In vitro and in vivo investigation of a thyroid hormone system-specific interaction with triazoles.

Alterations in thyroid hormones (TH) and thyroid-stimulating hormone levels are frequently found following exposure to chemicals of concern. Dysregulation of TH levels can severely perturb physiological growth, metabolism, differentiation, homeostasis in the adult and developmental processes in utero. A frequently identified mode of action for this interaction is the induction of hepatic detoxification mechanisms (e.g. SULTs and UGTs), which lead to TH conjugation and elimination and therefore interfere with hormonal homeostasis, fulfilling the endocrine disruptors (EDs) definition. A short-term study in rats with dietary exposure to cyproconazole, epoxiconazole and prochloraz was conducted and hepatocyte hypertrophy, hepatic UGT activity and Phase 1/2 gene expression inductions were observed together with changes in TH levels and thyroid follicular hypertrophy and hyperplasia. To test for specific interaction with the thyroid hormone system, in vitro assays were conducted covering thyroidal I-uptake (NIS), TH transmembranal transport via MCT8 and thyroid peroxidase (TPO) function. Assays for iodothyronine deiodinases (DIO1-DIO3) and iodotyrosine deiodinase (DEHAL1) were included, and from the animal experiment, Dio1 and Dehal1 activities were measured in kidney and liver as relevant local indicators and endpoints. The fungicides did not affect any TH-specific KEs, in vitro and in vivo, thereby suggesting hepatic conjugation as the dominant MoA.

Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),1 Lichtenstein et al. (2020),2 and Knebel et al. (2019).3.

Basic concepts of mixture toxicity and relevance for risk evaluation and regulation.

Exposure to multiple substances is a challenge for risk evaluation. Currently, there is an ongoing debate if generic "mixture assessment/allocation factors" (MAF) should be introduced to increase public health protection. Here, we explore concepts of mixture toxicity and the potential influence of mixture regulation concepts for human health protection. Based on this analysis, we provide recommendations for research and risk assessment. One of the concepts of mixture toxicity is additivity. Substances may act additively by affecting the same molecular mechanism within a common target cell, for example, dioxin-like substances. In a second concept, an "enhancer substance" may act by increasing the target site concentration and aggravating the adverse effect of a "driver substance". For both concepts, adequate risk management of individual substances can reliably prevent adverse effects to humans. Furthermore, we discuss the hypothesis that the large number of substances to which humans are exposed at very low and individually safe doses may interact to cause adverse effects. This commentary identifies knowledge gaps, such as the lack of a comprehensive overview of substances regulated under different silos, including food, environmentally and occupationally relevant substances, the absence of reliable human exposure data and the missing accessibility of ratios of current human exposure to threshold values, which are considered safe for individual substances. Moreover, a comprehensive overview of the molecular mechanisms and most susceptible target cells is required. We conclude that, currently, there is no scientific evidence supporting the need for a generic MAF. Rather, we recommend taking more specific measures, which focus on compounds with relatively small ratios between human exposure and doses, at which adverse effects can be expected.

New approach methodologies in human regulatory toxicology - Not if, but how and when!

The predominantly animal-centric approach of chemical safety assessment has increasingly come under pressure. Society is questioning overall performance, sustainability, continued relevance for human health risk assessment and ethics of this system, demanding a change of paradigm. At the same time, the scientific toolbox used for risk assessment is continuously enriched by the development of "New Approach Methodologies" (NAMs). While this term does not define the age or the state of readiness of the innovation, it covers a wide range of methods, including quantitative structure-activity relationship (QSAR) predictions, high-throughput screening (HTS) bioassays, omics applications, cell cultures, organoids, microphysiological systems (MPS), machine learning models and artificial intelligence (AI). In addition to promising faster and more efficient toxicity testing, NAMs have the potential to fundamentally transform today's regulatory work by allowing more human-relevant decision-making in terms of both hazard and exposure assessment. Yet, several obstacles hamper a broader application of NAMs in current regulatory risk assessment. Constraints in addressing repeated-dose toxicity, with particular reference to the chronic toxicity, and hesitance from relevant stakeholders, are major challenges for the implementation of NAMs in a broader context. Moreover, issues regarding predictivity, reproducibility and quantification need to be addressed and regulatory and legislative frameworks need to be adapted to NAMs. The conceptual perspective presented here has its focus on hazard assessment and is grounded on the main findings and conclusions from a symposium and workshop held in Berlin in November 2021. It intends to provide further insights into how NAMs can be gradually integrated into chemical risk assessment aimed at protection of human health, until eventually the current paradigm is replaced by an animal-free "Next Generation Risk Assessment" (NGRA).

