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Abstract Genetically modified maize DP51291 was developed to confer control against susceptible corn rootworm pests and tolerance to glufosinate‐containing herbicide; these properties were achieved by introducing the ipd072Aa, pmi and mo‐pat expression cassettes. The molecular characterisation data and bioinformatic analyses do not identify issues requiring food/feed safety assessment. None of the identified differences in the agronomic/phenotypic and compositional characteristics tested between maize DP51291 and its conventional counterpart needs further assessment, except for phosphorus in forage and manganese, proline, oleic acid (C18:1) and linoleic acid (C18:2) in grain, which do not raise safety and nutritional concerns. The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of the IPD072Aa, PAT and PMI proteins as expressed in maize DP51291 and finds no evidence that the genetic modification would change the overall allergenicity of maize DP51291. In the context of this application, the consumption of food and feed from maize DP51291 does not represent a nutritional concern in humans and animals. The GMO Panel concludes that maize DP51291 is as safe as its conventional counterpart and non‐GM maize varieties tested, and no post‐market monitoring of food/feed is considered necessary. In the case of accidental release of viable maize DP51291 grains into the environment, this would not raise environmental safety concerns. The post‐market environmental monitoring plan and reporting intervals are in line with the intended uses of maize DP51291. The GMO Panel concludes that maize DP51291 is as safe as its conventional counterpart and the tested non‐GM maize varieties with respect to potential effects on human and animal health and the environment.

Abstract Genetically modified Komagataella phaffii strain MXY0541 was developed to produce soy leghemoglobin by introducing the LGB2 coding sequence encoding leghemoglobin from soybean (Glycine max). The molecular characterisation data and bioinformatic analyses do not raise any safety concerns. The safety of soy leghemoglobin as a food additive has already been assessed by the EFSA FAF Panel (EFSA‐Q‐2022‐00031). The GMO Panel does not identify safety concerns regarding the toxicity and allergenicity of soy leghemoglobin protein as expressed in K. phaffii, and finds no evidence that the genetic modification would change its overall allergenicity. The GMO Panel concludes that the LegH Prep derived from genetically modified K. phaffii strain MXY0541 is safe for human consumption with regard to the effects of the genetic modification. No environmental impact from the use of this product is expected regarding the recombinant DNA sequences possibly remaining in the product. The GMO Panel concludes that LegH Prep from genetically modified K. phaffii strain MXY0541 is safe with respect to potential effects on human health and the environment at the proposed use and use level as far as the impact of the genetic modification is concerned. The overall conclusion is that the genetic modification does not lead to safety issues.

Abstract Following a request from the European Commission, the GMO Panel assessed additional information related to the application for authorisation of food and feed containing, consisting of and produced from genetically modified soybean MON × MON 87708 × MON 89788 (EFSA‐GMO‐NL‐2015‐126). The applicant conducted a 90‐day feeding study on GM soybean MON 87705 and provided a proposal for post‐market monitoring considering the altered fatty acid profile of GM soybean MON 87705 × MON 87708 × MON 89788, to fulfil the deficiencies identified by EFSA GMO Panel, addressing elements that remained inconclusive from a previous EFSA scientific opinion issued in 2020. The GMO Panel concludes that the 90‐day feeding study on GM soybean MON 87705 is in line with the requirements of Regulation (EU) No 503/2013 and that no treatment‐related adverse effects were observed in rats after feeding diets containing soybean MON 87705 meals at 30% or 15% for 90 days. The GMO Panel reiterates the recommendation for a PMM for food in accordance with Regulation (EC) No 1829/2003 and Regulation (EU) No 503/2013 and concludes that the proposal provided by the applicant is in line with the recommendations described for the PMM plan of soybean MON 87705 × MON 87708 × MON 89788 in the adopted scientific opinion. Taking into account the previous assessment and the new information, the GMO Panel concludes that soybean MON 87705 × MON 87708 × MON 89788, as assessed in the scientific opinion on application EFSA‐GMO‐NL‐2015‐126 and in the supplementary toxicity study, is as safe as its non‐GM comparator and the non‐GM reference varieties tested and does not represent a nutritional concern in humans and animals, within the scope of this application.

