Toxicological evaluation of microbial secondary metabolites in the context of European active substance approval for plant protection products.

Risk assessment (RA) of microbial secondary metabolites (SM) is part of the EU approval process for microbial active substances (AS) used in plant protection products (PPP). As the number of potentially produced microbial SM may be high for a certain microbial strain and existing information on the metabolites often are low, data gaps are frequently identified during the RA. Often, RA cannot conclusively clarify the toxicological relevance of the individual substances. This work presents data and RA conclusions on four metabolites, Beauvericin, 2,3-deepoxy-2,3-didehydro-rhizoxin (DDR), Leucinostatin A and Swainsonin in detail as examples for the challenging process of RA. To overcome the problem of incomplete assessment reports, RA of microbial AS for PPP is in need of new approaches. In view of the Next Generation Risk Assessment (NGRA), the combination of literature data, omic-methods, in vitro and in silico methods combined in adverse outcome pathways (AOPs) can be used for an efficient and targeted identification and assessment of metabolites of concern (MoC).

Thresholds of adversity for endocrine disrupting substances: a conceptual case study.

For endocrine disrupting chemicals (EDC) the existence of "safe exposure levels", that is exposure levels that do not present an appreciable risk to human health is most controversially discussed, as is the existence of health-based reference values. Concerns have been especially raised that EDCs might not possess a threshold level such that no exposure level to EDCs can be considered safe. To explore whether or not threshold levels can be identified, we performed a screening exercise on 14 pesticidal and biocidal active substances previously identified as EDCs in the European Union. The respective substances are ideal subjects for case studies to review for endocrine activity and disruptive potential following well-defined regulatory assessment based on solid data to effectually establish adversity as consequence of endocrine disruption. Dimethomorph, metiram and propiconazole for which the weight of evidence demonstrating endocrine disruption was the strongest were used as subjects for further study. Epoxiconazole was additionally selected as its effects on the endocrine system are extensive. For all four substances, analysis of the toxicological data clearly indicated thresholds of adversity below which no adverse effects mediated through an endocrine mechanism were observed. Particular emphasis was placed on mechanistic considerations including homeostasis and the concept of adversity. As a proof of concept this study provides evidence that like other substances of toxicological concern EDCs have threshold levels for adversity. While for some EDCs the respective thresholds might indeed be very low this shows that, data allowing, for other EDCs sufficiently protective reference values can be derived.

Application of AOPs to assist regulatory assessment of chemical risks - Case studies, needs and recommendations.

While human regulatory risk assessment (RA) still largely relies on animal studies, new approach methodologies (NAMs) based on in vitro, in silico or non-mammalian alternative models are increasingly used to evaluate chemical hazards. Moreover, human epidemiological studies with biomarkers of effect (BoE) also play an invaluable role in identifying health effects associated with chemical exposures. To move towards the next generation risk assessment (NGRA), it is therefore crucial to establish bridges between NAMs and standard approaches, and to establish processes for increasing mechanistically-based biological plausibility in human studies. The Adverse Outcome Pathway (AOP) framework constitutes an important tool to address these needs but, despite a significant increase in knowledge and awareness, the use of AOPs in chemical RA remains limited. The objective of this paper is to address issues related to using AOPs in a regulatory context from various perspectives as it was discussed in a workshop organized within the European Union partnerships HBM4EU and PARC in spring 2022. The paper presents examples where the AOP framework has been proven useful for the human RA process, particularly in hazard prioritization and characterization, in integrated approaches to testing and assessment (IATA), and in the identification and validation of BoE in epidemiological studies. Nevertheless, several limitations were identified that hinder the optimal usability and acceptance of AOPs by the regulatory community including the lack of quantitative information on response-response relationships and of efficient ways to map chemical data (exposure and toxicity) onto AOPs. The paper summarizes suggestions, ongoing initiatives and third-party tools that may help to overcome these obstacles and thus assure better implementation of AOPs in the NGRA.

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.

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.

Metabolism-Disrupting Chemicals Affecting the Liver: Screening, Testing, and Molecular Pathway Identification.

