The profile of steroid hormones in human fetal and adult ovaries.

BACKGROUND: Reproduction in women is at risk due to exposure to chemicals that can disrupt the endocrine system during different windows of sensitivity throughout life. Steroid hormone levels are fundamental for the normal development and function of the human reproductive system, including the ovary. This study aims to elucidate steroidogenesis at different life-stages in human ovaries. METHODS: We have developed a sensitive and specific LC-MS/MS method for 21 important steroid hormones and measured them at different life stages: in media from cultures of human fetal ovaries collected from elective terminations of normally progressing pregnancy and in media from adult ovaries from Caesarean section patients, and follicular fluid from women undergoing infertility treatment. Statistically significant differences in steroid hormone levels and their ratios were calculated with parametric tests. Principal component analysis (PCA) was applied to explore clustering of the ovarian-derived steroidogenic profiles. RESULTS: Comparison of the 21 steroid hormones revealed clear differences between the various ovarian-derived steroid profiles. Interestingly, we found biosynthesis of both canonical and "backdoor" pathway steroid hormones and corticosteroids in first and second trimester fetal and adult ovarian tissue cultures. 17α-estradiol, a less potent naturally occurring isomer of 17ß-estradiol, was detected only in follicular fluid. PCA of the ovarian-derived profiles revealed clusters from: adult ovarian tissue cultures with relatively high levels of androgens; first trimester and second trimester fetal ovarian tissue cultures with relatively low estrogen levels; follicular fluid with the lowest androgens, but highest corticosteroid, progestogen and estradiol levels. Furthermore, ratios of specific steroid hormones showed higher estradiol/ testosterone and estrone/androstenedione (indicating higher CYP19A1 activity, p < 0.01) and higher 17-hydroxyprogesterone/progesterone and dehydroepiandrosterone /androstenedione (indicating higher CYP17A1 activity, p < 0.01) in fetal compared to adult ovarian tissue cultures. CONCLUSIONS: Human ovaries demonstrate de novo synthesis of non-canonical and "backdoor" pathway steroid hormones and corticosteroids. Elucidating the steroid profiles in human ovaries improves our understanding of physiological, life-stage dependent, steroidogenic capacity of ovaries and will inform mechanistic studies to identify endocrine disrupting chemicals that affect female reproduction.

Exploring the Relationship Among Lipid Profile Changes, Growth, and Reproduction in Folsomia candida Exposed to Teflubenzuron Over Time.

The integration of untargeted lipidomics approaches in ecotoxicology has emerged as a strategy to enhance the comprehensiveness of environmental risk assessment. Although current toxicity tests with soil microarthropods focus on species performance, that is, growth, reproduction, and survival, understanding the mechanisms of toxicity across all levels of biological organization, from molecule to community is essential for informed decision-making. Our study focused on the impacts of sublethal concentrations of the insecticide teflubenzuron on the springtail Folsomia candida. Untargeted lipidomics was applied to link changes in growth, reproduction, and the overall stress response with lipid profile changes over various exposure durations. The accumulation of teflubenzuron in organisms exposed to the highest test concentration (0.035 mg a.s. kg-1 soil dry wt) significantly impacted reproductive output without compromising growth. The results suggested a resource allocation shift from reproduction to size maintenance. This hypothesis was supported by lipid shifts on day 7, at which point reductions in triacylglycerol and diacylglycerol content corresponded with decreased offspring production on day 21. The hypermetabolism of fatty acids and N-acylethanolamines on days 2 and 7 of exposure indicated oxidative stress and inflammation in the animals in response to teflubenzuron bioaccumulation, as measured using high-performance liquid chromatography-tandem mass spectrometry. Overall, the changes in lipid profiles in comparison with phenotypic adverse outcomes highlight the potential of lipid analysis as an early-warning tool for reproductive disturbances caused by pesticides in F. candida. Environ Toxicol Chem 2024;43:1149-1160. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

A comprehensive untargeted metabolomics study in zebrafish embryos exposed to perfluorohexane sulfonate (PFHxS).