Phycotoxins are responsible for foodborne intoxications. Symptoms depend on the ingested toxins but mostly imply gastro-intestinal and neurological disorders. Importantly, humans are exposed to combinations of several phycotoxins, resulting in possible mixture effects. Most previous studies, however, have been focused on single toxin effects. Thus, the aim of this study was to examine the effects of binary mixtures of three main phycotoxins, okadaic acid (OA), azaspiracid-1 (AZA1) and yessotoxin (YTX), on human intestinal Caco-2 cells. The focus was placed on cell viability studies and inflammation responses using a multi-parametric approach to assess cell population (nuclei staining), cell metabolism/viability (reductase activity and lysosomal integrity), and release of inflammation markers (e.g., interleukins). Mixture effects were evaluated using the concentration addition (CA) and independent action (IA) models. Our assays show that none of the toxins had an impact on the cell population in the tested concentration range. Only OA modulated reductase activity, while all three toxins had strong effects on lysosomal integrity. Furthermore, all toxins triggered the release of interleukin 8 (IL-8), with OA being most potent. Mixture effect analysis showed additivity in most cases. However, supra-additivity was observed in regards to IL-6 and IL-8 release for combinations implying high concentrations of OA. This study extends the knowledge on mixture effects of phycotoxins in human cells.

Background: Effective communication is crucial for broad acceptance and applicability of alternative methods in 3R biomedical research and preclinical testing. 3D bioprinting is used to construct intricate biological structures towards functional liver models, specifically engineered for deployment as alternative models in drug screening, toxicological investigations, and tissue engineering. Despite a growing number of reviews in this emerging field, a comprehensive study, systematically assessing practices and reporting quality for bioprinted liver models is missing. Methods: In this systematic scoping review we systematically searched MEDLINE (Ovid), EMBASE (Ovid) and BioRxiv for studies published prior to June 2nd, 2022. We extracted data on methodological conduct, applied bioinks, the composition of the printed model, performed experiments and model applications. Records were screened for eligibility and data were extracted from included articles by two independent reviewers from a panel of seven domain experts specializing in bioprinting and liver biology. We used RAYYAN for the screening process and SyRF for data extraction. We used R for data analysis, and R and Graphpad PRISM for visualization. Results: Through our systematic database search we identified 1042 records, from which 63 met the eligibility criteria for inclusion in this systematic scoping review. Our findings revealed that extrusion-based printing, in conjunction with bioinks composed of natural components, emerged as the predominant printing technique in the bioprinting of liver models. Notably, the HepG2 hepatoma cell line was the most frequently employed liver cell type, despite acknowledged limitations. Furthermore, 51% of the printed models featured co-cultures with non-parenchymal cells to enhance their complexity. The included studies offered a variety of techniques for characterizing these liver models, with their primary application predominantly focused on toxicity testing. Among the frequently analyzed liver markers, albumin and urea stood out. Additionally, Cytochrome P450 (CYP) isoforms, primarily CYP3A and CYP1A, were assessed, and select studies employed nuclear receptor agonists to induce CYP activity. Conclusion: Our systematic scoping review offers an evidence-based overview and evaluation of the current state of research on bioprinted liver models, representing a promising and innovative technology for creating alternative organ models. We conducted a thorough examination of both the methodological and technical facets of model development and scrutinized the reporting quality within the realm of bioprinted liver models. This systematic scoping review can serve as a valuable template for systematically evaluating the progress of organ model development in various other domains. The transparently derived evidence presented here can provide essential support to the research community, facilitating the adaptation of technological advancements, the establishment of standards, and the enhancement of model robustness. This is particularly crucial as we work toward the long-term objective of establishing new approach methods as reliable alternatives to animal testing, with extensive and versatile applications.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

