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

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.

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

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

G × E interactions as a basis for toxicological uncertainty.

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 gene sequence, the regulation of the gene expression and function should also be accounted for. The widened concept of past and present "gene expression" influences is summarized here as Ge. Also, the concept of "environment" needs some re-consideration in situations where exposure timing (Et) is pivotal: prolonged or repeated exposure to the insult (chemical, physical, life style) affects Ge. This implies that it changes the model system. The interaction of Ge with Et might be denoted as Ge × Et. 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).

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

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.

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

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.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

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

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.

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

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 anticipated 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 practical applicability of NAMs and 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.

The EU-ToxRisk method documentation, data processing and chemical testing pipeline for the regulatory use of new approach methods.

Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.

Harnessing the power of novel animal-free test methods for the development of COVID-19 drugs and vaccines.

The COVID-19-inducing virus, SARS-CoV2, is likely to remain a threat to human health unless efficient drugs or vaccines become available. Given the extent of the current pandemic (people in over one hundred countries infected) and its disastrous effect on world economy (associated with limitations of human rights), speedy drug discovery is critical. In this situation, past investments into the development of new (animal-free) approach methods (NAM) for drug safety, efficacy, and quality evaluation can be leveraged. For this, we provide an overview of repurposing ideas to shortcut drug development times. Animal-based testing would be too lengthy, and it largely fails, when a pathogen is species-specific or if the desired drug is based on specific features of human biology. Fortunately, industry has already largely shifted to NAM, and some public funding programs have advanced the development of animal-free technologies. For instance, NAM can predict genotoxicity (a major aspect of carcinogenicity) within days, human antibodies targeting virus epitopes can be generated in molecular biology laboratories within weeks, and various human cell-based organoids are available to test virus infectivity and the biological processes controlling them. The European Medicines Agency (EMA) has formed an expert group to pave the way for the use of such approaches for accelerated drug development. This situation illustrates the importance of diversification in drug discovery strategies and clearly shows the shortcomings of an approach that invests 95% of resources into a single technology (animal experimentation) in the face of challenges that require alternative approaches.

Internationalization of read-across as a validated new approach method (NAM) for regulatory toxicology.

Read-across (RAx) translates available information from well-characterized chemicals to a substance for which there is a toxicological data gap. The OECD is working on case studies to probe general applicability of RAx, and several regulations (e.g., EU-REACH) already allow this procedure to be used to waive new in vivo tests. The decision to prepare a review on the state of the art of RAx as a tool for risk assessment for regulatory purposes was taken during a workshop with international experts in Ranco, Italy in July 2018. Three major issues were identified that need optimization to allow a higher regulatory acceptance rate of the RAx procedure: (i) the definition of similarity of source and target, (ii) the translation of biological/toxicological activity of source to target in the RAx procedure, and (iii) how to deal with issues of ADME that may differ between source and target. The use of new approach methodologies (NAM) was discussed as one of the most important innovations to improve the acceptability of RAx. At present, NAM data may be used to confirm chemical and toxicological similarity. In the future, the use of NAM may be broadened to fully characterize the hazard and toxicokinetic properties of RAx compounds. Concerning available guidance, documents on Good Read-Across Practice (GRAP) and on best practices to perform and evaluate the RAx process were identified. Here, in particular, the RAx guidance, being worked out by the European Commission's H2020 project EU-ToxRisk together with many external partners with regulatory experience, is given.

Biology-inspired microphysiological systems to advance patient benefit and animal welfare in drug development

The first microfluidic microphysiological systems (MPS) entered the academic scene more than 15 years ago and were considered an enabling technology to human (patho)biology in vitro and, therefore, provide alternative approaches to laboratory animals in pharmaceutical drug development and academic research. Nowadays, the field generates more than a thousand scientific publications per year. Despite the MPS hype in academia and by platform providers, which says this technology is about to reshape the entire in vitro culture landscape in basic and applied research, MPS approaches have neither been widely adopted by the pharmaceutical industry yet nor reached regulated drug authorization processes at all. Here, 46 leading experts from all stakeholders - academia, MPS supplier industry, pharmaceutical and consumer products industries, and leading regulatory agencies - worldwide have analyzed existing challenges and hurdles along the MPS-based assay life cycle in a second workshop of this kind in June 2019. They identified that the level of qualification of MPS-based assays for a given context of use and a communication gap between stakeholders are the major challenges for industrial adoption by end-users. Finally, a regulatory acceptance dilemma exists against that background. This t4 report elaborates on these findings in detail and summarizes solutions how to overcome the roadblocks. It provides recommendations and a roadmap towards regulatory accepted MPS-based models and assays for patients' benefit and further laboratory animal reduction in drug development. Finally, experts highlighted the potential of MPS-based human disease models to feedback into laboratory animal replacement in basic life science research.