Genotoxicity assessment: opportunities, challenges and perspectives for quantitative evaluations of dose-response data.

Genotoxicity data are mainly interpreted in a qualitative way, which typically results in a binary classification of chemical entities. For more than a decade, there has been a discussion about the need for a paradigm shift in this regard. Here, we review current opportunities, challenges and perspectives for a more quantitative approach to genotoxicity assessment. Currently discussed opportunities mainly include the determination of a reference point (e.g., a benchmark dose) from genetic toxicity dose-response data, followed by calculation of a margin of exposure (MOE) or derivation of a health-based guidance value (HBGV). In addition to new opportunities, major challenges emerge with the quantitative interpretation of genotoxicity data. These are mainly rooted in the limited capability of standard in vivo genotoxicity testing methods to detect different types of genetic damage in multiple target tissues and the unknown quantitative relationships between measurable genotoxic effects and the probability of experiencing an adverse health outcome. In addition, with respect to DNA-reactive mutagens, the question arises whether the widely accepted assumption of a non-threshold dose-response relationship is at all compatible with the derivation of a HBGV. Therefore, at present, any quantitative genotoxicity assessment approach remains to be evaluated case-by-case. The quantitative interpretation of in vivo genotoxicity data for prioritization purposes, e.g., in connection with the MOE approach, could be seen as a promising opportunity for routine application. However, additional research is needed to assess whether it is possible to define a genotoxicity-derived MOE that can be considered indicative of a low level of concern. To further advance quantitative genotoxicity assessment, priority should be given to the development of new experimental methods to provide a deeper mechanistic understanding and a more comprehensive basis for the analysis of dose-response relationships.

Applying genomics in regulatory toxicology: a report of the ECETOC workshop on omics threshold on non-adversity.

In a joint effort involving scientists from academia, industry and regulatory agencies, ECETOC's activities in Omics have led to conceptual proposals for: (1) A framework that assures data quality for reporting and inclusion of Omics data in regulatory assessments; and (2) an approach to robustly quantify these data, prior to interpretation for regulatory use. In continuation of these activities this workshop explored and identified areas of need to facilitate robust interpretation of such data in the context of deriving points of departure (POD) for risk assessment and determining an adverse change from normal variation. ECETOC was amongst the first to systematically explore the application of Omics methods, now incorporated into the group of methods known as New Approach Methodologies (NAMs), to regulatory toxicology. This support has been in the form of both projects (primarily with CEFIC/LRI) and workshops. Outputs have led to projects included in the workplan of the Extended Advisory Group on Molecular Screening and Toxicogenomics (EAGMST) group of the Organisation for Economic Co-operation and Development (OECD) and to the drafting of OECD Guidance Documents for Omics data reporting, with potentially more to follow on data transformation and interpretation. The current workshop was the last in a series of technical methods development workshops, with a sub-focus on the derivation of a POD from Omics data. Workshop presentations demonstrated that Omics data developed within robust frameworks for both scientific data generation and analysis can be used to derive a POD. The issue of noise in the data was discussed as an important consideration for identifying robust Omics changes and deriving a POD. Such variability or "noise" can comprise technical or biological variation within a dataset and should clearly be distinguished from homeostatic responses. Adverse outcome pathways (AOPs) were considered a useful framework on which to assemble Omics methods, and a number of case examples were presented in illustration of this point. What is apparent is that high dimension data will always be subject to varying processing pipelines and hence interpretation, depending on the context they are used in. Yet, they can provide valuable input for regulatory toxicology, with the pre-condition being robust methods for the collection and processing of data together with a comprehensive description how the data were interpreted, and conclusions reached.

Toxicokinetic and toxicodynamic mixture effects of plant protection products: A case study.