Perfluorohexane sulfonate (PFHxS) is one of the short-chain perfluoroalkyl substances (PFASs), and frequently detected in the environment, humans, and wildlife, but a detailed mechanism of toxicity has been not studied yet. In this study, a comprehensive set of polar metabolites was determined in i) the developing zebrafish embryo (4, 24, 48, 72, and 120 h post fertilization (hpf)), and ii) in the developing zebrafish after exposure to four concentrations of PFHxS (0.3, 1, 3, and 10 µM) from 24to 120 hpf. The temporal (developmental stages) distribution of individual metabolites (541 metabolites) in zebrafish provided comprehensive information about the biological roles of various metabolites in developing vertebrates such as genetic processes, energy metabolism, protein metabolism, and glycerophospholipid metabolism. PFHxS in zebrafish embryo showed time- and concentration- dependent bioaccumulation, and no baseline toxicity was expected at the test concentrations. However, effects on many metabolites were already observed at the lowest tested concentration (0.3 µM), and these effects were more pronounced at later stages of developmental (72 and 120 hpf). In addition to oxidative stress, the effects of PFHxS on zebrafish embryos were related to the disruption of the fatty acid oxidation (FAO), sugar metabolism, and other metabolic pathways. This study gave new and comprehensive information on the underlying mechanism of the toxicity of PFHxS.

A Rapid Screening Method for the Detection of Additives in Electronics and Plastic Consumer Products Using AP-MALDI-qTOF-MS.

A novel method was developed and optimized for the fast-screening analysis of additives in electronics and plastic consumer products using atmospheric pressure matrix-assisted laser desorption ionization (AP-MALDI) coupled with a high-resolution quadrupole time-of-flight (qTOF) mass spectrometer (MS). To simplify sample preparation and increase sample throughput, an innovative 48 well graphene nanoplatelets (GNP) doped AP-MALDI target plate was developed. The GNP incorporated in the target plate fulfilled the role of the MALDI matrix and, therefore, sample extracts could be directly transferred to the AP-MALDI 48 well target plate and analyzed without a subsequent matrix addition. The homogeneously dispersed and immobilized GNP target plates also provided increased signal intensity and reproducibility. Furthermore, analytical standards of various plastic additives and plastic products with known concentrations of additives were studied to assess the AP-MALDI ionization mechanisms and method capability. The analysis time was 15 s per measurement using an automated sequence. The GNP-doped target plates exhibited high desorption/ionization of low molecular weight molecules (<1000 Da) and can be used in both positive and negative ionization modes. The AP-MALDI-qTOF-MS method was applied to screen for additives in various electronics and plastic consumer products. Suspect screening was performed using a database containing 1366 compounds. A total of 56 additives including antioxidants, flame retardants, plasticizers, UV-stabilizers, and UV-filters were identified (confidence level 4). Identification of certain plastic additives in plastic children's toys may indicate that they are recycled from waste electronic and electronic equipment (WEEE).

Identification of biomarkers and outcomes of endocrine disruption in human ovarian cortex using In Vitro Models.

Endocrine disrupting chemicals (EDCs) are raising concerns about adverse effects on fertility in women. However, there is a lack of information regarding mechanisms and effects in humans. Our study aims to identify mechanisms of endocrine disruption using two EDCs, diethylstilbestrol (DES) and ketoconazole (KTZ)1. Human ovarian cortical tissue obtained from Caesarean section patients was exposed to 10-9 M - 10-5 M KTZ and 10-10 M - 10-6 M DES in vitro for 6 days. Follicle survival and growth were studied via histology analysis and liquid-chromatography-mass spectrometry-based steroid quantification. RNA-sequencing was performed on COV434, KGN, and primary ovarian cells that were exposed for 24 h. Significantly lower unilaminar follicle densities were observed in DES 10-10 M group, whereas low KTZ exposure reduced secondary follicle density. KTZ 10-5 M reduced levels of pregnenolone and progesterone. RNA-sequencing revealed that 445 and 233 differentially expressed genes (false discovery rate < 0.1) altogether in DES and KTZ exposed groups. Gene set variation analysis showed that both chemicals modulated pathways that are important for folliculogenesis and steroidogenesis. We selected stearoyl-CoA desaturase (SCD) and 7-dehydrocholesterol reductase (DHCR7) for further validation. Up-regulation of both genes in response to KTZ was confirmed by qPCR and in situ RNA hybridization. Further validation with immunofluorescence focused on the expression of SCD in growing follicles in exposed ovarian tissue. In conclusion, SCD may serve as a potential novel human-relevant biomarker of EDC exposure and effects on ovaries.