The marine biotoxin okadaic acid (OA) is produced by dinoflagellates and enters the human food chain by accumulating in the fatty tissue of filter-feeding shellfish. Consumption of highly contaminated shellfish can lead to diarrheic shellfish poisoning. However, apart from the acute effects in the intestine, OA can also provoke toxic effects in the liver, as it is able to pass the intestinal barrier into the blood stream. However, molecular details of OA-induced hepatotoxicity are still insufficiently characterized, and especially at the proteomic level data are scarce. In this study, we used human HepaRG liver cells and exposed them to non-cytotoxic OA concentrations for 24 hours. Global changes in protein expression were analyzed using 2-dimensional gel electrophoresis in combination with mass-spectrometric protein identification. The results constitute the first proteomic analysis of OA effects in human liver cells and indicate, amongst others, that OA affects the energy homeostasis, induces oxidative stress, and induces cytoskeletal changes.

Objective: Both obesity and exposure to chemicals may induce non-alcoholic fatty liver disease (NAFLD). Pregnane X Receptor (PXR) is a central target of metabolism disrupting chemicals and disturbs hepatic glucose and lipid metabolism. We hypothesized that the metabolic consequences of PXR activation may be modified by existing obesity and associated metabolic dysfunction. Methods: Wildtype and PXR knockout male mice were fed high-fat diet to induce obesity and metabolic dysfunction. PXR was activated with pregnenolone-16α-carbonitrile. Glucose metabolism, hepatosteatosis, insulin signaling, glucose uptake, liver glycogen, plasma and liver metabolomics, and liver, white adipose tissue, and muscle transcriptomics were investigated. Results: PXR activation aggravated obesity-induced liver steatosis by promoting lipogenesis and inhibiting fatty acid disposal. Accordingly, hepatic insulin sensitivity was impaired and circulating alanine aminotransferase level increased. Lipid synthesis was facilitated by increased liver glucose uptake and utilization of glycogen reserves resulting in dissociation of hepatosteatosis and hepatic insulin resistance from the systemic glucose tolerance and insulin sensitivity. Furthermore, glucagon-induced hepatic glucose production was impaired. PXR deficiency did not protect from the metabolic manifestations of obesity, but the liver transcriptomics and metabolomics profiling suggest diminished activation of inflammation and less prominent changes in the overall metabolite profile. Conclusions: Obesity and PXR activation by chemical exposure have a synergistic effect on NAFLD development. To support liver fat accumulation the PXR activation reorganizes glucose metabolism that seemingly improves systemic glucose metabolism. This implies that obese individuals, already predisposed to metabolic diseases, may be more susceptible to harmful metabolic effects of PXR-activating drugs and environmental chemicals.

Plastic particles are found almost ubiquitously in the environment and can get ingested orally by humans. We have used food-relevant microplastics (2 µm polylactic acid), submicroplastics (250 nm polylactic acid and 366 nm melamine formaldehyde resin) and nanoplastics (25 nm polymethylmethacrylate) to study material- and size-dependent uptake and transport across the human intestinal barrier and liver. Therefore, different Transwell™-based in vitro (co-)culture models were used: Differentiated Caco-2 cells mimicking the intestinal enterocyte monolayer, an M-cell model complementing the Caco-2 monoculture with antigen uptake-specialized cells, a mucus model complementing the barrier with an intestinal mucus layer, and an intestinal-liver co-culture combining differentiated Caco-2 cells with differentiated HepaRG cells. Using these complex barrier models, uptake and transport of particles were analyzed based on the fluorescence of the particles using confocal microscopy and a fluorescence-based quantification method. Additionally, the results were verified by Time-of-Flight - Secondary Ion Mass Spectrometry (ToF-SIMS) analysis. Furthermore, an effect screening at the mRNA level was done to investigate oxidative stress response, inflammation and changes to xenobiotic metabolism in intestinal and hepatic cells after exposure to plastic particles. Oxidative stress and inflammation were additionally analyzed using a flow-cytometric assay for reactive oxygen species and cytokine measurements. The results reveal a noteworthy uptake into and transport of microplastic and submicroplastic particles across the intestinal epithelium. Particularly, we show a pronounced uptake of particles into liver cells after crossing of the intestinal epithelium, using the intestinal-liver co-culture. The particles evoke some alterations in xenobiotic metabolism, but did not cause increased oxidative stress or inflammatory response on protein level. Taken together, these complex barrier models can be applied on micro-, submicro- and nanoplastics and reveal information in particle uptake, transport and cellular impact.

