Comparative case study on NAMs: towards enhancing specific target organ toxicity analysis.

Traditional risk assessment methodologies in toxicology have relied upon animal testing, despite concerns regarding interspecies consistency, reproducibility, costs, and ethics. New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, hold promise by providing mechanistic information rather than assessing organ pathology. However, NAMs face limitations, like lacking a whole organism and restricted toxicokinetic interactions. This is an inherent challenge when it comes to the use of omics data from in vitro studies for the prediction of organ toxicity in vivo. One solution in this context are comparative in vitro-in vivo studies as they allow for a more detailed assessment of the transferability of the respective NAM data. Hence, hepatotoxic and nephrotoxic pesticide active substances were tested in human cell lines and the results subsequently related to the biology underlying established effects in vivo. To this end, substances were tested in HepaRG and RPTEC/tERT1 cells at non-cytotoxic concentrations and analyzed for effects on the transcriptome and parts of the proteome using quantitative real-time PCR arrays and multiplexed microsphere-based sandwich immunoassays, respectively. Transcriptomics data were analyzed using three bioinformatics tools. Where possible, in vitro endpoints were connected to in vivo observations. Targeted protein analysis revealed various affected pathways, with generally fewer effects present in RPTEC/tERT1. The strongest transcriptional impact was observed for Chlorotoluron in HepaRG cells (increased CYP1A1 and CYP1A2 expression). A comprehensive comparison of early cellular responses with data from in vivo studies revealed that transcriptomics outperformed targeted protein analysis, correctly predicting up to 50% of in vivo effects.

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.

Corrigendum: Development of new approach methods for the identification and characterization of endocrine metabolic disruptors-a PARC project.

[This corrects the article DOI: 10.3389/ftox.2023.1212509.].

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.

Acceptance criteria for new approach methods in toxicology and human health-relevant life science research - part I.

Every test procedure, scientific and non-scientific, has inherent uncertainties, even when performed according to a standard operating procedure (SOP). In addition, it is prone to errors, defects, and mistakes introduced by operators, laboratory equipment, or materials used. Adherence to an SOP and comprehensive validation of the test method cannot guarantee that each test run produces data within the acceptable range of variability and with the precision and accuracy determined during the method validation. We illustrate here (part I) why controlling the validity of each test run is an important element of experimental design. The definition and application of acceptance criteria (AC) for the validity of test runs is important for the setup and use of test methods, particularly for the use of new approach methods (NAM) in toxicity testing. AC can be used for decision rules on how to handle data, e.g., to accept the data for further use (AC fulfilled) or to reject the data (AC not fulfilled). The adherence to AC has important requirements and consequences that may seem surprising at first sight: (i) AC depend on a test method's objectives, e.g., on the types/concentrations of chemicals tested, the regulatory context, the desired throughput; (ii) AC are applied and documented at each test run, while validation of a method (including the definition of AC) is only performed once; (iii) if AC are altered, then the set of data produced by a method can change. AC, if missing, are the blind spot of quality assurance: Test results may not be reliable and comparable. The establishment and uses of AC will be further detailed in part II of this series.

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.

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.

Innovative tools and methods for toxicity testing within PARC work package 5 on hazard assessment.

New approach methodologies (NAMs) have the potential to become a major component of regulatory risk assessment, however, their actual implementation is challenging. The European Partnership for the Assessment of Risks from Chemicals (PARC) was designed to address many of the challenges that exist for the development and implementation of NAMs in modern chemical risk assessment. PARC's proximity to national and European regulatory agencies is envisioned to ensure that all the research and innovation projects that are initiated within PARC agree with actual regulatory needs. One of the main aims of PARC is to develop innovative methodologies that will directly aid chemical hazard identification, risk assessment, and regulation/policy. This will facilitate the development of NAMs for use in risk assessment, as well as the transition from an endpoint-based animal testing strategy to a more mechanistic-based NAMs testing strategy, as foreseen by the Tox21 and the EU Chemical's Strategy for Sustainability. This work falls under work package 5 (WP5) of the PARC initiative. There are three different tasks within WP5, and this paper is a general overview of the five main projects in the Task 5.2 'Innovative Tools and methods for Toxicity Testing,' with a focus on Human Health. This task will bridge essential regulatory data gaps pertaining to the assessment of toxicological prioritized endpoints such as non-genotoxic carcinogenicity, immunotoxicity, endocrine disruption (mainly thyroid), metabolic disruption, and (developmental and adult) neurotoxicity, thereby leveraging OECD's and PARC's AOP frameworks. This is intended to provide regulatory risk assessors and industry stakeholders with relevant, affordable and reliable assessment tools that will ultimately contribute to the application of next-generation risk assessment (NGRA) in Europe and worldwide.

Development of new approach methods for the identification and characterization of endocrine metabolic disruptors-a PARC project.