Metabolite alterations in zebrafish embryos exposed to hydroxylated polybrominated diphenyl ethers.

Hydroxylated polybrominated diphenyl ethers (OH-PBDEs) are formed by metabolism from the flame retardants polybrominated diphenyl ethers (PBDEs). In the aquatic environment, they are also produced naturally. OH-PBDEs are known for their potential to disrupt energy metabolism, the endocrine system, and the nervous system. This is the first study focusing on the effects of OH-PBDEs at the metabolite level in vivo. The aim of the current study was to investigate the metabolic effects of exposure to OH-PBDEs using metabolomics, and to identify potential biomarker(s) for energy disruption of OH-PBDEs. Zebrafish (Danio rerio) embryos were exposed to two different concentrations of 6-OH-BDE47 and 6-OH-BDE85 and a mixture of these two compounds. In total, 342 metabolites were annotated and 79 metabolites were affected in at least one exposure. Several affected metabolites, e.g. succinic acid, glutamic acid, glutamine, tyrosine, tryptophan, adenine, and several fatty acids, could be connected to known toxic mechanisms of OH-PBDEs. Several phospholipids were strongly up-regulated with up to a six-fold increase after exposure to 6-OH-BDE47, a scarcely described effect of OH-PBDEs. Based on the observed metabolic effects, a possible connection between disruption of the energy metabolism, neurotoxicity and potential immunotoxicity of OH-PBDEs was suggested. Single compound exposures to 6-OH-BDE47 and 6-OH-BDE85 showed little overlap in the affected metabolites. This shows that compounds of similar chemical structure can induce different metabolic effects, possibly relating to their different toxic mechanisms. There were inter-concentration differences in the metabolic profiles, indicating that the metabolic effects were concentration dependent. After exposure to the mixture of 6-OH-BDE47 and 6-OH-BDE85, a new metabolic profile distinct from the profiles obtained from the single compounds was observed. Succinic acid was up-regulated at the highest, but still environmentally relevant, concentration of 6-OH-BDE47, 6-OH-BDE85, and the mixture. Therefore, succinic acid is suggested as a potential biomarker for energy disruption of OH-PBDEs.

Fate-directed risk assessment of chemical mixtures: a case study for cedarwood essential oil.

The environmental risk assessment of UVCBs (i.e., substances of unknown or variable composition, complex reaction products, or biological materials) is challenging due to their inherent complexity. A particular problem is that UVCBs can contain constituents with unidentified chemical structures and/or have variable composition of constituents from batch to batch. Moreover, the composition of a UVCB in the environment is not the same as that of the UVCB in a product, meaning that a risk assessment based on environmental exposure to the UVCB in a product does not represent the actual environmental risk. Here we propose an in silico fate-directed risk assessment framework for UVCBs using cedarwood oil as a case study. The framework uses Monte Carlo simulations and the mass-balance models SimpleTreat and RAIDAR to provide quantitative information on whether unidentified constituents within the physical-chemical property space of a UVCB can be the decisive factor for the environmental risk of the entire UVCB. Thereby the framework provides a robust decision tool to evaluate if a UVCB risk assessment requires additional tests or if the data on known constituents is representative for the risk of the entire UVCB. In the case of cedarwood oil, it could be shown that a risk assessment based on the known constituents (representing around 70% of the overall UVCB by weight) is representative for the environmental risk of the entire UVCB - reducing the need for additional testing and test animals.

Fate of Per- and Polyfluoroalkyl Substances from Durable Water-Repellent Clothing during Use.

To make outdoor clothing water- or dirt-repellent, durable water-repellent (DWR) coatings based on side-chain fluorinated polymers (SFPs) are used. During use of outdoor clothing, per- and polyfluoroalkyl substances (PFASs) can be emitted from the DWR to the environment. In this study, the effects of aging, washing, and tumble drying on the concentration of extractable PFASs in the DWR of perfluorohexane-based short-chain SFPs (FC-6 chemistry) and of perfluorooctane-based long-chain SFPs (FC-8 chemistry) were assessed. For this purpose, polyamide (PA) and polyester (PES) fabrics were coated with FC-6- and FC-8-based DWRs. Results show that aging of the coated fabrics causes an increase in concentration and formation of perfluoroalkyl acids (PFAAs). The effect of aging on the volatile PFASs depends on the type of fabric. Washing causes a decrease in PFAA concentrations, and in general, volatile PFASs are partly washed out of the textiles. However, washing can also increase the extractable concentration of volatile PFASs in the fabrics. This effect becomes stronger by a combination of aging and washing. Tumble drying does not affect the PFAS concentrations in textiles. In conclusion, aging and washing of fabrics coated with the DWR based on SFPs release PFASs to the environment.