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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).

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.

Carcinogenic chemicals, or their metabolites, can be classified as genotoxic or non-genotoxic carcinogens (NGTxCs). Genotoxic compounds induce DNA damage, which can be detected by an established in vitro and in vivo battery of genotoxicity assays. For NGTxCs, DNA is not the primary target, and the possible modes of action (MoA) of NGTxCs are much more diverse than those of genotoxic compounds, and there is no specific in vitro assay for detecting NGTxCs. Therefore, the evaluation of the carcinogenic potential is still dependent on long-term studies in rodents. This 2-year bioassay, mainly applied for testing agrochemicals and pharmaceuticals, is time-consuming, costly and requires very high numbers of animals. More importantly, its relevance for human risk assessment is questionable due to the limited predictivity for human cancer risk, especially with regard to NGTxCs. Thus, there is an urgent need for a transition to new approach methodologies (NAMs), integrating human-relevant in vitro assays and in silico tools that better exploit the current knowledge of the multiple processes involved in carcinogenesis into a modern safety assessment toolbox. Here, we describe an integrative project that aims to use a variety of novel approaches to detect the carcinogenic potential of NGTxCs based on different mechanisms and pathways involved in carcinogenesis. The aim of this project is to contribute suitable assays for the safety assessment toolbox for an efficient and improved, internationally recognized hazard assessment of NGTxCs, and ultimately to contribute to reliable mechanism-based next-generation risk assessment for chemical carcinogens.

Okadaic acid (OA) is an algae-produced lipophilic marine biotoxin that accumulates in the fatty tissue of filter-feeding shellfish. Ingestion of contaminated shellfish leads to the diarrheic shellfish poisoning syndrome. Furthermore, several other effects of OA like genotoxicity, liver toxicity and tumor-promoting properties have been observed, probably linked to the phosphatase-inhibiting properties of the toxin. It has been shown that at high doses OA can disrupt the physical barrier of the intestinal epithelium. As the intestine and the liver do not only constitute a physical, but also a metabolic barrier against xenobiotic exposure, we here investigated the impact of OA on the expression of cytochrome P450 (CYP) enzymes and transporter proteins in human HepaRG cells liver cells in vitro at non-cytotoxic concentrations. The interplay of OA with known CYP inducers was also studied. Data show that the expression of various xenobiotic-metabolizing CYPs was downregulated after exposure to OA. Moreover, OA was able to counteract the activation of CYPs by their inducers. A number of transporters were also mainly downregulated. Overall, we demonstrate that OA has a significant effect on xenobiotic metabolism barrier in liver cells, highlighting the possibility for interactions of OA exposure with the metabolism of drugs and xenobiotics.

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.

Abstract The continuously increasing use of plastics is supposed to result in a rising exposure of MNPs to humans. Available data on human health risks of microplastics after oral uptake increased immensely in the past years and indicates very likely only low risks after oral consumption. Concerning nanoplastics, uptake, transport and potential adverse effects after oral uptake are less well understood. This study aims to investigate differences between microplastic particles and particles in the submicron- and nanoscaled size derived from food-relevant polymers with a particle size range consistent with higher potential for cellular uptake, fate, and effects when applied to human intestinal and liver cells. This work includes the development of cellular and subcellular detection methods for synthetic polymeric particles in the micro- and nanometer-range, using Scanning Electron Microscopy, Small-Angle X-ray and Dynamic Light Scattering methods, Asymmetric Flow Field Flow Fractionation, octanol-water fractionation, fluorescence microscopy and flow cytometry. Polylactic acid (250 nm and 2 μm (polydisperse)), melamine formaldehyde (366 nm) and polymethylmethacrylate (25 nm) were thoroughly characterized. The submicro- and nanoplastic test particles showed an increased uptake and transport quantity through intestinal cells. Both types of particles resulted in observed differences of uptake behavior, most likely influenced by different lipophilicity, which varied between the polymeric test materials. Toxic effects were detected after 24 h only in overload situations for the particles in the submicrometer range. This study provides further evidence for gastrointestinal uptake of submicro- and nanoplastics and points towards differences regarding bioavailability between microplastics and smaller plastic particles that may result following the ingestion of contaminated food and beverages. Furthermore, the results reinforce the importance for studying nanoplastics of different materials of varying size, surface properties, polymer composition and hydrophobicity.

