New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

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).

Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment.

Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85-92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86-92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97-101%), akin to a Matrigel-based liver model (83-102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.

Structural Elucidation of Agrochemical Metabolic Transformation Products Based on Infrared Ion Spectroscopy to Improve In Silico Toxicity Assessment.

Toxicological assessments of newly developed agrochemical agents consider chemical modifications and their metabolic and biotransformation products. To carry out an in silico hazard assessment, understanding the type of chemical modification and its location on the original compound can greatly enhance the reliability of the evaluation. Here, we present and apply a method based on liquid chromatography-mass spectrometry (LC-MS) enhanced with infrared ion spectroscopy (IRIS) to better delineate the molecular structures of transformation products before in silico toxicology evaluation. IRIS facilitates the recording of IR spectra directly in the mass spectrometer for features selected by retention time and mass-to-charge ratio. By utilizing quantum-chemically predicted IR spectra for candidate molecular structures, one can either derive the actual structure or significantly reduce the number of (isomeric) candidate structures. This approach can assist in making informed decisions. We apply this method to a plant growth stimulant, digeraniol sinapoyl malate (DGSM), that is currently under development. Incubation of the compound in Caco-2 and HepaRG cell lines in multiwell plates and analysis by LC-MS reveals oxidation, glucuronidation, and sulfonation metabolic products, whose structures were elucidated by IRIS and used as input for an in silico toxicology assessment. The toxicity of isomeric metabolites predicted by in silico tools was also assessed, which revealed that assigning the right metabolite structure is an important step in the overall toxicity assessment of the agrochemical. We believe this identification approach can be advantageous when specific isomers are significantly more hazardous than others and can help better understand metabolic pathways.

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.

New approach methodologies to facilitate and improve the hazard assessment of non-genotoxic carcinogens-a PARC project.

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.

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.

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.

Risk assessment and toxicological research on micro- and nanoplastics after oral exposure via food products.

Plastics are used ubiquitously and have become part of our everyday life. The global production of plastics is rising, which in consequence is leading to increasing amounts of plastics being released into the environment. Recently, the issue of human exposure to micro- and nanoplastic particles and potentially resulting toxicological consequences has been broached, triggered by the discovery of microplastics in foodstuff. In addition to dietary exposure via contaminated food and beverages, other exposure paths such as via air and cosmetics, have to be considered. Currently there is no legislation for microplastics and nanoplastics as contaminants in food. Substantial data gaps with respect to exposure as well as toxicity of such particles impede the risk assessment. Within this EU-FORA fellowship project, a comprehensive data mining approach was followed, focusing on up-to-date knowledge on the occurrence and possible toxic effects associated with micro- and nanoplastics after oral exposure, especially via food products and beverages, in order to provide a basis for risk assessment and to identify important research gaps. The fellowship project was further complemented by practical work aimed at the determination of in vitro toxicity of micro-sized polylactic acid particles.

Quantitative allergenicity risk assessment of food products containing yellow mealworm (Tenebrio molitor).

Insect-based foods are starting to enter the EU market, raising concerns about their safety. Allergic consumers might be exposed to even a greater risk, since insects have proven to trigger allergic symptoms, particularly in patients sensitised to crustaceans. Current legislation does not enforce producers to include insects in the list of allergenic ingredients. Food allergenicity risk assessment (FARA) is still at its infancy, and the debate on the need to define allergen thresholds is open. In this paper, we aimed at applying the concepts of stochastic quantitative FARA to describe present and future scenarios of exposure to foods containing Tenebrio molitor, the yellow mealworm. According to our risk characterisation, mealworm-based food products represent a major risk for individuals allergic to crustaceans to develop symptoms after the consumption of a dose lower than a serving size. Moreover, other allergic consumers might be at risk. A correct labelling of insect containing foods would help safeguarding the health of EU allergic consumers. Quantitatively assessing the risk of allergenicity provides a clear description of the problem, facilitating the decisional process of the risk manager, supporting the implementation of effective allergen management procedures and limiting the phenomenon of uninformative precautionary labelling.

