Humans are chronically exposed to mixtures of environmental contaminants. Exposure to endocrine-disrupting chemicals (EDCs) contributes to increased health impairment observed globally. This study aimed to evaluate the endocrine-disruptive and oxidative stress potential of a human-relevant, complex chemical mixture in vitro. By testing chemical class subgroup mixtures, the identity of toxicological drivers and mixture additivity could be investigated. A 50-component mixture was compiled based on Swedish human blood concentrations (xHBC), consisting of six subgroup mixtures: polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (PCB mixture), brominated flame retardants (BFR mixture), per- and polyfluoroalkyl substances (PFAS mixture), pesticide mixture, synthetic phenolic contaminants (phenol mixture), and phthalate mixture. These were tested in four chemically activated luciferase gene expression (CALUX) assays: dioxin responsive (DR-), estrogen receptor α (ERα-), androgen receptor. (AR-), and nuclear factor erythroid 2-related factor 2 (Nrf2)-CALUX, along with an adipocyte cell assay. The total mixture caused significant agonistic activity in DR- and ER-, and antagonistic activity in AR-CALUX at 0.1–15 xHBC, depending on the assay. Mixture additivity was assessed in ERα-, DR-, and anti-AR-CALUX using subgroup mixtures and the concentration addition (CA) model. The total mixture followed the CA model in ERα-, anti-AR- and DR-CALUX. The toxicological drivers of these activities were mainly the PCB and phenol mixture. A significant increase in differentiated adipocytes was observed at 100 xHBC. These results raise concerns regarding potential health effects on the endocrine system. The additive effects at human-relevant concentrations observed in this study motivate considering mixtures in regulatory contexts to protect the well-being of future generations.

Background: Humans and companion animals are continually exposed to mixtures of synthetic chemicals from household products, textiles, food, and personal care items. Epidemiological studies of emerging contaminants typically rely on invasive blood sampling, whereas saliva presents a non-invasive and cost-effective alternative matrix that reflects both local and systemic physiological changes. In this pilot study, paired dog saliva and blood serum were analysed for two chemical groups of concern, that is, synthetic phenolic antioxidants (SPAs) and per- and polyfluoroalkyl substances (PFAS), to investigate whether saliva can be used for chemical exposome studies. Methods: Blood serum and saliva samples of a total of 30 dogs were included in this study. All dogs were privately owned, representing different breeds, and were presented to private veterinary clinics in the Murcia Region, Spain. Samples were analysed using acetonitrile for denaturation and extraction, and clean-up using enhanced matrix removal powder. The extracts were analysed using complementary liquid and gas chromatography coupled with mass spectrometers. Dogs were divided into three groups (n = 10 per group): healthy normal-weight dogs, healthy obese dogs, and dogs diagnosed with mammary tumours. Results: Fourteen SPAs (including five metabolites) and eight PFAS were quantified in the dog’s saliva and/or serum. Generally, the levels were higher in serum than in saliva. None of the dogs were free of contaminants. The SPA levels were dominated by the 2,4-di-tert-butylphenol (2,4-DBP), found in 93% of saliva and 100% of serum samples, ranging from <LOQ-860 ng/g in saliva and 130–2,100 ng/g in serum. The levels of PFAS were dominated by perfluorononanoic acid (PFNA) and perfluorosulfonic acid (PFOS), which were quantified in more than 70% of the samples. No correlation between levels and the three groups, or the paired serum and saliva samples could be established. Conclusion: This study confirmed that SPAs and PFAS can be found in the saliva of dogs. The levels of PFAS in dog’s serum were similar to those generally reported in human blood. Furthermore, this study confirms that dogs are exposed to SPAs at concerningly high levels, given the limited knowledge regarding their toxicity.