Migration of hazardous contaminants from WEEE contaminated polymeric toy material by mouthing.

This study evaluated the migration of brominated flame retardants (BFRs), phosphate flame retardants (PFRs), bisphenols (BPA, BPF), and phthalate ester-based plasticizers from recycled polymeric toy material, containing waste electrical and electronic equipment (WEEE), in artificial saliva simulating 1 h of mouthing. In total 12 parts of 9 different toys were tested in triplicate after confirming WEEE specific contamination. Up to 11 contaminants were detected in saliva from one toy sample. The highest migration rate up to 128 ng/(cm2 x h) was found for BPA followed by bis(2-ethylhexyl) phthalate (DEHP) and diisobutyl phthalate (DIBP) with migration rates up to 25.5 and 8.27 ng/(cm2 x h), respectively. In addition to DecaBDE, which was detected in 3 saliva samples at migration rates between 0.09 and 0.31 ng/(cm2 x h), the decaBDE replacements 2,4,6-tris(2,4,6-tribromophenoxy)-1,3,5-triazine (TTBP-TAZ), decabromodiphenyl ethane (DBDPE), resorcinol bis(diphenyl phosphate) (RDP), and hexabromocyclododecane (HBCDD) were detected as well with comparable migration rates. 2,4,6-tribromphenol (246-TBP) reached migration rates up to 1.15 ng/(cm2 x h) in correspondence to the presence of TTBP-TAZ. Tetrabromobisphenol A (TBBPA), BPA, 246-TBP, DEHP, DIBP and triphenyl phosphate (TPHP) were predominantly observed in saliva with a detection frequency between 50 and 75%. Daily intake (DI) values were calculated for relevant analytes and compared to tolerable daily intake (TDI) values. The highest DI values of 72.4, 14.3, 5.74, 2.28 and 2.09 ng/(kg BW x day), were obtained for BPA, DEHP, DIBP, TBBPA, and TPHP, respectively. None of them exceed the TDI value or respective reference dose (RfD).

Using comprehensive lipid profiling to study effects of PFHxS during different stages of early zebrafish development.

PFHxS (Perfluorohexane sulfonic acid) is one of the short-chain perfluoroalkyl substances (PFASs) which are widely used in many industrial and consumer applications. However, limited information is available on the molecular mechanism of PFHxS toxicity (e.g. lipid metabolism). This study provides in-depth information on the lipid regulation of zebrafish embryos with and without PFHxS exposure. Lipid changes throughout zebrafish development (4 to 120 h post fertilization (hpf)) were closely associated with lipid species and lipid composition (fatty acyl chains). A comprehensive lipid analysis of four different PFHxS exposures (0, 0.3, 1, 3, and 10 µM) at different zebrafish developmental stages (24, 48, 72, and 120 hpf) was performed. Data on exposure concentration, lipids, and developmental stage showed that all PFHxS concentrations dysregulated the lipid metabolism and these were developmental-dependent. The pattern of significantly changed lipids revealed that PFHxS caused effects related to oxidative stress, inflammation, and impaired fatty acid ß-oxidation. Oxidative stress and inflammation caused the remodeling of glycerophospholipid (phosphatidylcholine (PC) and phosphatidylethanolamine (PE)), with increased incorporation of omega-3 PUFA and a decreased incorporation of omega-6 PUFA.

Chlorinated paraffins and tris (1-chloro-2-propyl) phosphate in spray polyurethane foams - A source for indoor exposure?

