Consideration of pathways for immunotoxicity of per- and polyfluoroalkyl substances (PFAS).

BACKGROUND: Per- and polyfluoroalkyl substances (PFAS) are of public health concern, because of their ubiquitous and extremely persistent occurrence, and depending on their structure, their bio-accumulative, mobile and toxic properties. Human health effects associated with exposure to PFAS include adverse effects on the immune system. In 2020, EFSA (the European Food Safety Authority) defined adverse effects on the immune system as the most critical effect for human health risk assessment, based on reduced antibody responses to childhood vaccines and similar effects observed in experimental animal studies. Likewise, the U.S. EPA (Environmental Protection Agency) considers PFAS-induced immunotoxicity, especially in children, as the critical effect for risk assessment. However, the mechanisms by which antibody concentrations are impacted are not completely understood. Furthermore, other targets of the immune system functions have been reported in the literature. OBJECTIVE: The aim of this review is to explore PFAS-associated immune-related effects. This includes, relevant mechanisms that may underlie the observed effects on the immune system, immunosuppression as well as immunoenhancement, such as i) modulation of cell signalling and nuclear receptors, such as NF-κB and PPARs; ii) alteration of calcium signalling and homoeostasis in immune cells; iii) modulation of immune cell populations; iv) oxidative stress and v) impact on fatty acid metabolism & secondary effects on the immune system. METHODS: A literature research was conducted using three databases (Web of Science, PubMed, and Scopus), which were searched in July 2021 for relevant studies published in the time frame from 2018 to 2021. In total, 487 publications were identified as potentially eligible and following expert-based judgement, articles relevant for mechanisms of PFAS induced immunotoxicity are discussed. CONCLUSIONS: Taken together, we show that there is substantial evidence from both in vitro and in vivo experimental as well as epidemiological studies, supporting that various PFAS, not only PFOA and PFOS, affect multiple aspects of the immune system. Timing of exposure is critical, because the developing immune system is especially vulnerable to toxic insults, resulting in a higher risk of particularly adverse immune effects but also other organs later in life.

Prenatal exposure to per- and polyfluoroalkyl substances and pregnancy outcome in Austria.

Per- and polyfluoroalkyl substances (PFAS) are a large group of persistent industrial chemicals that can harm reproductive health. PFAS levels were analysed to determine the current sources of exposure and possible associations between prenatal PFAS exposure and adverse pregnancy outcome. Samples from 136 mother-newborn pairs recruited between 2017 and 2019 were analysed for the presence of 31 target PFAS in maternal serum, umbilical cord serum, and placental tissue by high-performance liquid chromatography coupled to a tandem mass spectrometer. Questionnaires and medical records were used to survey sources of exposure and pregnancy outcome, including small for gestational age (SGA), fetal growth restriction (FGR), preeclampsia (PE), preterm birth, large for gestational age (LGA) and gestational diabetes mellitus (GDM). Data were analysed for individual PFAS and sum4PFAS (sum of perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexane sulfonate (PFHxS), and perfluorooctane sulfonate (PFOS) serum levels) in logistic regression analyses and categorical regression analyses. Compared to data from a previous Viennese study in 2010-12, sum4PFAS levels were generally lower. Sum4PFAS serum levels of three women (2.2%) exceeded 6.9 µg/L, a level that corresponds to the recently established tolerable weekly intake (TWI) of EFSA for nursing mothers aged 35 years; in the 2010/2012 study it was 13.6%. The large contribution of unidentified extractable organofluorine (EOF) fractions to total PFAS exposure is a concern. Study site, mean maternal corpuscular hemoglobin (MCH), use of facial lotion, and owning upholstered furniture were significantly influencing maternal exposure. While no effect of sum4PFAS on pregnancy outcome could be detected, we found highest placental PFDA levels in SGA births. PFHxS levels in umbilical cord and placenta were highest in preterm births. Further studies are needed to elucidate the relationship of prenatal PFAS exposure and pregnancy outcome, in particular to confirm whether and how placental PFDA levels may contribute to an increased risk for SGA.

The transplacental transfer efficiency of per- and polyfluoroalkyl substances (PFAS): a first meta-analysis.