Per- and polyfluoroalkyl substances (PFAS) have been detected worldwide, from the deep seas to polar regions. A previous review showed that PFAS are risk drivers of the chemical mixture present in human blood. This study focused on establishing the PFAS exposure of a Swedish subpopulation and investigated whether the exposure poses a risk of adverse health effects. Human serum from 60 blood donors in Stockholm, Sweden, was analyzed. A target method including 32 PFAS analytes and over 270 suspect features was used to detect and quantify PFAS. Twenty-six PFAS were quantified, and 7 suspect PFAS features (6 H-PFCAs and PFECHS) were semi-quantified. Nine mixture risk assessment (MRA) strategies were used to assess the risk of health outcomes. Fifteen effect levels were derived and used, along with 15 already established values. The certainty of various derivation techniques was discussed. The MRAs showed that the entire studied population exceeded some of the risk thresholds, with effects including high cholesterol and immune suppression. However, the certainty was lower when deriving these two effect levels. The MRA, using human biomonitoring guidance values (high certainty), concluded that for 63 % of the individuals, a risk for adverse health effects cannot be excluded. This study has demonstrated that there is a reason for concern regarding PFAS exposure in the general population of Sweden. To our knowledge, this is the first time the H-PFCAs have been semi-quantified in human blood using a reference standard.

Early identification of new and emerging risk chemicals (NERCs) is critical in protecting human and environmental health while chemical invention and production is growing on a global market. Chemicals market data is information on the production, import, and use of chemicals in materials and products. By integration of chemicals market data with suspect screening strategies NERCs could potentially be detected early. In silico tools play an important role in this integration to identify blind spots in current analytical approaches and in identification of the potentially most hazardous chemicals. This chapter starts with a brief presentation of the term “chemicals market data.” The integrated approach is then presented in three steps: (1) Data collection and curation, (2) Scoring, ranking, and filtering, and (3) Suspect screening. Each step is first presented conceptually and then exemplified with use cases from the authors. The use of chemicals market data provides a solid basis for identification of true NERCs with confidence, and true and false negative findings can more confidently be distinguished. Chemicals market data should be provided to authorities and researchers so that early warning systems for NERCs can be installed and analytical blind spots identified and addressed.

Background: Growing evidence links chemical exposure to declining reproductive function in both humans and dogs. Our aim was to investigate the exposure of a wide range of per- and polyfluoroalkyl substances (PFAS) in dog serum and to investigate the association between PFAS exposure and endocrine parameters as well as semen quality.

Method: Semen samples (n=65) were collected from Bernese mountain dogs during 2020. Sperm motility was evaluated under a phase-contrast microscope (100×, 200×). Total sperm count was calculated using a Bürker chamber. Sperm morphology was evaluated using standard protocols in wet preparations of semen fixed in buffered formalin and stained with carbolfuchsin-eosin. Serum was analyzed using a combined targeted and suspect screening approach for quantitative analysis of 50 PFAS. Following extraction, instrumental analysis was performed using an ultra-high-performance liquid chromatograph coupled to a Q ExactiveOrbitrap mass spectrometer. PFAS concentrations were associated with semen quality and endocrine biomarkers using Least Absolute Shrinkage and Selection Operator (LASSO) regression.

Results: In all samples, PFOA, PFNA, PDFA, PFPeS, PFHxS and PFOS could be detected, although PFPeS levels were not above the quantification limit. The levels of the dominant congeners were on average (5^th-95^th percentile) PFOA 0.44 (0.05-1.3) ng/g serum, PFHxS 0.39 (0.05-0.96) ng/g serum, and PFOS 2.1 (0.35-6.4) ng/g serum. Fifteen suspect PFAS congeners were identified, where perflouro-4-ethylcyclohexanesulfonate (PFECHS), H-PFOA, H-PFNA, and H-PFDA were found in > 60% of the samples. Significant associations were found between PFBS motility (β = 136.56, p = 0.03) and free androgen index (β = 0,931, p=0.02).

