RISK-HUNT3R

Institutions
  • WG Leist (In vitro Toxikologie und Biomedizin)
Publications
  Leist, Marcel; Buettner, Andrea; Diel, Patrick; Eisenbrand, Gerhard; Epe, Bernd; Först, Petra; Grune, Tilman; Haller, Dirk; Heinz, Volker; Hengstler, Jan G. (2024): Controversy on health-based guidance values for bisphenol A : the need of criteria for studies that serve as a basis for risk assessment Archives of Toxicology. Springer. 2024, 98(7), S. 1967-1973. ISSN 0340-5761. eISSN 1432-0738. Verfügbar unter: doi: 10.1007/s00204-024-03778-3

Controversy on health-based guidance values for bisphenol A : the need of criteria for studies that serve as a basis for risk assessment

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Since 2006, the responsible regulatory bodies have proposed five health-based guidance values (HBGV) for bisphenol A (BPA) that differ by a factor of 250,000. This range of HBGVs covers a considerable part of the range from highly toxic to relatively non-toxic substances. As such heterogeneity of regulatory opinions is a challenge not only for scientific risk assessment but also for all stakeholders, the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG) analyzed the reasons for the current discrepancy and used this example to suggest improvements for the process of HBGV recommendations. A key aspect for deriving a HBGV is the selection of appropriate studies that allow the identification of a point of departure (PoD) for risk assessment. In the case of BPA, the HBGV derived in the 2023 EFSA assessment was based on a study that reported an increase of Th17 cells in mice with a benchmark dose lower bound (BMDL 40 ) of 0.53 µg/kg bw/day. However, this study does not comply with several criteria that are important for scientific risk assessment: (1) the selected end-point, Th17 cell frequency in the spleen of mice, is insufficiently understood with respect to health outcomes. (2) It is unclear, by which mechanism BPA may cause an increase in Th17 cell frequency. (3) It is unknown, if an increase of Th17 cell frequency in rodents is comparably observed in humans. (4) Toxicokinetics were not addressed. (5) Neither the raw data nor the experimental protocols are available. A further particularly important criterion (6) is independent data confirmation which is not available in the present case. Previous studies using other readouts did not observe immune-related adverse effects such as inflammation, even at doses orders of magnitude higher than in the Th17 cell-based study. The SKLM not only provides here key criteria for the use of such studies, but also suggests that the use of such a “checklist” requires a careful and comprehensive scientific judgement of each item. It is concluded that the Th17 cell-based study data do not represent an adequate basis for risk assessment of BPA.

Origin (projects)

  Renner, Nadine; Schöb, Franziska; Pape, Regina; Suciu, Ilinca; Spreng, Anna-Sophie; Ückert, Anna-Katharina; Cöllen, Eike; Bovio, Federica; Leist, Marcel; Schildknecht, Stefan (2024): Modeling ferroptosis in human dopaminergic neurons : Pitfalls and opportunities for neurodegeneration research Redox Biology. Elsevier. 2024, 73, 103165. eISSN 2213-2317. Available under: doi: 10.1016/j.redox.2024.103165

Modeling ferroptosis in human dopaminergic neurons : Pitfalls and opportunities for neurodegeneration research

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The activation of ferroptosis is being pursued in cancer research as a strategy to target apoptosis-resistant cells. By contrast, in various diseases that affect the cardiovascular system, kidneys, liver, and central and peripheral nervous systems, attention is directed toward interventions that prevent ferroptotic cell death. Mechanistic insights into both research areas stem largely from studies using cellular in vitro models. However, intervention strategies that show promise in cellular test systems often fail in clinical trials, which raises concerns regarding the predictive validity of the utilized in vitro models.



In this study, the human LUHMES cell line, which serves as a model for human dopaminergic neurons, was used to characterize factors influencing the activation of ferroptosis. Erastin and RSL-3 induced cell death that was distinct from apoptosis. Parameters such as the differentiation state of LUHMES cells, cell density, and the number and timing of medium changes were identified as determinants of sensitivity to ferroptosis activation. In differentiated LUHMES cells, interventions at mechanistically divergent sites (iron chelation, coenzyme Q10, peroxidase mimics, or inhibition of 12/15-lipoxygenase) provide almost complete protection from ferroptosis. LUHMES cells allowed the experimental modulation of intracellular iron concentrations and demonstrated a correlation between intracellular iron levels, the rate of lipid peroxidation, as well as the sensitivity of the cells to ferroptotic cell death.