In this study, we investigated chemical additives present in new and used spray polyurethane foams (SPFs) and assessed the dermal transfer through direct contact. This first study shows that cured do-it-yourself spray one-component SPFs (OCFs) often contain chlorinated paraffins (C14-C37), and tris (1-chloro-2-propyl) phosphate (TCIPP), ranging 0.2-50%, and 0.9-30% w/w, respectively. Six OCFs contained CP levels ranging 22-50% w/w, whereas nine OCFs used for similar applications only contained CP levels ranging 2-17% w/w. It is unclear if the combination CPs/TCIPP is meant to improve the flame retardancy of products, and could suggest an unnecessary use of high CPs/TCIPP concentrations in OCFs. The two-component SPFs (TCFs) contained only TCIPP with levels ranging from 7.0% to 9.0%. The CPs and TCIPP were easily transferred from cured OCFs to the hands. Levels up to 590 µg per hand for CPs and up to 2.7 µg per hand for TCIPP were found. After end-of-life, it is challenging to recycle used SPFs. They may, therefore, end up at landfills where the TCIPP/CPs may leach into the environment. Therefore, further investigation is needed to assess potential exposure risks associated with general and occupational use, and the impact of landfill leaching on the environment.

Progress towards an OECD reporting framework for transcriptomics and metabolomics in regulatory toxicology.

Omics methodologies are widely used in toxicological research to understand modes and mechanisms of toxicity. Increasingly, these methodologies are being applied to questions of regulatory interest such as molecular point-of-departure derivation and chemical grouping/read-across. Despite its value, widespread regulatory acceptance of omics data has not yet occurred. Barriers to the routine application of omics data in regulatory decision making have been: 1) lack of transparency for data processing methods used to convert raw data into an interpretable list of observations; and 2) lack of standardization in reporting to ensure that omics data, associated metadata and the methodologies used to generate results are available for review by stakeholders, including regulators. Thus, in 2017, the Organisation for Economic Co-operation and Development (OECD) Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) launched a project to develop guidance for the reporting of omics data aimed at fostering further regulatory use. Here, we report on the ongoing development of the first formal reporting framework describing the processing and analysis of both transcriptomic and metabolomic data for regulatory toxicology. We introduce the modular structure, content, harmonization and strategy for trialling this reporting framework prior to its publication by the OECD.

Cross platform solutions to improve the zebrafish polar metabolome coverage using LC-QTOF MS: Optimization of separation mechanisms, solvent additives, and resuspension solvents.

Untargeted metabolomics has been widely used for studies with zebrafish embryos. Until now, the number of analytical approaches to determine metabolites in zebrafish is limited, and there is a lack of consensus on the best platforms for comprehensive metabolomics analysis of zebrafish embryos. In addition, the capacity of these methods to detect metabolites is unsatisfactory and the confidence level for identifying compounds is relatively low. To improve the metabolome coverage, we mainly focused on the optimization of separation mechanisms, mobile phase additives, and resuspension solvents based on liquid chromatography (LC) coupling to high-resolution mass spectrometry (HRMS) techniques. Moreover, the procedures for optimizing methods were assessed when taking metabolite profiles in both positive and negative ionization modes into account. Four LC columns were studied: C18, T3, PFP, and HILIC. In positive ionization mode, it was strongly recommended to employ the HILIC approach operated at the neutral condition, which led to the presence of more than 4700 features and the annotation of 151 metabolites, mainly zwitterionic and basic compounds, in comparison to reverse phase (RP)-based methods with less than 1000 features. In negative ionization mode, the PFP column operated at 0.02% acetic acid showed the best performance in terms of metabolite coverage: 3100 metabolic features were detected and 218 metabolites were annotated in zebrafish embryos. Metabolite profiles mainly contained acidic and zwitterionic compounds. HILIC-based platforms were complementary to RP columns when analyzing highly polar metabolites. Additionally, it was preferable to reconstitute zebrafish extracts in 100% water for analysis of metabolites on RP columns, with a 20-30% increase in the number of identified metabolites compared to a 50% water in methanol solution. However, water/methanol (1:9, v/v), as resuspension solution, was advantageous over water/methanol (1:1, v/v) for HILIC analysis showing an 8-15% increase in detected metabolites. In total 336 polar metabolites were annotated by the combination of the optimized HILIC (positive) and PFP (negative) approaches. The largest metabolome coverage of polar metabolites in zebrafish embryos was obtained when three approaches were combined (negative PFP and HILIC, and HILIC positive) resulting in more than 420 annotated compounds.

A deep dive into fat: Investigating blubber lipidomic fingerprint of killer whales and humpback whales in northern Norway.