Per- and polyfluorinated substances (PFAS), ubiquitously present in the environment and biota, are transferred to the fetus via the placenta. PFAS can be distinguished, among other things, by their different carbon chain lengths and functional groups. The aim of this study was to provide comprehensive evidence on PFAS transfer rates across the human placental barrier by means of a meta-analysis based upon a systematic review. The available literature up to April 2021 was reviewed and transplacental transfer efficiencies (TTEs) of PFAS assessed. A total of 39 studies reporting data on 20 PFAS were included in the systematic review. Of these, 20 studies with data on 19 compounds were included in the meta-analysis. Comprehensive Meta-Analysis (CMA v3.0) was used for quantitative, statistical analyses with random effects models. A curvilinear relationship was found with short and long chains of perfluorocarboxylic acids (PFCAs) exhibiting higher TTE than compounds with intermediate chain length. Among the less well studied PFAS, perfluorohexanoic acid (PFHxA), 6:2 fluorotelomersulfonic acid (6:2 FTS) and perfluorobutanoic acid (PFBA) stood out the most with a high TEEs. The dependence of TTEs on chain length and functional group is clearly shown in this first meta-analysis on PFAS transfer across the human placenta. More data on effects of less well studied PFAS in pregnant women and neonates are needed to assess the potential risk for fetal exposure.

Systemic PFOS and PFOA exposure and disturbed lipid homeostasis in humans: what do we know and what not?

Associations between per- and polyfluoroalkyl substances (PFASs) and increased blood lipids have been repeatedly observed in humans, but a causal relation has been debated. Rodent studies show reverse effects, i.e. decreased blood cholesterol and triglycerides, occurring however at PFAS serum levels at least 100-fold higher than those in humans. This paper aims to present the main issues regarding the modulation of lipid homeostasis by the two most common PFASs, PFOS and PFOA, with emphasis on the underlying mechanisms relevant for humans. Overall, the apparent contrast between human and animal data may be an artifact of dose, with different molecular pathways coming into play upon exposure to PFASs at very low versus high levels. Altogether, the interpretation of existing rodent data on PFOS/PFOA-induced lipid perturbations with respect to the human situation is complex. From a mechanistic perspective, research on human liver cells shows that PFOS/PFOA activate the PPARα pathway, whereas studies on the involvement of other nuclear receptors, like PXR, are less conclusive. Other data indicate that suppression of the nuclear receptor HNF4α signaling pathway, as well as perturbations of bile acid metabolism and transport might be important cellular events that require further investigation. Future studies with human-relevant test systems would help to obtain more insight into the mechanistic pathways pertinent for humans. These studies shall be designed with a careful consideration of appropriate dosing and toxicokinetics, so as to enable biologically plausible quantitative extrapolations. Such research will increase the understanding of possible perturbed lipid homeostasis related to PFOS/ PFOA exposure and the potential implications for human health.

Extractable Organofluorine Analysis in Pooled Human Serum and Placental Tissue Samples from an Austrian Subpopulation-A Mass Balance Analysis Approach.

Embryos and fetuses are of major concern due to their high vulnerability. Previous studies demonstrated that human exposure to per- and polyfluoroalkyl substances (PFAS) may be underestimated because only a limited number of known PFAS can be measured. This investigation studied the total PFAS exposure by measuring the extractable organofluorine (EOF) in pooled maternal serum, placental tissue, and cord serum samples (total number of pooled samples: n = 45). The EOF was analyzed using combustion ion chromatography, and the concentrations of known PFAS were determined using ultraperformance liquid chromatography coupled with a tandem mass spectrometer. Using a mass balance analysis approach, the amount of unknown PFAS was estimated between the levels of known PFAS and EOF. The EOF levels ranged from 2.85 to 7.17 ng F/mL (21 PFAS were quantified) in the maternal serum, from 1.02 to 1.85 ng F/g (23 PFAS were quantified) in the placental tissue, and from 1.2 to 2.10 ng F/mL (18 PFAS were quantified) in the cord serum. An average of 24, 51, and 9% of EOF is unidentified in the maternal serum, placental tissue, and cord serum, respectively. The results show that the levels of unidentified EOF are higher in the placental tissue, suggesting accumulation or potential transformation of precursors in the placenta.