These findings underscore the importance of understanding the various factors that influence ferroptosis activation and highlight the need for well-characterized in vitro models to enhance the reliability and predictive value of observations in ferroptosis research, particularly when translating findings into in vivo contexts.

Origin (projects)

  Pamies, David; Ekert, Jason; Zurich, Marie-Gabrielle; Frey, Olivier; Werner, Sophie; Piergiovanni, Monica; Freedman, Benjamin S.; Keong Teo, Adrian Kee; Hartung, Thomas; Leist, Marcel (2024): Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility Stem Cell Reports. Elsevier. 2024, 19(5), pp. 604-617. eISSN 2213-6711. Available under: doi: 10.1016/j.stemcr.2024.03.009

Recommendations on fit-for-purpose criteria to establish quality management for microphysiological systems and for monitoring their reproducibility

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Cell culture technology has evolved, moving from single-cell and monolayer methods to 3D models like reaggregates, spheroids, and organoids, improved with bioengineering like microfabrication and bioprinting. These advancements, termed microphysiological systems (MPSs), closely replicate tissue environments and human physiology, enhancing research and biomedical uses. However, MPS complexity introduces standardization challenges, impacting reproducibility and trust. We offer guidelines for quality management and control criteria specific to MPSs, facilitating reliable outcomes without stifling innovation. Our fit-for-purpose recommendations provide actionable advice for achieving consistent MPS performance.

Origin (projects)

  Tal, Tamara; Myhre, Oddvar; Fritsche, Ellen; Rüegg, Joëlle; Craenen, Kai; Aiello-Holden, Kiara; Agrillo, Caroline; Leist, Marcel; Pallocca, Giorgia; Bartmann, Kristina (2024): New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project Frontiers in Toxicology. Frontiers. 2024, 6, 1359507. eISSN 2673-3080. Available under: doi: 10.3389/ftox.2024.1359507

New approach methods to assess developmental and adult neurotoxicity for regulatory use: a PARC work package 5 project

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In the European regulatory context, rodent in vivo studies are the predominant source of neurotoxicity information. Although they form a cornerstone of neurotoxicological assessments, they are costly and the topic of ethical debate. While the public expects chemicals and products to be safe for the developing and mature nervous systems, considerable numbers of chemicals in commerce have not, or only to a limited extent, been assessed for their potential to cause neurotoxicity. As such, there is a societal push toward the replacement of animal models with in vitro or alternative methods. New approach methods (NAMs) can contribute to the regulatory knowledge base, increase chemical safety, and modernize chemical hazard and risk assessment. Provided they reach an acceptable level of regulatory relevance and reliability, NAMs may be considered as replacements for specific in vivo studies. The European Partnership for the Assessment of Risks from Chemicals (PARC) addresses challenges to the development and implementation of NAMs in chemical risk assessment. In collaboration with regulatory agencies, Project 5.2.1e (Neurotoxicity) aims to develop and evaluate NAMs for developmental neurotoxicity (DNT) and adult neurotoxicity (ANT) and to understand the applicability domain of specific NAMs for the detection of endocrine disruption and epigenetic perturbation. To speed up assay time and reduce costs, we identify early indicators of later-onset effects. Ultimately, we will assemble second-generation developmental neurotoxicity and first-generation adult neurotoxicity test batteries, both of which aim to provide regulatory hazard and risk assessors and industry stakeholders with robust, speedy, lower-cost, and informative next-generation hazard and risk assessment tools.

Origin (projects)

    Cöllen, Eike; Tanaskov, Yaroslav; Holzer, Anna-Katharina; Dipalo, Michele; Schäfer, Jasmin; Kraushaar, Udo; Leist, Marcel (2024): Elements and development processes for test methods in toxicology and human health-relevant life science research Alternatives to Animal Experimentation : ALTEX. Springer. 2024, 41(1), pp. 142-148. ISSN 1868-596X. eISSN 1868-8551. Available under: doi: 10.14573/altex.2401041

Elements and development processes for test methods in toxicology and human health-relevant life science research

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Many laboratory procedures generate data on properties of chemicals, but they cannot be equated with toxicological “test methods”. This apparent discrepancy is not limited to in vitro testing, using animal-free new approach methods (NAM), but also applies to animal-based testing approaches. Here, we give a brief overview of the differences between data generation and the setup or use of a complete test method. While there is excellent literature available on this topic for specialists (GIVIMP guidance; ToxTemp overview), a brief overview and easily-accessible entry point may be useful for a broader community. We provide a single figure to summarize all test method elements and processes required in the development (setup and adaptation) of a test method. The exposure scheme, the endpoint, and the test system are briefly outlined as fundamental elements of any test method. A rationale is provided, why they are not sufficient. We then explain the importance and role of purpose definition (including some information on what is modelled) and the prediction model, aka data interpretation procedure, which depends on the purpose definition, as further essential elements. This connection exemplifies that all fundamental elements are interdependent, and none can be omitted. Finally, discussion is provided on validation as a measure to provide confidence in the reliability, performance, and relevance of a test method. In this sense, validation may be considered a sixth fundamental element for practical use of test methods.