In cetaceans, blubber is the primary and largest lipid body reservoir. Our current understanding about lipid stores and uses in cetaceans is still limited, and most studies only focused on a single narrow snapshot of the lipidome. We documented an extended lipidomic fingerprint in two cetacean species present in northern Norway during wintertime. We were able to detect 817 molecular lipid species in blubber of killer whales (Orcinus orca) and humpback whales (Megaptera novaeangliae). The profiles were largely dominated by triradylglycerols in both species and, to a lesser extent, by other constituents including glycerophosphocholines, phosphosphingolipids, glycerophosphoethanolamines, and diradylglycerols. Through a unique combination of traditional statistical approaches, together with a novel bioinformatic tool (LION/web), we showed contrasting fingerprint composition between species. The higher content of triradylglycerols in humpback whales is necessary to fuel their upcoming half a year fasting and energy-demanding migration between feeding and breeding grounds. In adipocytes, we assume that the intense feeding rate of humpback whales prior to migration translates into an important accumulation of triacylglycerol content in lipid droplets. Upstream, the endoplasmic reticulum is operating at full capacity to supply acute lipid storage, consistent with the reported enrichment of glycerophosphocholines in humpback whales, major components of the endoplasmic reticulum. There was also an enrichment of membrane components, which translates into higher sphingolipid content in the lipidome of killer whales, potentially as a structural adaptation for their higher hydrodynamic performance. Finally, the presence of both lipid-enriched and lipid-depleted individuals within the killer whale population in Norway suggests dietary specialization, consistent with significant differences in δ15N and δ13C isotopic ratios in skin between the two groups, with higher values and a wider niche for the lipid-enriched individuals. Results suggest the lipid-depleted killer whales were herring specialists, while the lipid-enriched individuals might feed on both herrings and seals.

Combining High-Resolution Gas Chromatographic Continuous Fraction Collection with Nuclear Magnetic Resonance Spectroscopy: Possibilities of Analyzing a Whole GC Chromatogram.

This study presents, for the first time, the successful application of analyzing a whole gas chromatography (GC) chromatogram by nuclear magnetic resonance (NMR) spectroscopy using a continuous repeatable and stable (n = 280) high-resolution (HR) GC fractionation platform with a 96-well plate. Typically with GC- or liquid chromatography-mass spectrometry analysis, (isomer) standards and/or additional NMR analysis are needed to confirm the identification and/or structure of the analyte of interest. In the case of complex substances (e.g., UVCBs), isomer standards are often unavailable and NMR spectra too complex to achieve this. This proof of concept study shows that a HR GC fractionation collection platform was successfully applied to separate, purify, and enrich isomers in complex substances from a whole GC chromatogram, which would facilitate NMR analysis. As a model substance, a chlorinated paraffin (CP) mixture (>8,000 isomers) was chosen. NMR spectra were obtained from all 96 collected fractions, which provides important information for unravelling their full structure. As a proof of concept, a spectral interpretation of a few NMR spectra was made to assign sub-structures. More research is ongoing for the full characterization of CP isomers using multivariate statistical analysis. For the first time, up to only a few CP isomers per fraction were isolated from a highly complex mixture. These may be further purified and certified as standards, which are urgently needed, and can also be used for persistency, bioaccumulation, or toxicity studies.

Analysis of recycled rubber: Development of an analytical method and determination of polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds in rubber matrices.

Recycled crumb rubber (CR) is rich in compounds with unrecognized toxic potency; this study aims at the development of an analytical method that would allow identification and quantification of a very wide range of organic compounds extractable from the complex rubber matrix. The analytical set-up includes target analysis of polycyclic aromatic hydrocarbons (PAHs) and methyl-PAHs and suspect screening of raw extracts to tentatively identify primary organic compounds present, but not included in the standard target analysis of recycled rubber, followed by analytical method development and target analysis of identified compounds. Analyzed samples included weathered and new CR originating from football turf granulates, rubber mats, and end-of-life car tires (ELTs). The developed analytical method involves sonication extraction, followed by solid phase extraction (SPE) fractionation that enables simple and efficient separation of analytes with broad polarity scale. The application of SPE fractionation resolves coelution problems and simplifies the chromatograms. This analytical approach allowed to identify and quantify 46 sample specific compounds, including several heterocyclic PAHs like 2-methylthiobenzothiazole, benzonapthothiophenes, benzonaphthofuranes and aromatic amines like diphenylamine and N-phenyl-2-naphthylamine, which to our knowledge were not determined before. The PAHs concentrations determined in CR tiles purchased in Dutch and Spanish shops exceed the EU limits for articles marketed for use by the public. Furthermore, sets of methylated PAHs, dibenzothiazoles and aromatic amines were identified and quantified, and several other compounds were tentatively identified. The obtained results stress the need for expanding the list of target compounds analyzed in CR and the need for longitudinal studies on weathering processes taking place in CR.