Comparison of extraction methods for per- and polyfluoroalkyl substances (PFAS) in human serum and placenta samples-insights into extractable organic fluorine (EOF).

Since the detection of per- and polyfluoroalkyl substances (PFAS) in humans and different environmental media in the last two decades, this substance group has attracted a lot of attention as well as increasing concerns. The fluorine mass balance approach, by comparing the levels of targeted PFAS after conversion to fluorine equivalents with those of extractable organic fluorine (EOF), showed the presence of unidentified organofluorine in different environmental samples. Out of the thousands of PFAS in existence, only a very small fraction is included in routine analysis. In recent years, liquid chromatography coupled with tandem-mass spectrometry (LC-MS/MS) has demonstrated the ability to analytically cover a wide spectrum of PFAS. In contrast, conventional extraction methods developed 10 to 15 years ago were only evaluated for a limited number of PFAS. The aim of the present study was to evaluate the advantages and disadvantages of three different extraction methods, adapted from the literatures without further optimization (ion-pair liquid-liquid extraction, solid-phase extraction (SPE), using hydrophilic-lipophilic (HLB) or weak anion exchange (WAX) sorbents), for human biomonitoring of 61 PFAS in serum and placental tissue samples. In addition, levels of EOF were compared among these extraction methods via spiked samples. Results showed that performance, in terms of recovery, differed between the extraction methods for different PFAS; different extraction methods resulted in different EOF concentrations indicating that the choice of extraction method is important for target PFAS and EOF analysis. Results of maternal serum samples, analyzed in two different laboratories using two different extraction methods, showed an accordance of 107.6% (± 21.3); the detected perfluoroalkyl acids (PFAAs) in maternal and cord serum samples were in the range of 0.076 to 2.9 ng/mL.Graphical abstract.

Persistent Organic Pollutants in Austrian Human Breast Milk Collected between 2013 and 2016.

Breast milk holds an immense nutritional value as it contains health-promoting substances in a unique, optimal form. Additionally, breast milk's significance extends to health and environmental protection, as it serves as an indicator of both maternal and infant exposure. In this study, breast milk samples collected in 2013 and in 2014-2016 from mothers in Vienna (Austria) were analysed for polybrominated diphenyl ethers (PBDE) and per- and polyfluoroalkyl substances (PFAS), as well as further substances which have been listed under the Stockholm Convention on Persistent Organic Pollutants (POPs) due to their persistent, bioaccumulative and toxic properties. The total concentration of the PBDE congeners in the samples (n = 18, sampled 2013) ranged from 0.055 to 52 ng/g lipid, and from 0.002 to 2.5 ng/g breast milk. In the pooled sample, the sum of PBDEs was detected at a level of 4.4 ng/g lipid. Based on the 2014-2016 study population, certain PFAS were detected in all samples (n = 40). Exposure to the sum of four specific PFAS including perfluorooctanesulphonate (PFOS), perfluorooctanoic acid (PFOA), perfluoro-n-nonanoic acid (PFNA) and perfluoro-1-hexanesulfonate (PFHxS) ranged between 0.014 and 0.12 ng/L breast milk. In the pooled sample, PFOS and PFOA were found in concentrations of 0.025 ng/g and of 0.045 ng/g, respectively. In addition, the first generation of POPs, mainly organochlorine compounds, was measured in a pooled sample of breast milk from participants sampled in 2014-2016 as part of the WHO/UNEP breast milk monitoring program and compared to the POPs measured in pooled samples collected in 1987/1988 and 1992/1993, respectively. Therefore, this paper demonstrates the effectiveness of the Stockholm Convention on POPs by comparing the Austrian results from the WHO/UNEP global breast milk study from 1987 to 2016. However, the data also show that, despite these reductions, health-relevant levels are still being reached, particularly in terms of children's health when the presence of the new generation of POPs, such as PBDEs and PFAS, in human breast milk is taken into account.

Results of the Austrian Children's Biomonitoring Survey 2020 part A: Per- and polyfluorinated alkylated substances, bisphenols, parabens and other xenobiotics.

