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SEURAT-1: Development of a Research Strategy for the Replacement of in vivo Repeated Dose Systemic Toxicity Testing


Last Updated: August 25, 2013
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Tilman Gocht - Eberhard Karls University Tuebingen, Institute for Experimental and Clinical Pharmacology and Toxicology, Michael Schwarz - Eberhard Karls University Tuebingen, Institute for Experimental and Clinical Pharmacology and Toxicology, Elisabet Berggren - European Commission, Joint Research Centre (JRC), Institute for Health and Consumer Protection (IHCP) and EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM), Maurice Whelan - European Commission, Joint Research Centre (JRC), Institute for Health and Consumer Protection (IHCP) and EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)

SEURAT-1 is a major European private-public research consortium that is working towards animal-free testing and the highest level of consumer protection, co-funded by Cosmetics Europe (EUR 25 million) and the European Commission under the 7th Framework Programme (EUR 25 million). A research strategy was formulated around harnessing knowledge about toxicological modes-of-action and an organisational model was developed that marries crowd-sourcing with individual excellence. The proof of the initiative will be in demonstrating the applicability of the concepts on which SEURAT-1 is built. Results in this field will be relevant for a wide range of industrial and medical sectors and will have a positive impact on the competitiveness and innovation of EU companies.

This paper is substantially a summary of the SEURAT-1 research strategy that was published in the first three Annual Reports of the SEURAT-1 Research Initiative (Schwarz & Gocht, 2011; Gocht & Schwarz, 2012; 2013).1

Background

A full ban on animal testing for cosmetics came into force on the 11th of March 2013 in the European Union. Since this date, cosmetic ingredients tested on animals cannot be marketed within the EU. Based on the Seventh Amendment of the Cosmetics Directive2, animal testing was already prohibited from 2004 for cosmetic products and from 2009 for cosmetic ingredients. For the most complex human health effects (repeated dose toxicity, including skin sensitisation and carcinogenicity, reproductive toxicity, and toxicokinetics) the deadline for the testing ban was extended to March 2013.

'Safety Evaluation Ultimately Replacing Animal Testing' (SEURAT) was presented under the Health Theme of the 7th Framework Programme by the Euro¬pean Commission in 2008 as the long-term target in safety testing. Cosmetics Europe and the European Commission agreed to support the research initiative 'Towards the replacement of in vivo repeated dose systemic toxicity testing', which could be considered as a the first step in the long-term SEURAT strategy, i.e., SEURAT-1. A tiered approach is foreseen, starting with innovative concepts for repeated dose systemic toxicity, and ending with animal-free Innovative Toxicity Testing (ITT), enabling robust safety assessment. SEURAT-1 started its activities in January 2011. This is the first EU-funded project that addresses the issue of alternatives to animal testing for prediction of repeated dose systemic toxicity.

Goals and Objectives

The goal of the five-year SEURAT-1 Research Initiative is to develop a consistent research strategy that will also be implemented, in the long term, in future research programmes. This includes establishing innovative scientific tools for a better understanding of repeated dose toxicity based on in vitro tests and identifying the gaps in knowledge that are to be bridged by future research work. The end result would be testing methods which, within the framework of safety assessment, have a higher predictive value, are faster and cheaper than those currently used, and significantly reduce the use of animal tests.

The cluster-level objectives of the SEURAT-1 Research Initiative are to

  • develop highly innovative tools and methodology that can ultimately support regulatory safety assessment;
  • formulate and implement a research strategy based on generating and applying knowledge of mode-of-action;
  • demonstrate proof-of-concept at multiple levels – theoretical, systems, and application;
  • provide the blueprint for expanding the applicability domains – chemical, toxicological, and regulatory.

The research work in the SEURAT-1 projects includes the development of organ-simulating devices, the use of human-based target cells, the identification of relevant endpoints and intermediate markers, the application of approaches from systems biology, computational modelling and estimation techniques, and integrated data analysis.