The recent ban of some organic UV-filters – such as oxybenzone or octinoxate – considered toxic for coral reef, has heightened the need for eco-friendly alternatives, especially those synthesized using green approaches that reduce the carbon footprint. In this context, several thiobarbituric acid derivatives were synthetized from bio-based p-hydroxybenzaldehydes (e.g. vanillin, syringaldehyde) through a high-yielding sustainable Knoevenagel condensation, and their UV-filtering activity, stability and antiradical properties were investigated. The results showed promising UVA and blue light coverage over time, as well as competitive EC50 values in comparison to commercial antioxidants (i.e. BHA and BHT). In order to evaluate the potential of these molecules as substitutes to current petroleum-based UV-filters, the potential toxicity and fate in environment of these new compounds were evaluated in silico. This screening did not show a critical potential for toxicity, making them promising candidates for further in vitro and in vivo assessment.

ABSTRACTWith an increasing demand for safe and natural products from both industries and consumers, paired with the recent ban of decried molecules (i.e. octinoxate, avobenzone or octocrylene) due to their high negative impact on humans and the environment (i.e. endocrine disruption, coral bleaching), safe bio-based alternatives are a necessary and promising surrogate to substitute current commercialized petroleum-based UV filters. In this context, a class of bio-based molecules, displaying interesting UV-B filtering properties and great photostability were developed from furfural and 5-hydroxymethylfurfural (HMF), using the Knoevenagel condensation with a set of green conditions to minimize the impact on environment. Furthermore, those furfural- and HMF-based molecules demonstrated antimicrobial properties as secondary activity, highly sought by industries. Some furan derivatives being recognized to exhibit toxicological risks, in silico and in vitro assays were conducted and demonstrated the absence of endocrine disruption activity for these new molecules.

Abstract 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.

Abstract Nanoparticles exhibit a specific diffusion and sedimentation behavior under cell culture conditions as used in nantoxicological in vitro testing. How a particular particle suspension behaves depends on the particular physicochemical characteristics of the particles and the cell culture system. Only a fraction of the nanoparticles applied to a cell culture will thus reach the cells within a given time frame. Therefore, dosimetric calculations are essential not only to determine the exact fraction of nanoparticles that has come into contact with the cells, but also to ensure experimental comparability and correct interpretation of results, respectively. Yet, the use of published dosimetry models is limited. Not the least because the correct application of these in silico tools usually requires bioinformatics knowledge, which often is perceived a hurdle. Moreover, not all models are freely available and accessible. In order to overcome this obstacle, we have now developed an easy-to-use interface for our recently published 3DSDD dosimetry model, called NanoPASS (NanoParticle Administration Sedimentation Simulator). The interface is freely available to all researchers. It will facilitate the use of in silico dosimetry in nanotoxicology and thus improve interpretation and comparability of in vitro results in the field.