In 85 Austrian school children aged 6-10 years, two multi-analyte LC-MS/MS methods were used to study the concentrations of 33 chemical substances in urine, including per- and polyfluorinated alkylated substances (PFAS), bisphenols, parabens, benzophenones, triclosan, polycyclic aromatic hydrocarbon metabolites, and cotinine. Each of the children was exposed to 14-21 substances simultaneously. Correlations were found between compounds of the same and of divergent substance groups supporting the strong need to consider multiple exposures and mixture effects. Eight compounds, including perfluorohexanoic acid (PFHxA), perfluorononanoic acid (PFOA), methyl paraben (n-MeP), ethyl paraben (n-EtP), propyl paraben (n-PrP), benzophenone-1 (BP-1), 2-naphthol, and 3-hydroxyphenanthrene were detected in all urine samples. In the PFAS group the medians of detectable substances ranged between <0.0005 µg/l for perfluorononanoic acid (PFNA) and 0.004 µg/l for PFHxA. For other environmental contaminants investigated, a maximum urinary level of 893 µg/l was identified for n-MeP. The highest median value was 2.5 µg/l for 2-naphthol. Daily intakes were calculated for bisphenol A (BPA), triclosan (TCS), and four parabens. These values did not exceed the tolerable or acceptable daily intakes currently in force. Based on a recently proposed TDI for BPA, daily intakes of all children exceeded this value. A cumulative risk assessment was conducted for four parabens not showing exceedances of acceptable exposures. The results demonstrate simultaneous exposure to several different chemicals, with the majority showing impact on the endocrine system being of particular concern with respect to mixture effects. Further assessments with a stronger focus on mixtures are warranted. The results also highlight the need of policy actions as foreseen in the EU Chemicals Strategy for Sustainability.

Internal relative potency factors based on immunotoxicity for the risk assessment of mixtures of per- and polyfluoroalkyl substances (PFAS) in human biomonitoring.

Relative potency factors (RPFs) for per- and polyfluoroalkyl substances (PFAS) have previously been derived based on liver effects in rodents for the purpose of performing mixture risk assessment with primary input from biomonitoring studies. However, in 2020, EFSA established a tolerable weekly intake for four PFAS assuming equal toxic potency for immune suppressive effects in humans. In this study we explored the possibility of deriving RPFs for immune suppressive effects using available data in rodents and humans. Lymphoid organ weights, differential blood cell counts, and clinical chemistry from 28-day studies in male rats from the National Toxicology Program (NTP) were combined with modeled serum PFAS concentrations to derive internal RPFs by applying dose-response modelling. Identified functional studies used diverse protocols and were not suitable for derivation of RPFs but were used to support immunotoxicity of PFAS in a qualitative manner. Furthermore, a novel approach was used to estimate internal RPFs based on epidemiological data by dose-response curve fitting optimization, looking at serum antibody concentrations and key cell populations from the National Health and Nutrition Examination Survey (NHANES). Internal RPFs were successfully derived for PFAS based on rat thymus weight, spleen weight, and globulin concentration. The available dose-response information for blood cell counts did not show a significant trend. Immunotoxic potency in serum was determined in the order PFDA > PFNA > PFHxA > PFOS > PFBS > PFOA > PFHxS. The epidemiological data showed inverse associations for the sum of PFOA, PFNA, PFHxS, and PFOS with serum antibody concentrations to mumps and rubella, but the data did not allow for deduction of reliable internal RPF estimates. The internal RPFs for PFAS based on decreased rat lymphoid organ weights are similar to those previously established for increased rat liver weight, strengthening the confidence in the overall applicability of these RPFs.

The Austrian children's biomonitoring survey 2020 Part B: Mycotoxins, phytotoxins, phytoestrogens and food processing contaminants.