Conclusion: For the first time, levels of a wide range of target and suspect PFAS are described in dog serum. PFAS levels in dog serum were similar to those in cats and humans, confirming that humans and pets, to a considerable extent, may share exposure to PFAS through the home environment. The study contributes to bridging the existing knowledge gap of exposure to endocrine disruptors and health effects in dogs, and thus to the research infrastructure bridging between species with the benefit of both humans and pets in a true One Health approach.

Endocrine disruptors (EDs) pose a serious threat to human health and the environment and require a comprehensive evaluation to be identified. The identification of EDs require a substantial amount of data, both in vitro and in vivo, due to the current scientific criteria in the EU. At the same time, the EU strives to reduce animal testing due to concerns regarding animal welfare and sensitivity of animal studies to adequately detect adverse effects relevant for human health. Perfluorooctane sulfonic acid (PFOS) is a persistent organic pollutant that is suspected to be an ED based on academic research, however it is not identified as such from a regulatory perspective. It has previously been shown that PFOS has the potential to cause neurotoxicity as well as affect the thyroid system, and it is known that specific thyroid hormone levels are critical in the development of the brain during. In this work, the aim was to evaluate a mechanism-based approach to identify ED properties of PFOS based on the Adverse Outcome Pathway (AOP) framework and using New Approach Methods (NAMs), by comparing this approach to an ED assessment based on the currently available guidance document. An AOP network (AOPN) was generated for the thyroid modality, and AOPs leading to developmental neurotoxicity (DNT) were identified. A literature search and screening process based on the AOPN, and systematic review methodology, was performed, followed by a rigorous Weight-of-Evidence (WoE) assessment. Evidence was mapped back onto the AOPN used for the literature search, to identify possible endocrine Modes-of-Action (MoAs) for PFOS and data gaps in the two assessments. It could be concluded that PFOS fulfils the criteria for ED classification in the standard ED assessment, but not in the mechanism-based assessment. The need for quantitative information, such as quantitative AOPs, for the mechanism-based approach is discussed. The possibility of a directly neurotoxic alternative MoA was also highlighted based on available in vitro data. Opportunities and challenges with implementing AOPs and NAMs into the regulatory assessment of EDs, and assessing hazard in the Next Generation Risk Assessment, is discussed. This case study exploring the mechanism-based approach to ED identification represents an important step toward more accurate and predictive assessment of EDs based on AOPs and NAMs, and to the Next Generation Risk Assessment (NGRA) concept.

Identification of stereo- and positional isomers detected with high-resolution mass spectrometry (HRMS) is often challenging due to near-identical fragmentation spectra (MS2), similar retention times, and collision cross-section values (CCS). Here we address this challenge on the example of hydroxylated polychlorinated biphenyls (OH-PCBs) with the aim to (1) distinguish between isomers of OH-PCBs using two-dimensional ion mobility spectrometry (2D-IMS) and (2) investigate the structure of the fragments of OH-PCBs and their fragmentation mechanisms by ion mobility spectrometry coupled to high-resolution mass spectrometry (IMS-HRMS). The MS2 spectra as well as CCS values of the deprotonated molecule and fragment ions were measured for 18 OH-PCBs using flow injections coupled to a cyclic IMS-HRMS. The MS2 spectra as well as the CCS values of the parent and fragment ions were similar between parent compound isomers; however, ion mobility separation of the fragment ions is hinting at the formation of isomeric fragments. Different parent compound isomers also yielded different numbers of isomeric fragment mobilogram peaks giving new insights into the fragmentation of these compounds and indicating new possibilities for identification. For spectral interpretation, Gibbs free energies and CCS values for the fragment ions of 4 '-OH-CB35, 4 '-OH-CB79, 2-OH-CB77 and 4-OH-CB107 were calculated and enabled assignment of structures to the isomeric mobilogram peaks of [M-H-HCl](-) fragments. Finally, further fragmentation of the isomeric fragments revealed different fragmentation pathways depending on the isomeric fragment ions.