Mutations in genes encoding key players in oncogenic signaling pathways trigger specific downstream gene expression profiles in the respective tumor cell populations. While regulation of genes related to cell growth, survival, and death has been extensively studied, much less is known on the regulation of drug-metabolizing enzymes (DMEs) by oncogenic signaling. Here, a comprehensive review of the available literature is presented summarizing the impact of the most relevant genetic alterations in human and rodent liver tumors on the expression of DMEs with a focus on phases I and II of xenobiotic metabolism. Comparably few data are available with respect to DME regulation by p53-dependent signaling, telomerase expression or altered chromatin remodeling. By contrast, DME regulation by constitutive activation of oncogenic signaling via the RAS/RAF/mitogen-activated protein kinase (MAPK) cascade or via the canonical WNT/β-catenin signaling pathway has been analyzed in greater depth, demonstrating mostly positive-regulatory effects of WNT/β-catenin signaling and negative-regulatory effects of MAPK signaling. Mechanistic studies have revealed molecular interactions between oncogenic signaling and nuclear xeno-sensing receptors which underlie the observed alterations in DME expression in liver tumors. Observations of altered DME expression and inducibility in liver tumors with a specific gene expression profile may impact pharmacological treatment options. Key words: Xenobiotic metabolism, Hepatocytes, WNT/β-catenin signaling, RAS/MAPK signaling, Gene mutation, Cytochrome P450

Okadaic acid (OA) is a marine biotoxin that is produced by algae and accumulates in filter-feeding shellfish, through which it enters the human food chain, leading to diarrheic shellfish poisoning (DSP) after ingestion. Furthermore, additional effects of OA have been observed, such as cytotoxicity. Additionally, a strong downregulation of the expression of xenobiotic-metabolizing enzymes in the liver can be observed. The underlying mechanisms of this, however, remain to be examined. In this study, we investigated a possible underlying mechanism of the downregulation of cytochrome P450 (CYP) enzymes and the nuclear receptors pregnane X receptor (PXR) and retinoid-X-receptor alpha (RXRα) by OA through NF-κB and subsequent JAK/STAT activation in human HepaRG hepatocarcinoma cells. Our data suggest an activation of NF-κB signaling and subsequent expression and release of interleukins, which then activate JAK-dependent signaling and thus STAT3. Moreover, using the NF-κB inhibitors JSH-23 and Methysticin and the JAK inhibitors Decernotinib and Tofacitinib, we were also able to demonstrate a connection between OA-induced NF-κB and JAK signaling and the downregulation of CYP enzymes. Overall, we provide clear evidence that the effect of OA on the expression of CYP enzymes in HepaRG cells is regulated through NF-κB and subsequent JAK signaling.

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.

1,2-unsaturated Pyrrolizidine Alkaloids (PAs) are secondary plant metabolites that occur as food contaminants. Upon consumption, they can cause severe liver damage. PAs have been shown to induce apoptosis, to have cytotoxic and genotoxic effects, and to impair bile acid homeostasis in the human hepatoma cell line HepaRG. The major mode of action of PAs is DNA- and protein-adduct formation. Beyond that, nuclear receptor activation has only been observed for one receptor and two PAs, yielding the possibility that other cellular mediators are involved in PA-mediated toxicity. Here, the mode of action of Senecionine (Sc), a prominent and ubiquitous representative of hepatotoxic PAs, was investigated by analyzing 7 hepatic microRNAs (miRNAs) in HepaRG cells. Ultimately, 11 target genes that were predicted with Ingenuity Pathway Analysis software (IPA) were found to be significantly downregulated, while their assigned miRNAs showed significant upregulation of gene expression. According to IPA, these targets are positively correlated with apoptosis and cellular death and are involved in diseases such as hepatocellular carcinoma. Subsequent antagomiR-inhibition analysis revealed a significant correlation between PA-induced miRNA-4434 induction and P21-Activated Kinase-1 (PAK1) downregulation. PAK1 downregulation is usually associated with cell cycle arrest, suggesting a new function of Sc-mediated toxicity in human liver cells.