This study assessed the levels of environment and food-related exposures in urine of Austrian school children aged six to ten (n = 85) focusing on mycotoxins, phytoestrogens, and food processing by-products using two multi-analyte LC-MS/MS methods. Out of the 55 biomarkers of exposure reported in this study, 22 were quantified in the first void urine samples. Mycotoxins frequently quantified included zearalenone (detection rate 100%; median 0.11 ng/mL), deoxynivalenol (99%; 15 ng/mL), alternariol monomethyl ether (75%; 0.04 ng/mL), and ochratoxin A (19%; 0.03 ng/mL). Several phytoestrogens, including genistein, daidzein, and its metabolite equol, were detected in all samples at median concentrations of 22 ng/mL, 43 ng/mL, and 14 ng/mL, respectively. The food processing by-product 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was detected in 4% of the samples (median 0.016 ng/mL). None of the investigated samples contained the tested phytotoxins that were rarely considered for human biomonitoring previously (pyrrolizidine alkaloids, tropane alkaloids, aristolochic acids). When relating estimated exposure to current health-based guidance values, 22% of the children exceeded the tolerable daily intake for deoxynivalenol, and the estimated MOE for OTA indicates possible health risks for some children. The results clearly demonstrate frequent low-level (co-)exposure and warrant further exposome-scale exposure assessments, especially in susceptible sub-populations and longitudinal settings.

HBM4EU results support the Chemicals' Strategy for Sustainability and the Zero-Pollution Action Plan.

One of the major goals of the European Human Biomonitoring Initiative (HBM4EU) was to bridge the gap between science and policy by consulting both policy makers and national scientists and generating evidence of the actual exposure of residents to chemicals and whether that exposure would be suggest a potential health risk. Residents' perspectives on chemical exposure and risk were also investigated. HBM4EU's research was designed to answer specific short-term and long-term policy questions at national and European levels, and for its results to directly support regulatory action on chemicals. A strategy was established to prioritise chemicals for analysis in human matrices, with a total of 18 substances/substance groups chosen to be investigated throughout the five-and a -half-year project. HBM4EU produced new evidence of human exposure levels, developed reference values for exposure, investigated determinants of exposure and derived health-based guidance values for those substances. In addition, HBM4EU promoted the use of human biomonitoring data in chemical risk assessment and developed innovative tools and methods linking chemicals to possible health impacts, such as effect biomarkers. Furthermore, HBM4EU advanced understand of effects from combined exposures and methods to identify emerging chemicals. With the aim of supporting policy implementation, science-to-policy workshops were organised, providing opportunities for joint reflection and dialogue on research results. I, and indicators were developed to assess temporal and spatial patterns in the exposure of European population. A sustainable human biomonitoring monitoring framework, producing comparable quality assured data would allow: the evaluation of time trends; the exploration of spatial trends: the evaluation of the influence of socio-economic conditions on chemical exposure. Therefore, such a framework should be included in the European Chemicals' Strategy for Sustainability and the data would support the Zero Pollution Action Plan.

PFASs: What can we learn from the European Human Biomonitoring Initiative HBM4EU.

Per- and polyfluoroalkyl substances (PFASs) were one of the priority substance groups selected which have been investigated under the ambitious European Joint programme HBM4EU (2017-2022). In order to answer policy relevant questions concerning exposure and health effects of PFASs in Europe several activities were developed under HBM4EU namely i) synthesis of HBM data generated in Europe prior to HBM4EU by developing new platforms, ii) development of a Quality Assurance/Quality Control Program covering 12 biomarkers of PFASs, iii) aligned and harmonized human biomonitoring studies of PFASs. In addition, some cohort studies (on mother-child exposure, occupational exposure to hexavalent chromium) were initiated, and literature researches on risk assessment of mixtures of PFAS, health effects and effect biomarkers were performed. The HBM4EU Aligned Studies have generated internal exposure reference levels for 12 PFASs in 1957 European teenagers aged 12-18 years. The results showed that serum levels of 14.3% of the teenagers exceeded 6.9 µg/L PFASs, which corresponds to the EFSA guideline value for a tolerable weekly intake (TWI) of 4.4 ng/kg for some of the investigated PFASs (PFOA, PFOS, PFNA and PFHxS). In Northern and Western Europe, 24% of teenagers exceeded this level. The most relevant sources of exposure identified were drinking water and some foods (fish, eggs, offal and locally produced foods). HBM4EU occupational studies also revealed very high levels of PFASs exposure in workers (P95: 192 µg/L in chrome plating facilities), highlighting the importance of monitoring PFASs exposure in specific workplaces. In addition, environmental contaminated hotspots causing high exposure to the population were identified. In conclusion, the frequent and high PFASs exposure evidenced by HBM4EU strongly suggests the need to take all possible measures to prevent further contamination of the European population, in addition to adopting remediation measures in hotspot areas, to protect human health and the environment. HBM4EU findings also support the restriction of the whole group of PFASs. Further, research and definition for additional toxicological dose-effect relationship values for more PFASs compounds is needed.