Authorisation of ready to use plant protection products (PPPs) usually relies on the testing of acute and local toxicity only. This is in stark contrast to the situation for active substances where the mandatory data set comprises a most comprehensive set of studies. While the combination of certain active ingredients and co-formulants may nevertheless result in increased toxicity of the final product such combinations have never been evaluated systematically for complex and long-term toxicological endpoints. We therefore investigated the effect of three frequently used co-formulants on the toxicokinetic and toxicodynamic of the representative active substance combination of tebuconazol (Teb) and prothioconazol (Pro) or of cypermethrin (Cpm) and piperonyl butoxide (Pip), respectively. With all four active substances being potential liver steatogens, cytotoxicity and triglyceride accumulation in HepaRG were used as primary endpoints. Concomitantly transcriptomics and biochemical studies were applied to interrogate for effects on gene expression or inhibition of CYP3A4 as key enzyme for functionalization. Some of the tested combinations clearly showed more than additive effects, partly due to CYP3A4 enzyme inhibition. Other effects comprised the modulation of the expression and activity of steatosis-related nuclear key receptors. Altogether, the findings highlight the need for a more systematic consideration of toxicodynamic and toxicokinetic mixture effects during assessment of PPPs.

Mixture effects of co-formulants and two plant protection products in a liver cell line.

Plant protection products (PPPs) consist of one or more active substances and several co-formulants. Active substances provide the functionality of the PPP and are consequently evaluated according to standard test methods set by legal data requirements before approval, whereas co-formulants' toxicity is not as comprehensively assessed. However, in some cases mixture effects of active substances and co-formulants might result in increased or different forms of toxicity. In a proof-of-concept study we hence built on previously published results of Zahn et al. (2018[38]) on the mixture toxicity of Priori Xtra® and Adexar® to specifically investigate the influence of co-formulants on the toxicity of these commonly used fungicides. Products, their respective active substances in combination as well as some co-formulants were applied to human hepatoma cell line (HepaRG) in several dilutions. Cell viability analysis, mRNA expression, abundance of xenobiotic metabolizing enzymes and intracellular concentrations of active substances determined by LC-MS/MS analyses demonstrated that the toxicity of the PPPs is influenced by the presence of co-formulants in vitro. PPPs were more cytotoxic than the mix of their active substances. Gene expression profiles of cells treated with the PPPs were similar to those treated with their respective mixture combinations with marked differences. Co-formulants can cause gene expression changes on their own. LC-MS/MS analyses revealed higher intracellular concentrations of active substances in cells treated with PPPs compared to those treated with the respective active substances' mix. Proteomic data showed co-formulants can induce ABC transporters and CYP enzymes. Co-formulants can contribute to the observed increased toxicity of PPPs compared to their active substances in combination due to kinetic interactions, necessitating a more comprehensive evaluation approach.

A Critical Scoping Review of Pesticide Exposure Biomonitoring Studies in Overhead Cultures.

The exposure of operators, workers, residents and bystanders to pesticides is of high potential concern. Yet, reports on pesticide residues in the environment and near treated fields often spark debates if such findings might indicate a health risk. Although the underlying models are considered conservative, there are only limited field data on systemic exposure available. As a first step to improve the situation, we conducted a scoping review of state-of-the-art pesticide exposure biomonitoring studies in operators, workers, residents or bystanders. In contrast to existing reviews, we focused on target cultures of potential high pesticide exposure such as tree-grown produce, vine or hops. The search was conducted in Web of Science, Scopus and PubMed. Out of 17 eligible articles, a total of 11 studies met our search criteria, and 6 of them quantified the systemic exposure of humans. The analysis revealed that exposure was mainly driven by application of pesticides and reentry work, resulting in a higher exposure of operators and workers than of residents and bystanders. In nearly all cases, the systemic exposure was below the relevant toxicological reference values. The studies were subsequently analyzed to identify key criteria for a reliable design of a biomonitoring study on pesticide exposure.

An approach for mixture testing and prioritization based on common kinetic groups.