Structure of the SEURAT-1 Research Initiative

The SEURAT-1 Research Initiative is designed as a coordinated cluster of six research projects, one of which is a ‘data handling and servicing project’, plus a ‘coordination and support project’ at the cluster level. This structure is illustrated in Figure 1.

Figure 1. Building blocks of the SEURAT-1 Research Initiative (project acronyms in the right circle) that were established based on the call for proposals under the HEALTH Theme of the 7th European RTD Framework Programme 'Towards the Replacement of in vivo Repeated Dose Systemic Toxicity Testing' (key words from the call in the left circle).

The following six integrated projects form the backbone of SEURAT-1 (project title and aim):

  • 'Stem Cells for Relevant efficient extended and normalized TOXicology' (SCR&Tox): Stem cell differentiation for providing human-based organ-specific target cells to assay toxicity pathways in vitro
  • 'Hepatic Microfluidic Bioreactor' (HeMiBio): Development of a hepatic microfluidic bioreactor mimicking the complex structure and function of the human liver
  • 'Detection of endpoints and biomarkers for repeated dose toxicity using in vitro systems' (DETECTIVE): Identification and investigation of human biomarkers in cellular models for repeated dose in vitro testing
  • 'Integrated In Silico Models for the Prediction of Human Repeated Dose Toxicity of COSMetics to Optimise Safety' (COSMOS): Delivery of an integrated suite of computational tools to predict the effects of long-term exposure to chemicals in humans based on in silico calculations
  • 'Predicting long-term toxic effects using computer models based on systems characterization of organotypic cultures' (NOTOX): Development of systems biological tools for organotypic human cell cultures suitable for long-term toxicity testing and the identification and analysis of pathways of toxicological relevance
  • 'Supporting Integrated Data Analysis and Servicing of Alternative Testing Methods in Toxicology' (ToxBank): Data management, cell and tissue banking, selection of standard reference compounds and chemical repository

Furthermore, a coordination action project was designed in order to facilitate cluster interaction and activities:

  • 'Coordination of projects on new approaches to replace current repeated dose systemic toxicity testing of cosmetics and chemicals' (COACH): Cluster level coordination and support action

The scientific management and coordination of the SEURAT-1 Research Initiative is strongly supported by the Scientific Expert Panel (SEP), which plays a key role in providing scientific advice regarding the research work and future orientation of the SEURAT-1 Research Initiative.

The SEURAT-1 Research Initiative combines the research efforts of over 70 European universities, public research institutes, and companies. The composition is unique, with toxicologists, biologists from different disciplines, pharmacists, chemists, bioinformaticians, and leading experts from other domains closely working together on common scientific objectives. The participation of small and medium-sized enterprises (SMEs) in SEURAT-1 is high, at more than 30%.

The SEURAT-1 Research Strategy

The Vision

The SEURAT-1 Research Initiative combines the research efforts of over 70 European universities, public research institutes, and companies. The composition is unique, with toxicologists, biologists from different disciplines, pharmacists, chemists, bioinformaticians, and leading experts from other domains closely working together on common scientific objectives. The participation of small and medium-sized enterprises (SMEs) in SEURAT-1 is high, at more than 30%.

The vision foresees safety assessment frameworks that optimally combine a range of reliable and robust experimental (in vitro) and computational tools in a purposeful manner to deliver the relevant information needed for decision making. These predictive toxicology tools will associate substances of concern with a new taxonomy of toxicological hazard categories, and they will predict the likelihood of any adverse health effects as a function of exposure, for different sub-populations. The uncertainty of these predictions will be sufficiently characterised as to facilitate effective risk management and communication, with the appropriate degree of precaution. The predictive tools will be widely available, affordable and reliable, so that every substance destined for commerce will be sufficiently evaluated in good time, at a reasonable cost, and in a consistent manner. To facilitate trade and the global market, safety assessment frameworks will be established and harmonised at international level, allowing them to be implemented in all jurisdictions. The knowledge gained from safety assessment of new substances will be fed back into the product development process, thereby improving human safety evaluation, driving innovation, increasing consumer choice, promoting sustainability, and improving industrial competitiveness.