Origin (projects)

  Smirnova, Lena; Hogberg, Helena T.; Leist, Marcel; Hartung, Thomas (2024): Revolutionizing developmental neurotoxicity testing : a journey from animal models to advanced in vitro systems Alternatives to Animal Experimentation : ALTEX. ALTEX Edition. 2024, 41(2), pp. 152-178. ISSN 1868-596X. Available under: doi: 10.14573/altex.2403281

Revolutionizing developmental neurotoxicity testing : a journey from animal models to advanced in vitro systems

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Developmental neurotoxicity (DNT) testing has seen enormous progress over the last two decades. Preceding even the publication of the animal-based OECD test guideline for DNT testing in 2007, a series of non-animal technology workshops and conferences that started in 2005 has shaped a community that has delivered a comprehensive battery of in vitro test methods (DNT IVB). Its data interpretation is now covered by a very recent OECD guidance (No. 377). Here, we overview the progress in the field, focusing on the evolution of testing strategies, the role of emerging technol­ogies, and the impact of OECD test guidelines on DNT testing. In particular, this is an example of the targeted development of an animal-free testing approach for one of the most complex hazards of chemicals to human health. These developments started literally from a blank slate, with no proposed alternative methods available. Over two decades, cutting-edge science enabled the design of a testing approach that spares animals and enables throughput to address this challenging hazard. While it is evident that the field needs guidance and regulation, the massive economic impact of decreased human cognitive capacity caused by chemical exposure should be prioritized more highly. Beyond this, the claim to fame of DNT in vitro testing is the enormous scientific progress it has brought for understanding the human brain, its development, and how it can be perturbed.

Origin (projects)

    Botham, Philip; Cronin, Mark T. D.; Currie, Richard; Doe, John; Funk-Weyer, Dorothee; Gant, Timothy W.; Leist, Marcel; Marty, Sue; van Ravenzwaay, Bennard; Westmoreland, Carl (2023): Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology Archives of Toxicology. Springer. 2023, 97(12), pp. 3075-3083. ISSN 0340-5761. eISSN 1432-0738. Available under: doi: 10.1007/s00204-023-03601-5

Analysis of health concerns not addressed by REACH for low tonnage chemicals and opportunities for new approach methodology

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In Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) the criterion for deciding the studies that must be performed is the annual tonnage of the chemical manufactured or imported into the EU. The annual tonnage may be considered as a surrogate for levels of human exposure but this does not take into account the physico-chemical properties and use patterns that determine exposure. Chemicals are classified using data from REACH under areas of health concern covering effects on the skin and eye; sensitisation; acute, repeated and prolonged systemic exposure; effects on genetic material; carcinogenicity; and reproduction and development. We analysed the mandated study lists under REACH for each annual tonnage band in terms of the information they provide on each of the areas of health concern. Using the European Chemicals Agency (ECHA) REACH Registration data base of over 20,000 registered substances, we found that only 19% of registered substances have datasets on all areas of health concern. Information limited to acute exposure, sensitisation and genotoxicity was found for 62%. The analysis highlighted the shortfall of information mandated for substances in the lower tonnage bands. Deploying New Approach Methodologies (NAMs) at this lower tonnage band to assess health concerns which are currently not covered by REACH, such as repeat and extended exposure and carcinogenicity, would provide additional information and would be a way for registrants and regulators to gain experience in the use of NAMs. There are currently projects in Europe aiming to develop NAM-based assessment frameworks and they could find their first use in assessing low tonnage chemicals once confidence has been gained by their evaluation with data rich chemicals.