The liver is the central metabolic organ of the body. The plethora of anabolic and catabolic pathways in the liver is tightly regulated by physiological signaling but may become imbalanced as a consequence of malnutrition or exposure to certain chemicals, so-called metabolic endocrine disrupters, or metabolism-disrupting chemicals (MDCs). Among different metabolism-related diseases, obesity and non-alcoholic fatty liver disease (NAFLD) constitute a growing health problem, which has been associated with a western lifestyle combining excessive caloric intake and reduced physical activity. In the past years, awareness of chemical exposure as an underlying cause of metabolic endocrine effects has continuously increased. Within this review, we have collected and summarized evidence that certain environmental MDCs are capable of contributing to metabolic diseases such as liver steatosis and cholestasis by different molecular mechanisms, thereby contributing to the metabolic syndrome. Despite the high relevance of metabolism-related diseases, standardized mechanistic assays for the identification and characterization of MDCs are missing. Therefore, the current state of candidate test systems to identify MDCs is presented, and their possible implementation into a testing strategy for MDCs is discussed.

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.

More than additive effects on liver triglyceride accumulation by combinations of steatotic and non-steatotic pesticides in HepaRG cells.

The liver is constantly exposed to mixtures of hepatotoxic compounds, such as food contaminants and pesticides. Dose addition is regularly assumed for mixtures in risk assessment, which however might not be sufficiently protective in case of synergistic effects. Especially the prediction of combination effects of substances which do not share a common adverse outcome (AO) might be problematic. In this study, the focus was on the endpoint liver triglyceride accumulation in vitro, an indicator of hepatic fatty acid changes. The hepatotoxic compounds difenoconazole, propiconazole and tebuconazole were chosen which cause hepatic fatty acid changes in vivo, whereas fludioxonil was chosen as a hepatotoxic substance not causing fatty acid changes. Triglyceride accumulation was analyzed for combinations of steatotic and non-steatotic pesticides in human HepaRG hepatocarcinoma cells. Investigations revealed a potentiation of triglyceride accumulation by mixtures of the steatotic compounds with the non-steatotic fludioxonil, as compared to the single compounds. Mathematical modeling of combination effects indicated more than additive effects for the tested combinations if the method by Chou was applied, and a decrease in EC50 values of the steatotic compounds when applied in mixtures. Use of an adverse outcome pathway (AOP)-driven testing strategy for liver steatosis showed interactions of the test compounds with the nuclear receptors AHR, CAR and PXR, as well as a downregulation of ACOX2. An ACOX2-dependent mechanism underlying the observed mixture effect could not be verified using a siRNA approach. By contrast, a toxicokinetic interaction was identified including an inhibition of the metabolic enzyme CYP3A4 by fludioxonil and a decreased metabolic conversion of the CYP3A4 substrate difenoconazole when used in mixture experiments. In conclusion, an interaction by a steatotic and a non-steatotic compound at the toxicokinetic level on the endpoint triglyceride accumulation in vitro was described.

Cross-species analysis of hepatic cytochrome P450 and transport protein expression.

Most drugs and xenobiotics are metabolized in the liver. Amongst others, different cytochrome P450 (CYP) enzymes catalyze the metabolic conversion of foreign compounds, and various transport proteins are engaged in the excretion of metabolites from the hepatocytes. Inter-species and inter-individual differences in the hepatic levels and activities of drug-metabolizing enzymes and transporters result from genetic as well as from environmental factors, and play a decisive role in determining the pharmacokinetic properties of a compound in a given test system. To allow for a meaningful comparison of results from metabolism studies, it is, therefore, of utmost importance to know about the specific metabolic properties of the test systems, especially about the levels of metabolic enzymes such as the CYPs. Using a targeted proteomics approach, we, therefore, compared the hepatic levels of important CYP enzymes and transporters in different experimental systems in vivo and in vitro, namely Wistar rats, C57/Bl6 mice, mice humanized for the two xeno-sensing receptors PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor), mice with human hepatocyte-repopulated livers, human HepaRG hepatocarcinoma cells, primary human hepatocytes, and human liver biopsies. In addition, the effects of xenobiotic inducers of drug metabolism on CYP enzymes and transporters were analyzed in selected systems. This study for the first time presents a comprehensive overview of similarities and differences in important drug metabolism-related proteins among the different experimental models.

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.

Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats.

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures.

Caenorhabditis elegans As a Promising Alternative Model for Environmental Chemical Mixture Effect Assessment-A Comparative Study.