Cross-sectional associations between exposure to per- and polyfluoroalkyl substances and body mass index among European teenagers in the HBM4EU aligned studies.

Per- and polyfluoroalkyl substances (PFAS) are widespread pollutants that may impact youth adiposity patterns. We investigated cross-sectional associations between PFAS and body mass index (BMI) in teenagers/adolescents across nine European countries within the Human Biomonitoring for Europe (HBM4EU) initiative. We used data from 1957 teenagers (12-18 yrs) that were part of the HBM4EU aligned studies, consisting of nine HBM studies (NEBII, Norway; Riksmaten Adolescents 2016-17, Sweden; PCB cohort (follow-up), Slovakia; SLO CRP, Slovenia; CROME, Greece; BEA, Spain; ESTEBAN, France; FLEHS IV, Belgium; GerES V-sub, Germany). Twelve PFAS were measured in blood, whilst weight and height were measured by field nurse/physician or self-reported in questionnaires. We assessed associations between PFAS and age- and sex-adjusted BMI z-scores using linear and logistic regression adjusted for potential confounders. Random-effects meta-analysis and mixed effects models were used to pool studies. We assessed mixture effects using molar sums of exposure biomarkers with toxicological/structural similarities and quantile g-computation. In all studies, the highest concentrations of PFAS were PFOS (medians ranging from 1.34 to 2.79 µg/L). There was a tendency for negative associations with BMI z-scores for all PFAS (except for PFHxS and PFHpS), which was borderline significant for the molar sum of [PFOA and PFNA] and significant for single PFOA [ß-coefficient (95% CI) per interquartile range fold change = -0.06 (-0.17, 0.00) and -0.08 (-0.15, -0.01), respectively]. Mixture assessment indicated similar negative associations of the total mixture of [PFOA, PFNA, PFHxS and PFOS] with BMI z-score, but not all compounds showed associations in the same direction: whilst [PFOA, PFNA and PFOS] were negatively associated, [PFHxS] associated positively with BMI z-score. Our results indicated a tendency for associations of relatively low PFAS concentrations with lower BMI in European teenagers. More prospective research is needed to investigate this potential relationship and its implications for health later in life.

Implementation of effect biomarkers in human biomonitoring studies: A systematic approach synergizing toxicological and epidemiological knowledge.

Human biomonitoring (HBM) studies have highlighted widespread daily exposure to environmental chemicals. Some of these are suspected to contribute to adverse health outcomes such as reproductive, neurological, and metabolic disorders, among other developmental and chronic impairments. One of the objectives of the H2020 European Human Biomonitoring Initiative (HBM4EU) was the development of informative effect biomarkers for application in a more systematic and harmonized way in large-scale European HBM studies. The inclusion of effect biomarkers would complement exposure data with mechanistically-based information on early and late adverse effects. For this purpose, a stepwise strategy was developed to identify and implement a panel of validated effect biomarkers in European HBM studies. This work offers an overview of the complete procedure followed, from comprehensive literature search strategies, selection of criteria for effect biomarkers and their classification and prioritization, based on toxicological data and adverse outcomes, to pilot studies for their analytical, physiological, and epidemiological validation. We present the example of one study that demonstrated the mediating role of the effect biomarker status of brain-derived neurotrophic factor BDNF in the longitudinal association between infant bisphenol A (BPA) exposure and behavioral function in adolescence. A panel of effect biomarkers has been implemented in the HBM4EU Aligned Studies as main outcomes, including traditional oxidative stress, reproductive, and thyroid hormone biomarkers. Novel biomarkers of effect, such as DNA methylation status of BDNF and kisspeptin (KISS) genes were also evaluated as molecular markers of neurological and reproductive health, respectively. A panel of effect biomarkers has also been applied in HBM4EU occupational studies, such as micronucleus analysis in lymphocytes and reticulocytes, whole blood comet assay, and malondialdehyde, 8-oxo-2'-deoxyguanosine and untargeted metabolomic profile in urine, to investigate, for example, biological changes in response to hexavalent chromium Cr(VI) exposure. The use of effect biomarkers in HBM4EU has demonstrated their ability to detect early biological effects of chemical exposure and to identify subgroups that are at higher risk. The roadmap developed in HBM4EU confirms the utility of effect biomarkers, and support one of the main objectives of HBM research, which is to link exposure biomarkers to mechanistically validated effect and susceptibility biomarkers in order to better understand the public health implications of human exposure to environmental chemicals.