Origin (projects)

  Seidel, Florian; Kappenberg, Franziska; Fayyaz, Susann; Scholtz-Illigens, Andreas; Cherianidou, Anna; Derksen, Katharina; Nell, Patrick; Marchan, Rosemarie; Leist, Marcel; Hengstler, Jan G. (2023): Risk assessment of parabens in a transcriptomics-based in vitro test Chemico-Biological Interactions. Elsevier. 2023, 384, 110699. ISSN 0009-2797. eISSN 1872-7786. Available under: doi: 10.1016/j.cbi.2023.110699

Risk assessment of parabens in a transcriptomics-based in vitro test

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Parabens have been used for decades as preservatives in food, drugs and cosmetics. The majority however, were banned in 2009 and 2014 leaving only methyl-, ethyl-, propyl-, and butyl-derivates available for subsequent use. Methyl- and propylparaben have been extensively tested in vivo, with no resulting evidence for developmental and reproductive toxicity (DART). In contrast, ethylparaben has not yet been tested for DART in animal experiments, and it is currently debated if additional animal studies are warranted. In order to perform a comparison of the four currently approved parabens, we used a previously established in vitro test based on human induced pluripotent stem cells (iPSC) that are exposed to test substances during their differentiation to neuroectodermal cells. EC50 values for cytotoxicity were 906 μM, 698 μM, 216 μM and 63 μM for methyl-, ethyl-, propyl- and butylparaben, respectively, demonstrating that cytotoxicity increases with increasing alkyl chain length. Genome-wide analysis demonstrated that FDR-adjusted significant gene expression changes occurred only at cytotoxic or close to cytotoxic concentrations, for example 1720 differentially expressed genes (DEG) at 1000 μM ethylparaben, 1 DEG at 316 μM, and no DEG at 100 μM or lower concentrations. The highest concentration of ethylparaben that did not induce any cytotoxicity nor DEG was 1670-fold above the highest published concentrations reported in biomonitoring studies (60 nM ethylparaben in cord blood). In conclusion, cytotoxicity and gene expression alterations of ethylparaben occurred at concentrations of approximately three orders of magnitude above human blood concentrations; moreover, the substance fitted well into a scenario where toxicity increases with the alkyl chain length, and gene expression changes only occur at cytotoxic or close to cytotoxic concentrations. Therefore, no evidence was obtained suggesting that in vivo DART with ethylparaben would lead to different results as the methyl- or propyl derivates.

Origin (projects)

    Ückert, Anna-Katharina; Rütschlin, Sina; Gutbier, Simon; Hauer, Isa; Holzer, Anna-Katharina; Meyburg, Birthe; Mix, Ann-Kathrin; Hauck, Christof R.; Böttcher, Thomas; Leist, Marcel (2023): Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration Environment International. Elsevier. 2023, 180, 108229. ISSN 0160-4120. eISSN 1873-6750. Available under: doi: 10.1016/j.envint.2023.108229

Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration

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The causes of nigrostriatal cell death in idiopathic Parkinson’s disease are unknown, but exposure to toxic chemicals may play some role. We followed up here on suggestions that bacterial secondary metabolites might be selectively cytotoxic to dopaminergic neurons. Extracts from Streptomyces venezuelae were found to kill human dopaminergic neurons (LUHMES cells). Utilizing this model system as a bioassay, we identified a bacterial metabolite known as aerugine (C10H11NO2S; 2-[4-(hydroxymethyl)-4,5-dihydro-1,3-thiazol-2-yl]phenol) and confirmed this finding by chemical re-synthesis. This 2-hydroxyphenyl-thiazoline compound was previously shown to be a product of a wide-spread biosynthetic cluster also found in the human microbiome and in several pathogens. Aerugine triggered half-maximal dopaminergic neurotoxicity at 3-4 µM. It was less toxic for other neurons (10-20 µM), and non-toxic (at <100 µM) for common human cell lines. Neurotoxicity was completely prevented by several iron chelators, by distinct anti-oxidants and by a caspase inhibitor. In the Caenorhabditis elegans model organism, general survival was not affected by aerugine concentrations up to 100 µM. When transgenic worms, expressing green fluorescent protein only in their dopamine neurons, were exposed to aerugine, specific neurodegeneration was observed. The toxicant also exerted functional dopaminergic toxicity in nematodes as determined by the “basal slowing response” assay. Thus, our research has unveiled a bacterial metabolite with a remarkably selective toxicity toward human dopaminergic neurons in vitro and for the dopaminergic nervous system of Caenorhabditis elegans in vivo. These findings suggest that microbe-derived environmental chemicals should be further investigated for their role in the pathogenesis of Parkinson's disease.