The Strategy: A Mode-of-Action Backbone based on Mechanistic Understanding

The mode-of-action framework (Boobis et al., 2008) is based on the premise that any adverse human health effect caused by exposure to an exogenous substance can be described by a series of causally linked biochemical or biological key 'events' that result in a pathological endpoint or disease outcome. An 'adverse outcome pathway' is a very similar concept proposed by the computational toxicology community (Ankley et al., 2010), where the linking of a chemical with a pathway that leads to an adverse human health or ecological outcome is determined by its ability to trigger the associated 'molecular initiating event'. Another related framework is that of 'toxicity pathways' introduced by the NRC (Krewski et al., 2010), where the description of toxicological processes tends to focus on early events at the molecular and cellular level. Thus one can consider toxicological pathways as critical upstream elements of a more expansive mode-of-action or adverse outcome pathway description of how a chemical can compromise human health (Figure 2).

Figure 2. Schematic illustration of a sequence of events contributing to an Adverse Outcome Pathway, including the Mode-of-Action and Toxicity Pathways as sub-sequences.

Mode-of-action theory is still emerging but already a number of important principles have shaped the SEURAT research strategy. The first is that every toxicant can be associated with one or more mode-of-action categories. To facilitate this, however, a suitable ontology that describes all the possible modes of toxicological action needs to be developed by harvesting and organising the wealth of knowledge and information available from the literature on well-studied chemicals and pharmaceuticals. Systematically checking 'reference' chemicals against mode-of-action categories will help to challenge and refine the mode-of-action ontology as it emerges, and will identify a wide range of key biological events and pathways that should be represented in relevant experimental (in vitro) and computational models.

The framework assumes that many modes-of-action share common key biomolecular or biological events. Thus it is the particular chain of causally linked events that makes a mode-of-action unique. In the case where a substance is promiscuous and could trigger multiple modes-of-action, the concentration and persistence of the substance at the initiation sites will dictate the modes-of-action that will tend to dominate. Thus, for example, chronic low-dose effects may be quite different in many cases from high-dose acute effects (which is, according to Haber's Rule, not true for the so-called 'c x t-compounds', for which a toxicological effect is the result of the total dose over a period of time, such that even very small doses, given for prolonged periods of time, will produce the same toxic effect as a high dose given for only a short period of time). Special consideration needs be given, therefore, to characterising dose-response relationships, to describe how and when mode-of-action transitioning may occur for a single substance, depending on factors such as exposure dynamics, site of action, genetic and epigenetic predisposition, and inherent phenotypic vulnerabilities.

Another principle to be considered concerning mode-of-action theory is that many key events and pathways are common to many cell types throughout the human body. Thus, although the same substance can cause different pathological outcomes in different tissues, the upstream event, such as mitochondrial inhibition or generation of reactive oxygen species, may be common to the modes-of-action triggered at each site. On the other hand, certain modes-of-action involve key events or pathways that are associated with specific biological functions expressed by particular cell types, for example, the presence of metabolising enzymes in liver cells that may bioactivate exogenous chemicals to produce toxic metabolites, or the presence of cell membrane transporters required for the uptake of certain toxicants. Similarly, the presence of receptors for neurotransmitters in neuronal cells that can be targeted by toxicants is another example of cell-specific properties that can be implicated in a toxicological mode-of-action.

Biological Models. Although many toxicological modes-of-action are conserved across mammalian species, there will likely be many situations where, for example, rodent- or tumour-derived cell lines will fail to capture essential aspects of human biology. Attention needs to be given therefore to the development of experimental models based on properly conditioned human primary cells or differentiated stem cells. In addition, modelling a toxicological mode-of-action in a holistic fashion will require the emulation of downstream events that manifest themselves as pathology at the tissue level. Simple cell-based in vitro models will not be sufficient for this purpose and thus 3D tissue models will be needed to reproduce the more apical biological processes or endpoints.