Template for the description of cell-based toxicological test methods to allow evaluation and regulatory use of the data.

Only few cell-based test methods are described by Organisation for Economic Co-operation and Development (OECD) test guidelines or other regulatory references (e.g., the European Pharmacopoeia). The majority of toxicity tests still falls into the category of non-guideline methods. Data from these tests may nevertheless be used to support regulatory decisions or to guide strategies to assess compounds (e.g., drugs, agrochemicals) during research and development if they fulfill basic requirements concerning their relevance, reproducibility and predictivity. Only a method description of sufficient clarity and detail allows interpretation and use of the data. To guide regulators faced with increasing amounts of data from non-guideline studies, the OECD formulated Guidance Document 211 (GD211) on method documentation for the purpose of safety assessment. As GD211 is targeted mainly at regulators, it leaves scientists less familiar with regulation uncertain as to what level of detail is required and how individual questions should be answered. Moreover, little attention was given to the description of the test system (i.e., cell culture) and the steps leading to it being established in the guidance. To address these issues, an annotated toxicity test method template (ToxTemp) was developed (i) to fulfill all requirements of GD211, (ii) to guide the user concerning the types of answers and detail of information required, (iii) to include acceptance criteria for test elements, and (iv) to define the cells sufficiently and transparently. The fully annotated ToxTemp is provided here, together with reference to a database containing exemplary descriptions of more than 20 cell-based tests.

Insects in food and feed - allergenicity risk assessment and analytical detection.

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.

Nanomaterials: certain aspects of application, risk assessment and risk communication.

Development and market introduction of new nanomaterials trigger the need for an adequate risk assessment of such products alongside suitable risk communication measures. Current application of classical and new nanomaterials is analyzed in context of regulatory requirements and standardization for chemicals, food and consumer products. The challenges of nanomaterial characterization as the main bottleneck of risk assessment and regulation are presented. In some areas, e.g., quantification of nanomaterials within complex matrices, the establishment and adaptation of analytical techniques such as laser ablation inductively coupled plasma mass spectrometry and others are potentially suited to meet the requirements. As an example, we here provide an approach for the reliable characterization of human exposure to nanomaterials resulting from food packaging. Furthermore, results of nanomaterial toxicity and ecotoxicity testing are discussed, with concluding key criteria such as solubility and fiber rigidity as important parameters to be considered in material development and regulation. Although an analysis of the public opinion has revealed a distinguished rating depending on the particular field of application, a rather positive perception of nanotechnology could be ascertained for the German public in general. An improvement of material characterization in both toxicological testing as well as end-product control was concluded as being the main obstacle to ensure not only safe use of materials, but also wide acceptance of this and any novel technology in the general public.

[Nanomaterials in foodstuffs - toxicological properties and risk assessment]. / Nanomaterialien in Lebensmitteln ­ toxikologische Eigenschaften und Risikobewertung.

Nanomaterials measure below 100 nm in size in at least one dimension. In general, organic and inorganic nanoparticles can be distinguished. In addition, nanosized structures may differ substantially with regard to their shape, chemical composition and physical properties. They may originate either from natural processes or can be manufactured intentionally. The possible specific toxicological properties of nanoparticles that might be based on their enhanced reactivity due to an increased surface-to-volume ratio, as compared to larger particles, or which might result from preferential uptake in cells and tissues, are the subject of current toxicological research. Consumers are exposed to nanomaterials via the oral route by foodstuffs containing naturally formed, unknowingly incorporated or intentionally added nanoparticles. In the course of this work, an overview of the occurrence of nanomaterials in foodstuffs, including the legal definition and labeling requirements, will be given. Furthermore, specific characteristics of the oral uptake of nanomaterials will be presented alongside challenges for experimental investigation of gastrointestinal uptake and the effects of nanoscaled particles. These will be discussed in relation to potentially relevant toxicological properties and modes of action. Despite a plethora of available experimental studies, there are still knowledge gaps with regard to the exposure against orally ingested nanoparticles and with regard to possible toxicological consequences of such particles in order to allow for accurate risk assessment of the materials in foodstuffs and food packaging.