Origin (projects)

    Grillberger, Karin; Cöllen, Eike; Trivisani, Claudia Immacolata; Blum, Jonathan; Leist, Marcel; Ecker, Gerhard F. (2023): Structural Insights into Neonicotinoids and N-Unsubstituted Metabolites on Human nAChRs by Molecular Docking, Dynamics Simulations, and Calcium Imaging International Journal of Molecular Sciences. MDPI. 2023, 24(17), 13170. ISSN 1661-6596. eISSN 1422-0067. Available under: doi: 10.3390/ijms241713170

Structural Insights into Neonicotinoids and N-Unsubstituted Metabolites on Human nAChRs by Molecular Docking, Dynamics Simulations, and Calcium Imaging

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Neonicotinoid pesticides were initially designed in order to achieve species selectivity on insect nicotinic acetylcholine receptors (nAChRs). However, concerns arose when agonistic effects were also detected in human cells expressing nAChRs. In the context of next-generation risk assessments (NGRAs), new approach methods (NAMs) should replace animal testing where appropriate. Herein, we present a combination of in silico and in vitro methodologies that are used to investigate the potentially toxic effects of neonicotinoids and nicotinoid metabolites on human neurons. First, an ensemble docking study was conducted on the nAChR isoforms α7 and α3β4 to assess potential crucial molecular initiating event (MIE) interactions. Representative docking poses were further refined using molecular dynamics (MD) simulations and binding energy calculations using implicit solvent models. Finally, calcium imaging on LUHMES neurons confirmed a key event (KE) downstream of the MIE. This method was also used to confirm the predicted agonistic effect of the metabolite descyano-thiacloprid (DCNT).

Origin (projects)

    Butera, Alessio; Smirnova, Lena; Ferrando-May, Elisa; Hartung, Thomas; Brunner, Thomas; Leist, Marcel; Amelio, Ivano (2023): Deconvoluting gene and environment interactions to develop an “epigenetic score meter” of disease EMBO Molecular Medicine. Wiley. 2023, 15(9), e18208. ISSN 1757-4676. eISSN 1757-4684. Available under: doi: 10.15252/emmm.202318208

Deconvoluting gene and environment interactions to develop an “epigenetic score meter” of disease

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Human health is determined both by genetics (G) and environment (E). This is clearly illustrated in groups of individuals who are exposed to the same environmental factor showing differential responses. A quantitative measure of the gene–environment interactions (GxE) effects has not been developed and in some instances, a clear consensus on the concept has not even been reached; for example, whether cancer is predominantly emerging from “bad luck” or “bad lifestyle” is still debated. In this article, we provide a panel of examples of GxE interaction as drivers of pathogenesis. We highlight how epigenetic regulations can represent a common connecting aspect of the molecular bases. Our argument converges on the concept that the GxE is recorded in the cellular epigenome, which might represent the key to deconvolute these multidimensional intricated layers of regulation. Developing a key to decode this epigenetic information would provide quantitative measures of disease risk. Analogously to the epigenetic clock introduced to estimate biological age, we provocatively propose the theoretical concept of an “epigenetic score‐meter” to estimate disease risk.

Origin (projects)

    Suciu, Ilinca; Pamies, David; Peruzzo, Roberta; Wirtz, Petra H.; Pallocca, Giorgia; Hauck, Christof R.; Brunner, Thomas; Hartung, Thomas; Amelio, Ivano; Leist, Marcel (2023): G × E interactions as a basis for toxicological uncertainty Archives of Toxicology. Springer. 2023, 97(7), pp. 2035-2049. ISSN 0340-5761. eISSN 1432-0738. Available under: doi: 10.1007/s00204-023-03500-9

G × E interactions as a basis for toxicological uncertainty

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To transfer toxicological findings from model systems, e.g. animals, to humans, standardized safety factors are applied to account for intra-species and inter-species variabilities. An alternative approach would be to measure and model the actual compound-specific uncertainties. This biological concept assumes that all observed toxicities depend not only on the exposure situation (environment = E), but also on the genetic (G) background of the model (G  ×  E). As a quantitative discipline, toxicology needs to move beyond merely qualitative G  ×  E concepts. Research programs are required that determine the major biological variabilities affecting toxicity and categorize their relative weights and contributions. In a complementary approach, detailed case studies need to explore the role of genetic backgrounds in the adverse effects of defined chemicals. In addition, current understanding of the selection and propagation of adverse outcome pathways (AOP) in different biological environments is very limited. To improve understanding, a particular focus is required on modulatory and counter-regulatory steps. For quantitative approaches to address uncertainties, the concept of “genetic” influence needs a more precise definition. What is usually meant by this term in the context of G  ×  E are the protein functions encoded by the genes. Besides the g ene sequence, the regulation of the gene expression and function should also be accounted for. The widened concept of past and present “ g ene expression” influences is summarized here as G e . Also, the concept of “environment” needs some re-consideration in situations where exposure timing (E t ) is pivotal: prolonged or repeated exposure to the insult (chemical, physical, life style) affects G e . This implies that it changes the model system. The interaction of G e with E t might be denoted as G e  ×  E t . We provide here general explanations and specific examples for this concept and show how it could be applied in the context of New Approach Methodologies (NAM).