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

In this manuscript, which appeared in ALTEX (2019), 36(4), 682- 699, doi:10.14573/altex.1909271 , the affiliation of Hennicke Kamp should be Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany. Further, the reference to an article by Bal-Price et al. (2015) should have the following doi:10.1007/s00204-015-1464-2 .

Determination of benchmark concentrations and their statistical uncertainty for cytotoxicity test data and functional in vitro assays.

Many toxicological test methods, including assays of cell viability and function, require an evaluation of concentration-response data. This often involves curve fitting, and the resulting mathematical functions are then used to determine the concentration at which a certain deviation from the control value occurs (e.g. a decrease of cell viability by 15%). Such a threshold is called the benchmark response (BMR). For a toxicological test, it is often of interest to determine the concentration of test compound at which a pre-defined BMR of e.g. 10, 25 or 50% is reached. The concentration at which the modelled curve crosses the BMR is called the benchmark concentration (BMC). We present a user-friendly, web-based tool (BMCeasy), designed for operators without programming skills and profound statistical background, to determine BMCs and their confidence intervals. BMCeasy allows simultaneous analysis of viability plus a functional test endpoint, and it yields absolute BMCs with confidence intervals for any BMR. Besides an explanation of the algorithm underlying BMCeasy, this article also gives multiple examples of data outputs. BMCeasy was used within the EU-ToxRisk project for preparing data packages that were submitted to regulatory authorities, demonstrating the real-life applicability of the tool.

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.

Impairment of human neural crest cell migration by prolonged exposure to interferon-beta.

Human cell-based toxicological assays have been used successfully to detect known toxicants, and to distinguish them from negative controls. However, there is at present little experience on how to deal with hits from screens of compounds with yet unknown hazard. As a case study to this issue, we characterized human interferon-beta (IFNß) as potential developmental toxicant affecting neural crest cells (NCC). The protein was identified as a hit during a screen of clinically used drugs in the 'migration inhibition of neural crest' (MINC) assay. Concentration-response studies in the MINC combined with immunocytochemistry and mRNA quantification of cellular markers showed that IFNß inhibited NCC migration at concentrations as low as 20 pM. The effective concentrations found here correspond to levels found in human plasma, and they were neither cytostatic nor cytotoxic nor did they did they affect the differentiation state and overall phenotype of NCC. Data from two other migration assays confirmed that picomolar concentration of IFNß reduced the motility of NCC, while other interferons were less potent. The activation of JAK kinase by IFNß, as suggested by bioinformatics analysis of the transcriptome changes, was confirmed by biochemical methods. The degree and duration of pathway activation correlated with the extent of migration inhibition, and pharmacological block of this signaling pathway before, or up to 6 h after exposure to the cytokine prevented the effects of IFNß on migration. Thus, the reduction of vital functions of human NCC is a hitherto unknown potential hazard of endogenous or pharmacologically applied interferons.

Fingerprinting of neurotoxic compounds using a mouse embryonic stem cell dual luminescence reporter assay.

