Tolerable Daily Intake (t-TDI) of 4 µg/kg_bw/d proposed by the European Food Safety Authority. Taking into account age-dependent bioavailability differences, internal exposure of premature neonates hosted in intensive care units was reckoned close to the biologically effective dose (BED) resulting from translating the EFSA temporary total daily intake (t-TDI) into equivalent internal dose. Use of the ToxCast21 Biological Pathway Altering Dose (BPAD) as an alternative internal exposure reference value, resulted in increased margins of safety compared to the conventional exposure/risk characterization scheme.