Origin (projects)

    Rovida, Costanza; Busquet, Francois; Leist, Marcel; Hartung, Thomas (2023): REACH out-numbered! : The future of REACH and animal numbers Alternatives to Animal Experimentation : ALTEX. Springer. 2023, 40(3), pp. 367-388. ISSN 1868-596X. eISSN 1868-8551. Available under: doi: 10.14573/altex.2307121

REACH out-numbered! : The future of REACH and animal numbers

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The EU’s REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) Regulation requires animal testing only as a last resort. However, our study (Knight et al., 2023) in this issue reveals that approximately 2.9 million animals have been used for REACH testing for reproductive toxicity, developmental toxicity, and repeated-dose toxicity alone as of December 2022. Currently, additional tests requiring about 1.3 million more animals are in the works. As compliance checks continue, more animal tests are anticipated. According to the European Chemicals Agency (ECHA), 75% of read-across methods have been rejected during compliance checks. Here, we estimate that 0.6 to 3.2 million animals have been used for other endpoints, likely at the lower end of this range. The ongoing discussion about the grouping of 4,500 regis­tered petrochemicals can still have a major impact on these numbers. The 2022 amendment of REACH is estimated to add 3.6 to 7.0 million animals. This information comes as the European Parliament is set to consider changes to REACH that could further increase animal testing. Two proposals currently under discussion would likely necessitate new animal testing: extending the requirement for a chemical safety assessment (CSA) to Annex VII substances could add 1.6 to 2.6 million animals, and the registration of polymers adds a challenge comparable to the petrochemical discussion. These findings high­light the importance of understanding the current state of REACH animal testing for the upcoming debate on REACH revisions as an opportunity to focus on reducing animal use.

Origin (projects)

    Holzer, Anna-Katharina; Dreser, Nadine; Pallocca, Giorgia; Mangerich, Aswin; Stacey, Glyn; Dipalo, Michael; Rovida, Costanza; Wirtz, Petra H.; Hartung, Thomas; Leist, Marcel (2023): Acceptance criteria for new approach methods in toxicology and human health-relevant life science research – part I Alternatives to Animal Experimentation : ALTEX. Springer Spektrum. 2023, 40(4), pp. 706-712. eISSN 1868-596X. Available under: doi: 10.14573/altex.2310021

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

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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.

Origin (projects)

    Knight, Jean; Hartung, Thomas; Rovida, Costanza (2023): 4.2 million and counting… The animal toll for REACH systemic toxicity studies Alternatives to Animal Experimentation : ALTEX. Springer. 2023, 40(3), pp. 389-407. ISSN 1868-596X. eISSN 1868-8551. Available under: doi: 10.14573/altex.2303201

4.2 million and counting… The animal toll for REACH systemic toxicity studies

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The EU’s chemicals regulation, REACH, requires that most chemicals in the EU be evaluated for human health and ecosystem risks, with a mandate to minimize use of animal tests for these evaluations. The REACH process has been ongoing since about 2008, but a calculation of the resulting animal use is not publicly available. For this reason, we have undertaken a count of animals used for REACH. With EU legislators set to consider REACH revisions that could expand animal testing, we are releasing results for test categories counted to date: reproductive toxicity tests, developmental toxicity tests, and repeat­ed-dose toxicity tests for human health. The total animal count as of December 2022 for these categories is about 2.9 million. Additional tests involving about 1.3 million animals are currently required by a final proposal authorization or compliance check but not yet completed. The total, 4.2 million, for just these three test categories exceeds the original European Com­mission forecast of 2.6 million for all REACH tests. The difference is primarily because the European Commission estimate excluded offspring, which are most of the animals used for REACH. Other reasons for the difference are extra animals included in tests to ensure sufficient survive to meet the minimum test requirement; dose range-finding tests; extra test animal groups, e.g., for recovery analysis; and a high rejection rate of read-across studies. Given higher than forecast animal use, the upcoming debate on proposed REACH revisions is an opportunity to refocus on reducing animal numbers in keeping with the REACH mandate.