Abstract Background Even though a continuously high number of in vitro studies on nanoparticles are being published, the issue of correct dose matter is often not sufficiently taken into account. Due to their size, the diffusion of nanoparticles is slower, as compared to soluble chemicals, and they sediment slowly. Therefore, the administered dose of particles in in vitro experiments is not necessarily the same (effective) dose that comes into contact with the cellular system. This can lead to misinterpretations of experimental toxic effects and disturbs the meaningfulness of in vitro studies. In silico calculations of the effective nanoparticle dose can help circumventing this problem. Results This study addresses more complex in vitro models like the human intestinal cell line Caco-2 or the human liver cell line HepaRG, which need to be differentiated over a few weeks to reach their full complexity. During the differentiation time the cells grow up the wall of the cell culture dishes and therefore a three-dimensional-based in silico model of the nanoparticle dose was developed to calculate the administered dose received by different cell populations at the bottom and the walls of the culture dish. Moreover, the model can perform calculations based on the hydrodynamic diameter which is measured by light scattering methods, or based on the diffusion coefficient measured by nanoparticle tracking analysis (NTA). This 3DSDD (3D-sedimentation-diffusion-dosimetry) model was experimentally verified against existing dosimetry models and was applied to differentiated Caco-2 cells incubated with silver nanoparticles. Conclusions The 3DSDD accounts for the 3D distribution of cells in in vitro cell culture dishes and is therefore suitable for differentiated cells. To encourage the use of dosimetry calculating software, our model can be downloaded from the supporting information.

Abstract Diseases and toxins may lead to death of active liver tissue, resulting in a loss of total clearance capacity at the whole-body level. However, it remains difficult to study, whether the loss of metabolizing tissue is sufficient to explain loss of metabolic capacity of the liver or whether the surviving tissue undergoes an adaptive response to compensate the loss. To understand the cellular impact of toxic liver damage in an in vivo situation, we here used physiologically-based pharmacokinetic modelling to investigate pharmacokinetics of a specifically designed drug cocktail at three different sampling sites of the body in healthy mice and mice treated with carbon tetrachloride (CCl4). Liver zonation was explicitly quantified in the models through immunostaining of cytochrome P450s enzymes. Comparative analyses between the simulated decrease in clearance capacity and the experimentally measured loss in tissue volume indicated that CCl4-induced impairment of metabolic functions goes beyond the mere loss of metabolically active tissue. The here established integrative modelling strategy hence provides mechanistic insights into functional consequences of toxic liver damage in an in vivo situation, which would not have been accessible by conventional methods.

Abstract Insects and insect‐based food products have entered in the European market, carrying along issues of safety and the need of establishing a new legal framework. The consumption of massively reared insects can pose chemical and microbiological risks, and insect proteins are likely to represent a hazard for a subpopulation of allergic individuals. All insect‐based products are considered ‘Novel Food’ and fall under EU regulation 2015/2283, according to which a specific application to the European Commission, followed by EFSA scientific evaluation, is needed before the product is put on the market. The recent EU Regulation 2017/893, entered into force on 1 July 2017, allowed a shortlist of seven insect species to be included in the formulation of feeds for aquaculture. Previously, the addition of any insect to any feed for farmed animals was not allowed, due to the risk of prion‐derived diseases. The introduction of this new Regulation raises the issue to switch from a classical detection method based on microscopy to a more sophisticated and species‐specific method. The overall aims of this EU‐FORA project were (i) to set up a new next generation sequencing (NGS)‐based molecular method for the identification of insect DNA in feeds for aquaculture; and (ii) to carry out a conceptual work on a probabilistic quantitative risk assessment focused on the allergenicity of yellow mealworm (Tenebrio molitor) employed in foods.

Pyrrolizidine alkaloids (PAs) are a group of secondary plant metabolites being contained in various plant species. The consumption of contaminated food can lead to acute intoxications in humans and exert severe hepatotoxicity. The development of jaundice and elevated bile acid concentrations in blood have been reported in acute human PA intoxication, indicating a connection between PA exposure and the induction of cholestasis. Additionally, it is considered that differences in toxicity of individual PAs is based on their individual chemical structures. Therefore, we aimed to elucidate the structure-dependent disturbance of bile acid homeostasis by PAs in the human hepatoma cell line HepaRG. A set of 14 different PAs, including representatives of all major structural characteristics, namely, the four different necine bases retronecine, heliotridine, otonecine and platynecine and different grades of esterification, was analyzed in regard to the expression of genes involved in bile acid synthesis, metabolism and transport. Additionally, intra- and extracellular bile acid levels were analyzed after PA treatment. In summary, our data show significant structure-dependent effects of PAs on bile acid homeostasis. Especially PAs of diester type caused the strongest dysregulation of expression of genes associated with cholestasis and led to a strong decrease of intra- and extracellular bile acid concentrations.