In light of an ever-increasing exposure to chemicals, the topic of potential mixture toxicity has gained increased attention, particularly as the toxicological toolbox to address such questions has vastly improved. Routinely toxicological risk assessments will rely on the analysis of individual compounds with mixture effects being considered only in those specific cases where co-exposure is foreseeable, for example for pesticides or food contact materials. In the field of pesticides, active substances are summarized in so-called cumulative assessment groups (CAG) which are primarily based on their toxicodynamic properties, that is, respective target organs and mode of action (MoA). In this context, compounds causing toxicity by a similar MoA are assumed to follow a model of dose/concentration addition (DACA). However, the respective approach inherently falls short of addressing cases where there are dissimilar or independent MoAs resulting in wider toxicokinetic effects. Yet, the latter are often the underlying cause when effects deviate from the DACA model. In the present manuscript, we therefore suggest additionally to consider toxicokinetic effects (especially related to xenobiotic metabolism and transporter interaction) for the grouping of substances to predict mixture toxicity. In line with the concept of MoA-based CAGs, we propose common kinetics groups (CKGs) as an additional tool for grouping of chemicals and mixture prioritization. Fundamentals of the CKG concept are discussed, along with challenges for its implementation, and methodological approaches and examples are explored.

Commensal-Related Changes in the Epidermal Barrier Function Lead to Alterations in the Benzo[a]Pyrene Metabolite Profile and Its Distribution in 3D Skin.

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyrene (B[a]P) are among the most abundant environmental pollutants, resulting in continuous exposure of human skin and its microbiota. However, effects of the latter on B[a]P toxicity, absorption, metabolism, and distribution in humans remain unclear. Here, we demonstrate that the skin microbiota does metabolize B[a]P on and in human skin in situ, using a recently developed commensal skin model. In this model, microbial metabolism leads to high concentrations of known microbial B[a]P metabolites on the surface as well as in the epidermal layers. In contrast to what was observed for uncolonized skin, B[a]P and its metabolites were subject to altered rates of skin penetration and diffusion, resulting in up to 58% reduction of metabolites recovered from basal culture medium. The results indicate the reason for this altered behavior to be a microbially induced strengthening of the epidermal barrier. Concomitantly, colonized models showed decreased formation and penetration of the ultimate carcinogen B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), leading, in consequence, to fewer BPDE-DNA adducts being formed. Befittingly, transcript and expression levels of key proteins for repairing environmentally induced DNA damage such as xeroderma pigmentosum complementation group C (XPC) were also found to be reduced in the commensal models, as was expression of B[a]P-associated cytochrome P450-dependent monooxygenases (CYPs). The results show that the microbiome can have significant effects on the toxicology of external chemical impacts. The respective effects rely on a complex interplay between microbial and host metabolism and microbe-host interactions, all of which cannot be adequately assessed using single-system studies. IMPORTANCE Exposure to xenobiotics has repeatedly been associated with adverse health effects. While the majority of reported cases relate to direct substance effects, there is increasing evidence that microbiome-dependent metabolism of xenobiotic substances likewise has direct adverse effects on the host. This can be due to microbial biotransformation of compounds, interaction between the microbiota and the host's endogenous detoxification enzymes, or altered xenobiotic bioavailability. However, there are hardly any studies addressing the complex interplay of such interactions in situ and less so in human test systems. Using a recently developed microbially competent three-dimensional (3D) skin model, we show here for the first time how commensal influence on skin physiology and gene transcription paradoxically modulates PAH toxicity.

Effects of co-formulants on the absorption and secretion of active substances in plant protection products in vitro.

Currently, the authorisation process for plant protection products (PPPs) relies on the testing of acute and topological toxicity only. Contrastingly, the evaluation of active substances includes a more comprehensive set of toxicity studies. Nevertheless, mixture effects of active ingredients and co-formulants may result in increased toxicity. Therefore, we investigated effects of surface active co-formulants on the toxicity of two PPPs focussing on qualitative and quantitative toxicokinetic effects on absorption and secretion. The respective products are based on the active substances abamectin and fluroxypyr-meptyl and were tested for cytotoxicity in the presence or absence of the corresponding surfactants and co-formulants using Caco-2 cells. In addition, the effect of co-formulants on increased cellular permeation was quantified using LC-MS/MS, while potential kinetic mixture effects were addressed by fluorescence anisotropy measurements and ATPase assays. The results show that surface active co-formulants significantly increase the cytotoxicity of the investigated PPPs, leading to more than additive mixture effects. Moreover, analytical investigations show higher efflux ratios of both active substances and the metabolite fluroxypyr upon combination with certain concentrations of the surfactants. The results further point to a significant and concentration-dependent inhibition of Pgp transporters by most of the surfactants as well as to increased membrane fluidity. Altogether, these findings strongly support the hypothesis that surfactants contribute to increased cytotoxicity of PPPs and do so by increasing the bioavailability of the respective active substances.