Origin (projects)

    Kranaster, Petra; Blum, Jonathan; Dold, Jeremias E.G.A.; Wittmann, Valentin; Leist, Marcel (2023): Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells Journal of Neurochemistry. Wiley. 2023, 164(4), pp. 481-498. eISSN 1471-4159. Available under: doi: 10.1111/jnc.15737

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells

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Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of 'target-specific MGE', we found that in dopaminergic or sensory neurons > 60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.

Origin (projects)

    Neuhaus, Winfried; Reininger-Gutmann, Birgit; Rinner, Beate; Plasenzotti, Roberto; Wilflingseder, Doris; De Kock, Joery; Hartung, Thomas; Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2022): The Current Status and Work of Three Rs Centres and Platforms in Europe Alternatives to Laboratory Animals : ATLA. Sage. 2022, 50(6), pp. 381-413. ISSN 0261-1929. eISSN 2632-3559. Available under: doi: 10.1177/02611929221140909

The Current Status and Work of Three Rs Centres and Platforms in Europe

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The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Origin (projects)

    Holzer, Anna-Katharina; Karreman, Christiaan; Suciu, Ilinca; Furmanowsky, Lara-Seline; Wohlfarth, Harald; Loser, Dominik; Dirks, Wilhelm G.; Pardo González, Emilio; Leist, Marcel (2022): Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions Stem Cells Translational Medicine. Oxford University Press (OUP). 2022, 11(7), pp. 727-741. ISSN 2157-6564. eISSN 2157-6580. Available under: doi: 10.1093/stcltm/szac031

Generation of Human Nociceptor-Enriched Sensory Neurons for the Study of Pain-Related Dysfunctions

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In vitro models of the peripheral nervous system would benefit from further refinements to better support studies on neuropathies. In particular, the assessment of pain-related signals is still difficult in human cell cultures. Here, we harnessed induced pluripotent stem cells (iPSCs) to generate peripheral sensory neurons enriched in nociceptors. The objective was to generate a culture system with signaling endpoints suitable for pharmacological and toxicological studies. Neurons generated by conventional differentiation protocols expressed moderate levels of P2X3 purinergic receptors and only low levels of TRPV1 capsaicin receptors, when maturation time was kept to the upper practically useful limit of 6 weeks. As alternative approach, we generated cells with an inducible NGN1 transgene. Ectopic expression of this transcription factor during a defined time window of differentiation resulted in highly enriched nociceptor cultures, as determined by functional (P2X3 and TRPV1 receptors) and immunocytochemical phenotyping, complemented by extensive transcriptome profiling. Single cell recordings of Ca2+-indicator fluorescence from >9000 cells were used to establish the "fraction of reactive cells" in a stimulated population as experimental endpoint, that appeared robust, transparent and quantifiable. To provide an example of application to biomedical studies, functional consequences of prolonged exposure to the chemotherapeutic drug oxaliplatin were examined at non-cytotoxic concentrations. We found (i) neuronal (allodynia-like) hypersensitivity to otherwise non-activating mechanical stimulation that could be blocked by modulators of voltage-gated sodium channels; (ii) hyper-responsiveness to TRPV1 receptor stimulation. These findings and several other measured functional alterations indicate that the model is suitable for pharmacological and toxicological studies related to peripheral neuropathies.

Origin (projects)

    Pallocca, Giorgia; Moné, Martijn J.; Kamp, Hennicke; Luijten, Mirjam; Van de Water, Bob; Leist, Marcel (2022): Next-generation risk assessment of chemicals - Rolling out a human-centric testing strategy to drive 3R implementation : The RISK-HUNT3R project perspective Alternatives to Animal Experimentation : ALTEX. Springer Spektrum. 2022, 39(3), pp. 419-426. eISSN 1868-596X. Available under: doi: 10.14573/altex.2204051

Next-generation risk assessment of chemicals - Rolling out a human-centric testing strategy to drive 3R implementation : The RISK-HUNT3R project perspective