A key challenge of mixture toxicity testing is that a multitude of substances with even more combinations need to be tested in a broad dose range. Consequently testing in rodent bioassays, the current gold standard of toxicity testing, is hardly feasible. High-throughput compatible cell culture systems, however, suffer from limitations with respect to toxicokinetics, tissue interactions, and compensatory mechanisms. Therefore, simple organisms like the nematode Caenorhabditis elegans, combining relevant advantages of complex in vivo and fast in vitro assays might prove highly valuable within a testing strategy for mixtures. To investigate the comparability between results obtained with C. elegans and traditional rodent assays, we used five azole fungicides as well investigated model substances. Our findings suggest that azoles act additively in C. elegans which is in line with previous results in rats. Additionally, we show that toxicokinetics are one important factor for the differences in the relative toxicity of the azoles in both species. Importantly, we also demonstrate that in contrast to most rodent in vivo studies, C. elegans assays provide well-defined concentration-response relationships which are a very good basis for the prediction of mixture effects. We conclude that C. elegans may be an appropriate model for mixture toxicity testing at least within a first step to identify and prioritize relevant mixtures for further testing.

Pregnane X receptor mediates steatotic effects of propiconazole and tebuconazole in human liver cell lines.

Triazoles are commonly used fungicides which show liver toxicity in rodent studies. While hepatocellular hypertrophy is the most prominent finding, some triazoles have also been reported to cause hepatocellular steatosis. The aim of our study was to elucidate molecular mechanisms of triazole-mediated steatosis. Therefore, we used the two triazoles propiconazole (Pi) and tebuconazole (Te) as test compounds in in vitro assays using the human hepatocarcinoma cell lines HepG2 and HepaRG. Triglyceride accumulation was measured using the Adipored assay and by a gas-chromatographic method. Reporter gene analyses were used to assess the ability of Pi and Te to activate nuclear receptors, which are described as the molecular initiators in the adverse outcome pathway (AOP) for liver steatosis. The expression of steatosis-associated genes was investigated by RT-PCR. Mechanistic analyses of triazole-mediated steatosis were performed using HepaRG subclones that are deficient in different nuclear receptors. Pi and Te both interacted with the constitutive androstane receptor (CAR), the peroxisome proliferator-activated receptor alpha (PPARα), and the pregnane X receptor (PXR). Both compounds induced expression of steatosis-related genes and cellular triglyceride accumulation. The knockout of PXR in HepaRG cells, but not the CAR knockout, abolished triazole-induced triglyceride accumulation, thus underlining the crucial role of PXR in hepatic steatosis resulting from exposure to these fungicides. In conclusion, our findings provide new insight into the molecular mechanisms of steatosis induction by triazole fungicides and identify PXR as a critical mediator of this process.

Assessment of mixture toxicity of (tri)azoles and their hepatotoxic effects in vitro by means of omics technologies.

Consumers are constantly exposed to chemical mixtures such as multiple residues of different pesticides via the diet. This raises questions concerning potential combination effects, especially because these substances are tested for regulatory purposes on an individual basis. With approximately 500 active substances approved as pesticides, there are too many possible combinations to be tested in standard animal experiments generally requested for regulatory purposes. Therefore, the development of in vitro tools and alternative testing strategies for the assessment of mixture effects is extremely important. As a first step in the development of such in vitro tools, we used (tri)azoles as model substances in a set of different cell lines derived from the primary target organ of these substances, the liver (human: HepaRG, rat: H4IIE). Concentrations were reconciled with measured tissue concentrations obtained from in vivo experiments to ensure comparable effect levels. The effects of the substances were subsequently analyzed by transcriptomics and metabolomics techniques and compared to data from corresponding in vivo studies. The results show that similar toxicity pathways are affected by substances and combinations, thus indicating a similar mode of action and additive effects. Two biomarkers obtained by the approach, CAR and Cyp1A1, were used for mixture toxicity modeling and confirmed the concentration-additive effects, thus supporting the selected testing strategy and raising hope for the development of in vitro methods suitable to detect combination effects and prioritize mixtures of concern for further testing.

Propiconazole is an activator of AHR and causes concentration additive effects with an established AHR ligand.

Consumers are exposed to pesticide residues and other food contaminants via the diet. Both can exert adverse effects on different target organs via the activation of nuclear receptor pathways. Hepatotoxic effects of the widely used triazole fungicide propiconazole (Pi) are generally attributed to the activation of the constitutive androstane receptor (CAR) or the pregnane X receptor (PXR). We now investigated the effects of Pi on the aryl hydrocarbon receptor (AHR) and possible mixture toxicity when Pi is present in combination with BbF, an AHR ligand. In silico docking simulations indicate that Pi can bind to human AHR. Subsequent dual luciferase reporter gene assays in human HepG2 cells showed that Pi activates the AHR in vitro. This concentration-dependent activation was confirmed by real-time RT-PCR analyses of the model AHR target genes CYP1A1 and CYP1A2 in human HepaRG and HepG2 cells. In addition, induction of CYP1A1 protein levels and enzyme activity were recorded. Similarly, increased mRNA expression and enzyme activity of Cyp1a1 and Cyp1a2 was observed in livers of rats treated with Pi for 28 days via the diet. Gene expression analysis in AHR-knockout HepaRG cells showed no induction of CYP1A1 and CYP1A2, whereas gene expression in CAR-, and PXR-knockout cells was induced. Finally, mixture effects of Pi and BbF were analyzed in human cell lines: modeling of concentration-response curves revealed concentration additivity. In conclusion, our results demonstrate that the triazole Pi is an activator of AHR in silico, in vitro and in vivo and causes additive effects with an established AHR ligand.

Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice.

The agricultural fungicides cyproconazole and prochloraz exhibit hepatotoxicity in rodent studies and are tumorigenic following chronic exposure. Both substances are suspected to act via a CAR (constitutive androstane receptor)/PXR (pregnane-X-receptor)-dependent mechanism. Human relevance of these findings is under debate. A 28-day toxicity study was conducted in mice with humanized CAR and PXR (hCAR/hPXR) with two dose levels (50 or 500 ppm) of both substances, using the model CAR activator phenobarbital as a reference. Results were compared to wild-type mice. A treatment-related increase in liver weights was observed for all three substances at least at the high-dose level. Changes in the expression of classic CAR/PXR target genes such as Cyp2b10 were induced by cyproconazole and phenobarbital in both genotypes, while prochloraz treatment resulted in gene expression changes indicative of additional aryl hydrocarbon receptor activation, e.g. by up-regulation of Cyp1a1 expression. Cyproconazole-induced effects on CAR-dependent gene expression, liver weight, and hepatic lipid accumulation were more prominent in wild-type mice, where significant genotype differences were observed at the high-dose level. Moreover, high-dose cyproconazole-treated mice from the wild-type group responded with a marked increase in hepatocellular proliferation, while hCAR/hPXR mice did not. In conclusion, our data demonstrate that cyproconazole and PB induce CAR/PXR downstream effects in hepatocytes in vivo via both, the murine and human receptors. At high doses of cyproconazole, however, the responses were clearly more pronounced in wild-type mice, indicating increased sensitivity of rodents to CAR agonist-induced effects in hepatocytes.

Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement.

Endocrine disruption is a specific form of toxicity, where natural and/or anthropogenic chemicals, known as "endocrine disruptors" (EDs), trigger adverse health effects by disrupting the endogenous hormone system. There is need to harmonize guidance on the regulation of EDs, but this has been hampered by what appeared as a lack of consensus among scientists. This publication provides summary information about a consensus reached by a group of world-leading scientists that can serve as the basis for the development of ED criteria in relevant EU legislation. Twenty-three international scientists from different disciplines discussed principles and open questions on ED identification as outlined in a draft consensus paper at an expert meeting hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany on 11-12 April 2016. Participants reached a consensus regarding scientific principles for the identification of EDs. The paper discusses the consensus reached on background, definition of an ED and related concepts, sources of uncertainty, scientific principles important for ED identification, and research needs. It highlights the difficulty in retrospectively reconstructing ED exposure, insufficient range of validated test systems for EDs, and some issues impacting on the evaluation of the risk from EDs, such as non-monotonic dose-response and thresholds, modes of action, and exposure assessment. This report provides the consensus statement on EDs agreed among all participating scientists. The meeting facilitated a productive debate and reduced a number of differences in views. It is expected that the consensus reached will serve as an important basis for the development of regulatory ED criteria.

Relevance and reliability of experimental data in human health risk assessment of pesticides.

Evaluation of data relevance, reliability and contribution to uncertainty is crucial in regulatory health risk assessment if robust conclusions are to be drawn. Whether a specific study is used as key study, as additional information or not accepted depends in part on the criteria according to which its relevance and reliability are judged. In addition to GLP-compliant regulatory studies following OECD Test Guidelines, data from peer-reviewed scientific literature have to be evaluated in regulatory risk assessment of pesticide active substances. Publications should be taken into account if they are of acceptable relevance and reliability. Their contribution to the overall weight of evidence is influenced by factors including test organism, study design and statistical methods, as well as test item identification, documentation and reporting of results. Various reports make recommendations for improving the quality of risk assessments and different criteria catalogues have been published to support evaluation of data relevance and reliability. Their intention was to guide transparent decision making on the integration of the respective information into the regulatory process. This article describes an approach to assess the relevance and reliability of experimental data from guideline-compliant studies as well as from non-guideline studies published in the scientific literature in the specific context of uncertainty and risk assessment of pesticides.