The number of chemicals in the anthroposphere is increasing and some of them end up in humans. A literature search was made to assess which anthropogenic organic contaminants (OCs) that have been analysed in blood from the general population. The reviewed articles were used to create a database of studies [human blood database (HBDB), containing 559 OCs] reporting blood analyses made worldwide. All studies analysing blood from the Swedish population were compiled into a second database [Swedish exposure database (SEDB), containing 166 OCs] listing blood concentrations of OCs. Data from the SEDB showed decreasing levels of regulated chemicals in blood over time, indicating that regulation had made an impact. The Hazard Index (HI) approach was used as a qualitative mixture risk assessment of the OCs with established human biomonitoring guidance values (HBM-GVs) and blood levels in the SEDB. Nine HBM-GVs were found and the HI of the corresponding OCs/groups of OCs showed that a risk of adverse effects in the general population could not be excluded, which is a cause for concern considering that only a fraction of the analysed OCs in the SEDB were included. This study presents the OCs identified in human blood and concentration time trends. The study highlights the lack of HBM-GVs needed for mixture risk assessments to assess the combined risk of chemical exposure to the general population. 

Indoor environmental pollutants are a threat to human health. In the current study, we analysed 25 per- and polyfluoroalkyl substances (PFASs) in seven different size fraction of house dust including the two relevant for exposure via ingestion and inhalation. The highest PFAS concentration is found in the inhalable particulate fraction which is explained by the increased surface area as the particulate's sizes decrease. The estimated daily intake (EDI) of the individual PFAS and exposure pathways were calculated for children and adults. In addition, the total EDI for PFOA and its precursors was estimated. The polyfluoroalkyl phosphoric acid diesters (diPAP), followed by PFOA and PFHxA fluortelomer, showed the highest concentrations of PFAS analysed. The cumulative EDI of PFAS for children was 3.0 ng/kg bw per day, a worst-case scenario, which is 17 times higher than the calculated EDI for adults. For children, ingestion of dust was found to result in 800 times higher PFOA exposure than via inhalation. The contribution from PFOA precursors corresponded to only 1% of the EDI from dust indicating PFOA as the main source of exposure. The EDI's of PFOA and PFOS from dust were lower than the calculated EDI's from food ingestion reported by the Swedish Food Agency. Our data indicate that the EDI for the sum of four PFASs: PFOA, PFNA, PFHxS and PFOS from dust intake alone is close to the established tolerable weakly intake of 4.4 ng/kg bw in children, set by European Food Safety Authority (EFSA) in 2020. The combined EDI levels PFOA and PFOS from both dust and food exceeded the EFSA TWI for both children and adults. This study demonstrates that dust is a relevant exposure pathway for PFAS intake and that analysis of relevant particle size fractions is important for evaluation of dust as an exposure pathway.

Hydroxylated PCBs are an important class of metabolites of the widely distributed environmental contaminants polychlorinated biphenyls (PCBs). However, the absence of authentic standards is often a limitation when subject to detection, identification, and quantification. Recently, new strategies to quantify compounds detected with non-targeted LC/ESI/HRMS based on predicted ionization efficiency values have emerged. Here, we evaluate the impact of chemical space coverage and sample matrix on the accuracy of ionization efficiency-based quantification. We show that extending the chemical space of interest is crucial in improving the performance of quantification. Therefore, we extend the ionization efficiency-based quantification approach to hydroxylated PCBs in serum samples with a retraining approach that involves 14 OH-PCBs and validate it with an additional four OH-PCBs. The predicted and measured ionization efficiency values of the OH-PCBs agreed within the mean error of 2.1 × and enabled quantification with the mean error of 4.4 × or better. We observed that the error mostly arose from the ionization efficiency predictions and the impact of matrix effects was of less importance, varying from 37 to 165%. The results show that there is potential for predictive machine learning models for quantification even in very complex matrices such as serum. Further, retraining the already developed models provides a timely and cost-effective solution for extending the chemical space of the application area.