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In many industrial sectors, there is a need for reliable ways to evaluate the safety of chemicals with methods anchored to human biology and pathology. For this purpose, many animal-free new approach methods (NAMs) have been developed and implemented in various stages of risk assessment. Now it is time to assemble individual NAMs into a comprehensive next-generation risk assessment (NGRA) strategy. The European Horizon 2020 RISK-HUNT3R project ("Risk assessment of chemicals integrating human-centric next-generation testing strategies promoting the 3Rs") has been designed to promote a combination of computational toxicology, in vitro toxicology, and systems biology. It is assumed that this approach will lead to faster and more accurate risk assessment procedures. The RISK-HUNT3R NGRA strategy will be developed to address the implementation of a comprehensive NAM toolbox into the regulatory framework. Critical conceptual approaches of the project include i) the integration of human-relevant data on biotransformation and elimination, ii) the translation of high-content mode-of-action datasets into predictions of adverse outcomes, iii) development of quantitative adverse outcome pathways (qAOPs), and iv) quantification of uncertainties associated with the predictions based on NGRA strategies. Many of the project steps will be used iteratively to generate datasets with sufficient quality and certainty for NGRA. Scientists and regulators will work together on case studies to evaluate NAMs' practical applicability and the strategies to combine information therefrom. Here we delineate how the strategy will be deployed to establish an overall NGRA framework for chemicals, pesticides, food additives, and drugs.

Origin (projects)

    Pallocca, Giorgia; Leist, Marcel (2022): On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains Alternatives to Animal Experimentation : ALTEX. Springer Spektrum. 2022, 39(3), pp. 531-539. ISSN 1868-596X. eISSN 1868-8551. Available under: doi: 10.14573/altex.2207111

On the usefulness of animals as a model system (part II) : Considering benefits within distinct use domains

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In many countries, animal experiments can only be performed when their necessity has been demonstrated in a legal document. As the usefulness of animals in research is also a significant societal and political issue, criteria to structure debates and evaluations are needed. Here, background information is given on laboratory animal studies. Moreover, parameters that may be considered in judging their usefulness are suggested. The discussion is strictly focused on animals used as tools/test systems/models to provide information on humans. In this context, general features and performance characteristics of models are discussed. Examples are given for well-recognized criteria (e.g., robustness, relevance, predictivity) to judge the usefulness of predictive models. The main hypothesis put forward here is that a benefits evaluation (usefulness metrics) is only possible within sharply circumscribed "use domains". Examples are given for the research fields of drug and vaccine research, toxicology, disease pathogenesis, and basic biological research. Efficacy, safety, and quality studies are highlighted as "use domains" within the field of drug discovery and production. A further separation into individual diseases, drug targets or symptoms is suggested for, e.g., efficacy studies or pathophysiology. Finally, an outlook is given on the evaluation of model advantages and disadvantages to arrive at their "net benefit". Moreover, the need to compare the net benefits of animal models versus that of their alternatives is highlighted.

Origin (projects)

    Pallocca, Giorgia; Rovida, Costanza; Leist, Marcel (2022): On the usefulness of animals as a model system (part I) : Overview of criteria and focus on robustness Alternatives to Animal Experimentation : ALTEX. Springer Spektrum. 2022, 39(2), pp. 347-353. eISSN 1868-596X. Available under: doi: 10.14573/altex.2203291

On the usefulness of animals as a model system (part I) : Overview of criteria and focus on robustness

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Banning or reduction of the use of animals for laboratory experiments is a frequently-discussed societal and scientific issue. Moreover, the usefulness of animals needs to be considered in any decision process on the permission of specific animal studies. This complex issue is often simplified and generalized in the media around the question, "Are animals useful as a model?" To render an often emotional discussion about animal experimentation more rational, it is important to define "usefulness" in a structured and transparent way. To achieve such a goal, many sub-questions need to be asked, and the following aspects require clarification: (i) consistency of animal-derived data (robustness of the model system); (ii) scientific domain investigated (e.g., toxicology vs disease modelling vs therapy); (iii) measurement unit for "benefit" (inte-grating positive and negative aspects); (iv) benchmarking to alternatives; (v) definition of success criteria (how good is good enough); (vi) the procedure to assess benefit and necessity. This series of articles discusses the overall benchmarking process by specifying the six issues. The goal is to provide guidance on what needs to be clarified in scientific and political discussions. This framework should help in the future to structure available information, to identify and fill information gaps, and to arrive at rational decisions in various sub-fields of animal use. In part I of the series, we focus on the robustness of animal models. This describes the capacity of models to produce the same output/response when faced with the "same" input. Follow-up articles will cover the remaining usefulness aspects.

Origin (projects)

Funding sources
Name Finanzierungstyp Kategorie Project no.
ERC third-party funds research funding program 524/21
Further information
Period: 01.06.2021 – 31.05.2026