In past times, the analysis of endocrine disrupting properties of chemicals has mainly been focused on (anti-)estrogenic or (anti-)androgenic properties, as well as on aspects of steroidogenesis and the modulation of thyroid signaling. More recently, disruption of energy metabolism and related signaling pathways by exogenous substances, so-called metabolism-disrupting chemicals (MDCs) have come into focus. While general effects such as body and organ weight changes are routinely monitored in animal studies, there is a clear lack of mechanistic test systems to determine and characterize the metabolism-disrupting potential of chemicals. In order to contribute to filling this gap, one of the project within EU-funded Partnership for the Assessment of Risks of Chemicals (PARC) aims at developing novel in vitro methods for the detection of endocrine metabolic disruptors. Efforts will comprise projects related to specific signaling pathways, for example, involving mTOR or xenobiotic-sensing nuclear receptors, studies on hepatocytes, adipocytes and pancreatic beta cells covering metabolic and morphological endpoints, as well as metabolism-related zebrafish-based tests as an alternative to classic rodent bioassays. This paper provides an overview of the approaches and methods of these PARC projects and how this will contribute to the improvement of the toxicological toolbox to identify substances with endocrine disrupting properties and to decipher their mechanisms of action.

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).

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.

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.

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

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

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.

The use of NAMs and omics data in risk assessment.

The animal-centric approach so far predominantly employed in risk assessment has been questioned in recent years due to a number of shortcomings regarding performance, consistency, transferability of results, sustainability, costs and ethical reasons. Alternatives to animal testing, collectively termed NAMs, may have the potential to deliver sound, cost-effective, prompt and reliable information, but their regulatory acceptance has not been established yet. The main reasons behind this are mostly related to actual methodological obstacles, with particular reference to addressing complex endpoints such as repeated-dose toxicity, the issue of translating the concept of adversity to NAMs, and doubts of stakeholders about the level of chemical safety ensured by NAMs. With the aim of providing an updated view on major conceptual and methodological developments in the field of toxicology, a symposium and a workshop were organised by the German Federal Institute for Risk Assessment (Bundesinstitut für Risikobewertung, BfR) and Helmholtz Centre for Environmental Research on 15-17 November 2021 in Berlin. The conference, entitled 'Challenges in Public Health Protection in the 21st Century: New Methods, Omics and Novel Concepts in Toxicology' brought together eminent scientists with representatives from industry and regulatory authorities. The organisation, day-to-day operations and the reporting of the event main outcomes in a position paper were the main focus of the present EFSA EU-FORA work programme. Tasks pertaining to 'The use of NAMs and omics data in risk assessment' were implemented under the shared supervision of units 'Testing and Assessment Strategies Pesticides' and 'Effect-based Analytics and Toxicogenomics' of the German Federal Institute for Risk Assessment.

New Approach Methods for Hazard Identification: A Case Study with Azole Fungicides Affecting Molecular Targets Associated with the Adverse Outcome Pathway for Cholestasis.

Triazole fungicides such as propiconazole (Pi) or tebuconazole (Te) show hepatotoxicity in vivo, e.g., hypertrophy and vacuolization of liver cells following interaction with nuclear receptors such as PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor). Accordingly, azoles affect gene expression associated with these adverse outcomes in vivo but also in human liver cells in vitro. Additionally, genes indicative of liver cholestasis are affected in vivo and in vitro. We therefore analyzed the capability of Pi and Te to cause cholestasis in an adverse outcome pathway (AOP)-driven approach in hepatic cells of human origin in vitro, considering also previous in vivo studies. Bile salt export pump (BSEP) activity assays confirmed that both azoles are weak inhibitors of BSEP. They alternate the expression of various cholestasis-associated target genes and proteins as well as the mitochondrial membrane function. Published in vivo data, however, demonstrate that neither Pi nor Te cause cholestasis in rodent bioassays. This discrepancy can be explained by the in vivo concentrations of both azoles being well below their EC50 for BSEP inhibition. From a regulatory perspective, this illustrates that toxicogenomics and human in vitro models are valuable tools to detect the potential of a substance to cause a specific type of toxicity. To come to a sound regulatory conclusion on the in vivo relevance of such a finding, results will have to be considered in a broader context also including toxicokinetics in a weight-of-evidence approach.

Use of transcriptomics in hazard identification and next generation risk assessment: A case study with clothianidin.

Toxicological risk assessment is essential in the evaluation and authorization of different classes of chemical substances. Genotoxicity and mutagenicity testing are of highest priority and rely on established in vitro systems with bacterial and mammalian cells, sometimes followed by in vivo testing using rodent animal models. Transcriptomic approaches have recently also shown their value to determine transcript signatures specific for genotoxicity. Here, we studied how transcriptomic data, in combination with in vitro tests with human cells, can be used for the identification of genotoxic properties of test compounds. To this end, we used liver samples from a 28-day oral toxicity study in rats with the pesticidal active substances imazalil, thiacloprid, and clothianidin, a neonicotinoid-type insecticide with, amongst others, known hepatotoxic properties. Transcriptomic results were bioinformatically evaluated and pointed towards a genotoxic potential of clothianidin. In vitro Comet and γH2AX assays in human HepaRG hepatoma cells, complemented by in silico analyses of mutagenicity, were conducted as follow-up experiments to check if the genotoxicity alert from the transcriptomic study is in line with results from a battery of guideline genotoxicity studies. Our results illustrate the combined use of toxicogenomics, classic toxicological data and new approach methods in risk assessment. By means of a weight-of-evidence decision, we conclude that clothianidin does most likely not pose genotoxic risks to humans.