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.

Application of human biomonitoring data to support policy development, raise awareness and environmental public health protection among countries within the HBM4EU project.

Most countries have acknowledged the importance of assessing and quantifying their population's internal exposure from chemicals in air, water, soil, food and other consumer products due to the potential health and economic impact. Human biomonitoring (HBM) is a valuable tool which can be used to quantify such exposures and effects. Results from HBM studies can also contribute to improving public health by providing evidence of individuals' internal chemical exposure as well as data to understand the burden of disease and associated costs thereby stimulating the development and implementation of evidence-based policy. To have a holistic view on HBM data utilisation, a multi-case research approach was used to explore the use of HBM data to support national chemical regulations, protect public health and raise awareness among countries participating in the HBM4EU project. The Human Biomonitoring for Europe (HBM4EU) Initiative (https://www.hbm4eu.eu/) is a collaborative effort involving 30 countries, the European Environment Agency (EEA) and the European Commission (contracting authority) to harmonise procedures across Europe and advance research into the understanding of the health impacts of environmental chemical exposure. One of the aims of the project was to use HBM data to support evidence based chemical policy and make this information timely and directly available for policy makers and all partners. The main data source for this article was the narratives collected from 27 countries within the HBM4EU project. The countries (self-selection) were grouped into 3 categories in terms of HBM data usage either for public awareness, policy support or for the establishment HBM programme. Narratives were analysed/summarised using guidelines and templates that focused on ministries involved in or advocating for HBM; steps required to engage policy makers; barriers, drivers and opportunities in developing a HBM programme. The narratives reported the use of HBM data either for raising awareness or addressing environmental/public health issues and policy development. The ministries of Health and Environment were reported to be the most prominent entities advocating for HBM, the involvement of several authorities/institutions in the national hubs was also cited to create an avenue to interact, discuss and gain the attention of policy makers. Participating in European projects and the general population interest in HBM studies were seen as drivers and opportunities in developing HBM programmes. A key barrier that was cited by countries for establishing and sustaining national HBM programmes was funding which is mainly due to the high costs associated with the collection and chemical analysis of human samples. Although challenges and barriers still exist, most countries within Europe were already conversant with the benefits and opportunities of HBM. This article offers important insights into factors associated with the utilisation of HBM data for policy support and public awareness.

[The history of emerging substances in Austria]. / Eine Geschichte der Emerging Substances in Österreich.

Over time, many different groups of substances became the focus of interest, so their occurrence, behaviour and effects were studied. While in the 1990s, it was detergents and the formation of foam in water, later the causes of discolouration around tanneries were researched, as well as the presence of chemicals and pollutants near industrial plants. Organochlorine pesticides, brominated flame retardants, perfluoroalkyl substances or PFAS, organotin compounds are some examples of such Emerging substances. After pesticides and industrial chemicals, active pharmaceutical ingredients, cosmetics and personal care products have also become "Emerging substances". Ultimately, however, it is the effect of the substances-whether persistent, bioaccumulative, mobile, toxic or even endocrine disruption-that attracts attention and triggers legal regulations. As the substances and the methods for their detection changed, so did the corresponding legislation. This in turn led to the use of new or slightly modified substances and substance groups. Innovative methods such as non-targeted analytics and biological effect tests or bioassays are now being utilised to address the variety and combined effects of the existing substances. In order to ensure comprehensive groundwater and water protection, the investigation and assessment methods must be developed. Furthermore, the existing and newly acquired knowledge need to be translated into regulatory consequences more quickly. Beyond that, a comprehensive societal transformation with regard to the sustainable use of natural water resources is essential for environmentally sound and healthy development. This must therefore be implemented on many different levels; with knowledge transfer and awareness-raising also having a significant role to play.