Identification of neurotoxic drugs and environmental chemicals is an important challenge. However, only few tools to address this topic are available. The aim of this study was to develop a neurotoxicity/developmental neurotoxicity (DNT) test system, using the pluripotent mouse embryonic stem cell line CGR8 (ESCs). The test system uses ESCs at two differentiation stages: undifferentiated ESCs and ESC-derived neurons. Under each condition, concentration-response curves were obtained for three parameters: activity of the tubulin alpha 1 promoter (typically activated in early neurons), activity of the elongation factor 1 alpha promoter (active in all cells), and total DNA content (proportional to the number of surviving cells). We tested 37 compounds from the ESNATS test battery, which includes polypeptide hormones, environmental pollutants (including methylmercury), and clinically used drugs (including valproic acid and tyrosine kinase inhibitors). Different classes of compounds showed distinct concentration-response profiles. Plotting of the lowest observed adverse effect concentrations (LOAEL) of the neuronal promoter activity against the general promoter activity or against cytotoxicity, allowed the differentiation between neurotoxic/DNT substances and non-neurotoxic controls. Reporter activity responses in neurons were more susceptible to neurotoxic compounds than the reporter activities in ESCs from which they were derived. To relate the effective/toxic concentrations found in our study to relevant in vivo concentrations, we used a reverse pharmacokinetic modeling approach for three exemplary compounds (teriflunomide, geldanamycin, abiraterone). The dual luminescence reporter assay described in this study allows high-throughput, and should be particularly useful for the prioritization of the neurotoxic potential of a large number of compounds.

Identification of transcriptome signatures and biomarkers specific for potential developmental toxicants inhibiting human neural crest cell migration.

The in vitro test battery of the European research consortium ESNATS ('novel stem cell-based test systems') has been used to screen for potential human developmental toxicants. As part of this effort, the migration of neural crest (MINC) assay has been used to evaluate chemical effects on neural crest function. It identified some drug-like compounds in addition to known environmental toxicants. The hits included the HSP90 inhibitor geldanamycin, the chemotherapeutic arsenic trioxide, the flame-retardant PBDE-99, the pesticide triadimefon and the histone deacetylase inhibitors valproic acid and trichostatin A. Transcriptome changes triggered by these substances in human neural crest cells were recorded and analysed here to answer three questions: (1) can toxicants be individually identified based on their transcript profile; (2) how can the toxicity pattern reflected by transcript changes be compacted/dimensionality-reduced for practical regulatory use; (3) how can a reduced set of biomarkers be selected for large-scale follow-up? Transcript profiling allowed clear separation of different toxicants and the identification of toxicant types in a blinded test study. We also developed a diagrammatic system to visualize and compare toxicity patterns of a group of chemicals by giving a quantitative overview of altered superordinate biological processes (e.g. activation of KEGG pathways or overrepresentation of gene ontology terms). The transcript data were mined for potential markers of toxicity, and 39 transcripts were selected to either indicate general developmental toxicity or distinguish compounds with different modes-of-action in read-across. In summary, we found inclusion of transcriptome data to largely increase the information from the MINC phenotypic test.

Grouping of histone deacetylase inhibitors and other toxicants disturbing neural crest migration by transcriptional profiling.

Functional assays, such as the "migration inhibition of neural crest cells" (MINC) developmental toxicity test, can identify toxicants without requiring knowledge on their mode of action (MoA). Here, we were interested, whether (i) inhibition of migration by structurally diverse toxicants resulted in a unified signature of transcriptional changes; (ii) whether statistically-identified transcript patterns would inform on compound grouping even though individual genes were little regulated, and (iii) whether analysis of a small group of biologically-relevant transcripts would allow the grouping of compounds according to their MoA. We analyzed transcripts of 35 'migration genes' after treatment with 16 migration-inhibiting toxicants. Clustering, principal component analysis and correlation analyses of the data showed that mechanistically related compounds (e.g. histone deacetylase inhibitors (HDACi), PCBs) triggered similar transcriptional changes, but groups of structurally diverse toxicants largely differed in their transcriptional effects. Linear discriminant analysis (LDA) confirmed the specific clustering of HDACi across multiple separate experiments. Similarity of the signatures of the HDACi trichostatin A and suberoylanilide hydroxamic acid to the one of valproic acid (VPA), suggested that the latter compound acts as HDACi when impairing neural crest migration. In conclusion, the data suggest that (i) a given functional effect (e.g. inhibition of migration) can be associated with highly diverse signatures of transcript changes; (ii) statistically significant grouping of mechanistically-related compounds can be achieved on the basis of few genes with small regulations. Thus, incorporation of mechanistic markers in functional in vitro tests may support read-across procedures, also for structurally un-related compounds.