Microbially competent 3D skin: a test system that reveals insight into host-microbe interactions and their potential toxicological impact.

The skin`s microbiome is predominantly commensalic, harbouring a metabolic potential far exceeding that of its host. While there is clear evidence that bacteria-dependent metabolism of pollutants modulates the toxicity for the host there is still a lack of models for investigating causality of microbiome-associated pathophysiology or toxicity. We now report on a biologically characterised microbial-skin tissue co-culture that allows studying microbe-host interactions for extended periods of time in situ. The system is based on a commercially available 3D skin model. In a proof-of-concept, this model was colonised with single and mixed cultures of two selected skin commensals. Two different methods were used to quantify the bacteria on the surface of the skin models. While Micrococcus luteus established a stable microbial-skin tissue co-culture, Pseudomonas oleovorans maintained slow continuous growth over the 8-day cultivation period. A detailed skin transcriptome analysis showed bacterial colonisation leading to up to 3318 significant changes. Additionally, FACS, ELISA and Western blot analyses were carried out to analyse secretion of cytokines and growth factors. Changes found in colonised skin varied depending on the bacterial species used and comprised immunomodulatory functions, such as secretion of IL-1α/ß, Il-6, antimicrobial peptides and increased gene transcription of IL-10 and TLR2. The colonisation also influenced the secretion of growth factors such as VFGFA and FGF2. Notably, many of these changes have already previously been associated with the presence of skin commensals. Concomitantly, the model gained first insights on the microbiome's influence on skin xenobiotic metabolism (i.e., CYP1A1, CYP1B1 and CYP2D6) and olfactory receptor expression. The system provides urgently needed experimental access for assessing the toxicological impact of microbiome-associated xenobiotic metabolism in situ.

Characterization of Quinoline Yellow Dyes As Transient Aryl Hydrocarbon Receptor Agonists.

The aryl hydrocarbon receptor (AHR) and estrogen receptor alpha (ERα) are two ligand activated transcription factors that are targeted by a wide range of anthropogenic compounds. Crosstalk between both receptors is well established but little understood. We previously developed a dual color luciferase assay (i.e., XEER) which allows time dissolved monitoring of the activation of both receptors in situ. The system was now used in conjunction with HPLC-qTOF to identify several quinophthalone dyes as transient receptor agonists of the AHR. Altogether the approach identified three widely used dyes, that is the plastic colorant latyl yellow 3G (LY), the structurally related textile dye disperse yellow 64 (DY), and the cosmetic dye quinoline yellow (QY). The latter was the most potent agonist followed by LY and DY as confirmed by the XEER assay and CYP1A1 gene induction in MCF7 cells. In addition QY, LY, and DY also inhibited ER signaling in an AHR-dependent manner. This establishes some evidence for quinoline yellow dyes as potential disruptors of AHR/ER signaling, raising potential toxicological concern. Although none of the dyes featured any signs of genotoxicity in vitro, our data point to the need for a systematic approach when screening for substances of potential toxicological and endocrine relevance.

Aggregated aluminium exposure: risk assessment for the general population.