In vitro biotransformation and evaluation of potential transformation products of chlorinated paraffins by high resolution accurate mass spectrometry.

Chlorinated paraffins (CPs) are high production chemicals, which leads to their ubiquitous presence in the environment. To date, few studies have measured CPs in humans and typically at relatively low concentrations, despite indications that exposure may be high compared to various persistent organic pollutants. The aim of this study is to investigate the in vitro biotransformation of CPs by human liver fractions. We determined the changes of the CP concentrations after the enzymatic transformation with human liver microsomes using a two-tiered in vitro approach. CP concentrations decreased with human liver microsomes, with the decreases of 33-94% after incubating with different groups of enzymes for 2 h. The profiles of CP rapidly shifted after the incubation with human liver microsomes. In addition, the concentrations of CPs and the biotransformation products were tentatively measured using high-resolution mass spectrometric analysis, including very short CP (carbon chain length <10), alcohols, ketones, and carboxylic acids. C‒C bond cleavage is a potential transformation pathway for CPs, and ketones are potential products of CP biotransformation, especially for long-chain CPs (C>17). The ketone products may be investigated as CP exposure biomarker in biomonitoring studies.

Risk assessment of chlorinated paraffins in feed and food.

The European Commission asked EFSA for a scientific opinion on the risks for animal and human health related to the presence of chlorinated paraffins in feed and food. The data for experimental animals were reviewed and the CONTAM Panel identified the liver, kidney and thyroid as the target organs for the SCCP and MCCP mixtures tested in repeated dose toxicity studies. Decreased pup survival and subcutaneous haematoma/haemorrhage were also identified as critical effects for an MCCP mixture. For the LCCP mixtures tested, the liver was identified as the target organ. The Panel selected as reference points a BMDL 10 of 2.3 mg/kg bw per day for increased incidence of nephritis in male rats, and of 36 mg/kg bw per day for increased relative kidney weights in male and female rats for SCCPs and MCCPs, respectively. For LCCPs, a reference point relevant for humans could not be identified. Due to the limitations in the toxicokinetic and toxicological database, the Panel concluded that derivation of a health-based guidance value was not appropriate. Only limited data on the occurrence of SCCPs and MCCPs in some fish species were submitted to EFSA. No data were submitted for LCCPs. Thus, a robust exposure assessment and consequently a complete risk characterisation could not be performed. A preliminary risk characterisation based only on the consumption of fish was performed, and the calculated margins of exposure suggested no health concern for this limited scenario. The Panel noted that dietary exposure will be higher due to the contribution of CPs from other foods. The Panel was not able to identify reference points for farm animals, horses and companion animals. No occurrence data for feed were submitted to EFSA. Therefore, no risk characterisation could be performed for any of these animal species.

Improving the Environmental Risk Assessment of Substances of Unknown or Variable Composition, Complex Reaction Products, or Biological Materials.

Substances of unknown or variable composition, complex reaction products, or biological materials (UVCBs) pose unique risk assessment challenges to regulators and to product registrants. These substances can contain many constituents, sometimes partially unknown and/or variable, depending on fluctuations in their source material and/or manufacturing process. International regulatory agencies have highlighted the difficulties in characterizing UVCBs and assessing their toxicity and environmental fate. Several industrial sectors have attempted to address these issues by developing frameworks and characterization methods. Based on the output of a 2016 workshop, this critical review examines current practices for UVCB risk assessment and reveals a need for a multipronged and transparent approach integrating whole-substance and constituent-based information. In silico tools or empirical measurements can provide information on discrete and/or blocks of UVCB constituents with similar hazard properties. Read-across and/or whole-substance toxicity and fate testing using adapted emerging methods can provide whole-substance information. Continued collaboration of stakeholders representing government, industry, and academia will facilitate the development of practical testing strategies and guidelines for addressing regulatory requirements for UVCBs. Environ Toxicol Chem 2020;39:2097-2108. © 2020 Health and Environmental Sciences Institute. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.