1,2-unsaturated pyrrolizidine alkaloids (PAs) belong to a group of secondary plant metabolites. Exposure to PA-contaminated feed and food may cause severe hepatotoxicity. A pathway possibly involved in PA toxicity is the disturbance of bile acid homeostasis. Therefore, in this study, the influence of four structurally different PAs on bile acid homeostasis was investigated after single (24 h) and repeated (14 days) exposure using the human hepatoma cell line HepaRG. PAs induce a downregulation of gene expression of various hepatobiliary transporters, enzymes involved in bile acid synthesis, and conjugation, as well as several transcription regulators in HepaRG cells. This repression may lead to a progressive impairment of bile acid homeostasis, having the potential to accumulate toxic bile acids. However, a significant intracellular and extracellular decrease in bile acids was determined, pointing to an overall inhibition of bile acid synthesis and transport. In summary, our data clearly show that PAs structure-dependently impair bile acid homeostasis and secretion by inhibiting the expression of relevant genes involved in bile acid homeostasis. Furthermore, important biliary efflux mechanisms seem to be disturbed due to PA exposure. These mole-cular mechanisms may play an important role in the development of severe liver damage in PA-intoxicated humans.

Data from a comparative proteomic analysis of three human breast epithelial cell lines are presented. M13SV1 cells and their tumorigenic derivatives M13SV1-R2-2 and M13SV1-R2-N1 were used. Proteomic data were obtained using 2-dimensional gel electrophoresis and subsequent identification of proteins by MALDI-TOF mass spectrometry. In a second experiment, the three cell lines were treated with different concentrations of the estrogenic compounds β-estradiol or genistein and alterations in protein expression were monitored by 2-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. Presented data provide a comprehensive overview of proteomic differences between the three cell lines and their response to estrogenic stimulation.

Azole fungicides, especially triazole compounds, are widely used in agriculture and as pharmaceuticals. For a considerable number of agricultural azole fungicides, the liver has been identified as the main target organ of toxicity. A number of previous studies points towards an important role of nuclear receptors such as the constitutive androstane receptor (CAR), the pregnane-X-receptor (PXR), or the aryl hydrocarbon receptor (AHR), within the molecular pathways leading to hepatotoxicity of these compounds. Nuclear receptor-mediated hepatic effects may comprise rather adaptive changes such as the induction of drug-metabolizing enzymes, to hepatocellular hypertrophy, histopathologically detectable fatty acid changes, proliferation of hepatocytes, and the promotion of liver tumors. Here, we present a comprehensive review of the current knowledge of the interaction of major agricultural azole-class fungicides with the three nuclear receptors CAR, PXR, and AHR in vivo and in vitro. Nuclear receptor activation profiles of the azoles are presented and related to histopathological findings from classic toxicity studies. Important issues such as species differences and multi-receptor agonism and the consequences for data interpretation and risk assessment are discussed.

Abstract Heat processing of food gives rise to a plethora of chemical compounds whose toxicological effects are largely unknown. Due to a general lack of experimental toxicological data, assessing the risks associated with the consumption of these substances remains a challenge. Computer models that allow for an in silico prediction of physicochemical and toxicological characteristics, may be able to fill current data gaps and facilitate the risk assessment of toxicologically uncharacterised chemicals, their transformation products and their biological metabolites. The overall aims of the present project were for the fellow: (i) to get acquainted with the application of computational toxicological analyses tools in risk assessment based on results and experiences from previous research performed at the German Federal Institute for Risk Assessment (BfR); and (ii) to apply the newly gained skills on historic and novel data using updated and additional in silico tools. The project contributed to the continuous further education of the fellow in the use of computational toxicology tools, corroborated findings related to the safety of heat‐induced contaminants and laid the foundations for future collaborations between the fellow's home institution, the Institute of Marine Research (IMR) in Norway, and the BfR in Germany.