Aluminium is one of the most abundant elements in earth's crust and its manifold uses result in an exposure of the population from many sources. Developmental toxicity, effects on the urinary tract and neurotoxicity are known effects of aluminium and its compounds. Here, we assessed the health risks resulting from total consumer exposure towards aluminium and various aluminium compounds, including contributions from foodstuffs, food additives, food contact materials (FCM), and cosmetic products. For the estimation of aluminium contents in foodstuff, data from the German "Pilot-Total-Diet-Study" were used, which was conducted as part of the European TDS-Exposure project. These were combined with consumption data from the German National Consumption Survey II to yield aluminium exposure via food for adults. It was found that the average weekly aluminium exposure resulting from food intake amounts to approx. 50% of the tolerable weekly intake (TWI) of 1 mg/kg body weight (bw)/week, derived by the European Food Safety Authority (EFSA). For children, data from the French "Infant Total Diet Study" and the "Second French Total Diet Study" were used to estimate aluminium exposure via food. As a result, the TWI can be exhausted or slightly exceeded-particularly for infants who are not exclusively breastfed and young children relying on specially adapted diets (e.g. soy-based, lactose free, hypoallergenic). When taking into account the overall aluminium exposure from foods, cosmetic products (cosmetics), pharmaceuticals and FCM from uncoated aluminium, a significant exceedance of the EFSA-derived TWI and even the PTWI of 2 mg/kg bw/week, derived by the Joint FAO/WHO Expert Committee on Food Additives, may occur. Specifically, high exposure levels were found for adolescents aged 11-14 years. Although exposure data were collected with special regard to the German population, it is also representative for European and comparable to international consumers. From a toxicological point of view, regular exceedance of the lifetime tolerable aluminium intake (TWI/PTWI) is undesirable, since this results in an increased risk for health impairments. Consequently, recommendations on how to reduce overall aluminium exposure are given.

Chemical activation of estrogen and aryl hydrocarbon receptor signaling pathways and their interaction in toxicology and metabolism.

INTRODUCTION: Estrogen receptors (ERs) and the arylhydrocarbon receptor (AHR) are ligand-activated transcription factors that regulate the expression of genes involved in many physiological processes. With both receptors binding a broad range of natural and anthropogenic ligands, they are molecular targets for many substances, raising concerns for possible health effects. Areas covered: This review shall give a brief overview on the physiological functions of both receptors including their underlying molecular mechanisms. It summarizes the interaction of the respective signaling pathways including impacts on metabolism of endogenous estrogens, transcriptional interference, inhibitory crosstalk, and proteasomal degradation. Also addressed are the AHR dependent formation of estrogenic metabolites from polycyclic aromatic hydrocarbons and the possible impact of the ER/AHR crosstalk in the context of drug metabolism. Expert opinion: Despite decade-long research, the physiological role of the AHR and ER as well as the implications of their complex mutual crosstalk remain to be determined as do resulting potential impacts on human health. With more and more endogenous AHR ligands being discovered, future research should hence systematically address the potential impact of such substances on estrogen signaling. The intimate link between these two pathways and the genes regulated therein bears the potential for impacts on drug metabolism and human health.

Mineral oil in food, cosmetic products, and in products regulated by other legislations.

For a few years, mineral oils and their potential adverse health effects have been a constant issue of concern in many regulatory areas such as food, cosmetics, other consumer products, and industrial chemicals. Analytically, two fractions can be distinguished: mineral oil saturated hydrocarbons (MOSH) and mineral oil aromatic hydrocarbons (MOAH). This paper aims at assessing the bioaccumulative potential and associated histopathological effects of MOSH as well as the carcinogenic potential of MOAH for consumer-relevant mineral oils. It also covers the absorption, distribution, metabolism, and excretion of MOSH and MOAH upon oral and dermal exposures. The use and occurrence of consumer-relevant, highly refined mineral oils in food, cosmetics and medicinal products are summarized, and estimates for the exposure of consumers are provided. Also addressed are the challenges in characterizing the substance identity of mineral oil products under REACH. Evidence from more recent autopsy and biopsy studies, along with information on decreasing food contamination levels, indicates a low risk for adverse hepatic lesions that may arise from the retention of MOSH in the liver. With respect to MOAH, at present there is no indication of any carcinogenic effects in animals dermally or orally exposed to highly refined mineral oils and waxes. Such products are used not only in cosmetics but also in medicinal products and as additives in food contact materials. The safety of these mineral oil-containing products is thus indirectly documented by their prevalent and long-term use, with a simultaneous lack of clinical and epidemiological evidence for adverse health effects.