Characterization of Potential Adverse Outcome Pathways Related to Metabolic Outcomes and Exposure to Per- and Polyfluoroalkyl Substances Using Artificial Intelligence.

Human exposure to per- and polyfluoroalkyl substances (PFAS) has been associated with numerous adverse health effects, depending on various factors such as the conditions of exposure (dose/concentration, duration, route of exposure, etc.) and characteristics associated with the exposed target (e.g., age, sex, ethnicity, health status, and genetic predisposition). The biological mechanisms by which PFAS might affect systems are largely unknown. To support the risk assessment process, AOP-helpFinder, a new artificial intelligence tool, was used to rapidly and systematically explore all available published information in the PubMed database. The aim was to identify existing associations between PFAS and metabolic health outcomes that may be relevant to support building adverse outcome pathways (AOPs). The collected information was manually organized to investigate linkages between PFAS exposures and metabolic health outcomes, including dyslipidemia, hypertension, insulin resistance, and obesity. Links between PFAS exposure and events from the existing metabolic-related AOPs were also retrieved. In conclusion, by analyzing dispersed information from the literature, we could identify some associations between PFAS exposure and components of existing AOPs. Additionally, we identified some linkages between PFAS exposure and metabolic outcomes for which only sparse information is available or which are not yet present in the AOP-wiki database that could be addressed in future research.

Reduced Birth Weight and Exposure to Per- and Polyfluoroalkyl Substances: A Review of Possible Underlying Mechanisms Using the AOP-HelpFinder.

Prenatal exposure to per- and polyfluorinated substances (PFAS) may impair fetal growth. Our knowledge of the underlying mechanisms is incomplete. We used the Adverse Outcome Pathway (AOP)-helpFinder tool to search PubMed for studies published until March 2021 that examined PFAS exposure in relation to birth weight, oxidative stress, hormones/hormone receptors, or growth signaling pathways. Of these 1880 articles, 106 experimental studies remained after abstract screening. One clear finding is that PFAS are associated with oxidative stress in in vivo animal studies and in vitro studies. It appears that PFAS-induced reactive-oxygen species (ROS) generation triggers increased peroxisome proliferator-activated receptor (PPAR)γ expression and activation of growth signaling pathways, leading to hyperdifferentiation of pre-adipocytes. Fewer proliferating pre-adipocytes result in lower adipose tissue weight and in this way may reduce birth weight. PFAS may also impair fetal growth through endocrine effects. Estrogenic effects have been noted in in vivo and in vitro studies. Overall, data suggest thyroid-damaging effects of PFAS affecting thyroid hormones, thyroid hormone gene expression, and histology that are associated in animal studies with decreased body and organ weight. The effects of PFAS on the complex relationships between oxidative stress, endocrine system function, adipogenesis, and fetal growth should be further explored.

Citizens' Perception and Concerns on Chemical Exposures and Human Biomonitoring-Results from a Harmonized Qualitative Study in Seven European Countries.

Exposure to different chemicals is an inevitable part of our everyday lives. Within HBM4EU, focus group discussions were conducted to gather data on citizens' perceptions of chemical exposure and human biomonitoring. These discussions were hosted in Cyprus, Denmark, Hungary, Israel, Latvia, the Netherlands, and North Macedonia following a protocol developed in the first round of discussions. Results indicate the very high concern of European citizens regarding food safety and the environment. Focus group participants were well aware of potential uptake of chemicals through food consumption (e.g., preservatives, flavor enhancers, coloring agents, pesticides, fertilizers, metals), drinking water, or from polluted air and water. One of the positive aspects identified here, is the high interest of citizens in awareness and education on personal measures to control exposure. The promotion of personal behavioral changes requires active involvement of society (e.g., commuting habits, energy choices, waste disposal, dietary habits). Activities should focus on raising awareness of the general public, implementation of policy measures, and mainstreaming of related topics into the education system. Raising awareness of the general public may promote engagement of citizens, which in turn may empower them to put pressure on politicians to take effective actions. There is also a need for further research which might focus on the impact of country-specific situations and of the COVID-19 pandemic on the exposure of citizens to chemicals.