In SEURAT-1, these 3D tissue models will be produced experimentally in bioreactor systems, or virtually using computational biology approaches. Such models will not only allow the qualitative association of a chemical with one or more modes-of-action, but will also serve to quantify dose-response relationships. Complementing the cell and tissue models, computational chemistry, quantitative structure-activity relationships (QSARs), and chemoinformatics tools will provide the means to understand and predict key biochemical events such as protein binding and metabolic transformation. However, these advanced experimental and computational approaches may be limited if they are overly reductionist or simplistic, thus failing to capture aspects such as hormonal regulation, tissue innervation, immune surveillance, blood circulation, and metabolic turnover.

Describing Mode-of-Action. As yet there is no generally accepted practice for gathering mode-of-action knowledge and presenting it in a consistent and structured manner so that it can be effectively managed and transferred. However, the International Programme on Chemical Safety (IPCS) of the World Health Organisation (WHO) has published guidance (Boobis et al., 2008) on what type of information should be provided to describe a mode-of-action and, just as importantly, how the relevant evidence should be presented to demonstrate the validity of the description proposed. More recently, the Organisation for Economic Cooperation and Development (OECD) has followed this direction but gone somewhat further by proposing that mechanistic or mode-of-action information on a chemical can be captured using an analytical tool termed 'adverse outcome pathway', or 'AOP' (see above). Guidance has been issued on practical ways to describe an AOP using a recommended template, and also on how to present and evaluate scientific evidence to assess its completeness (OECD, 2012a). A supporting document summarising published definitions of relevant terms has also been provided, in order to facilitate more transparent communication between different scientific communities and as a step towards eventual harmonisation of vocabulary and definitions (OEDC, 2012b). The OECD has also established an AOP work programme to be led by the Advisory Group on Toxicogenomics and Molecular Screening, which will officially commence in 2013. Taking these international developments into consideration, the usefulness of WHO and OECD guidance to serve the needs of SEURAT-1 is currently being investigated.

Putting Theory into Practice

Adopting a mode-of-action toxicological framework means that one needs to learn by doing, with the starting point being identifying some 'prototype' modes-of-action that could be elaborated. Covering all potential modes-of-action is just not feasible based on existing knowledge. Hence, 'being selective' is the key word by starting this endeavour, 'selective' in terms of chemicals, modes-of action, and definition and design of case studies proving the underlying concept.

Chemical Selection. The first immediate sign of how the guiding principles of the research strategy outlined above have influenced the cluster is reflected in the approach adopted for the selection of reference chemicals to be used across the projects (Wiseman, 2012). Traditional approaches often set out to select reference chemicals that satisfy a heterogeneous set of criteria such as: belonging to certain chemical classes, used in selected commercial sectors or products, possessing particular physico-chemical properties, or associated with certain adverse health outcomes. Embracing the SEURAT-1 philosophy, the 'Gold Compound' Working Group committed instead to first identify and describe a range of known modes-of-action more commonly cited in repeated dose toxicity studies, and then to pick molecules for which there is ample mechanistic evidence of association with toxicological effects or pathways underpinning those modes-of-action. Not surprisingly, many of the reference chemicals are actually pharmaceuticals since these molecules typically have specific mechanisms or modes-of-action that are extensively described in the literature. It is precisely these mode-of-action related properties that make them reliable candidates for nomination as reference compounds, rather than their actual origin or commercial use. Therefore, once a test system is established to prospectively evaluate whether chemicals of interest are causing toxicity through a specific mode-of-action, those chemicals that are found to be positive can be considered as being similar with respect to their membership of the same toxicodynamic category or group. This categorisation or grouping therefore has a mode-of-action basis and is populated initially by the associated reference chemicals. This approach doesn't preclude the possibility that an actual chemical may be promiscuous in nature, being associated possibly with more than one mode-of-action. In this case, it is probable that toxicokinetic aspects will play a role in determining which mode-of-action will likely dominate under particular conditions. Moreover, promiscuity at the level of molecular initiation potentially triggering a range of possible modes-of-action may still be reduced to a reasonable number of categories, based on a reduced number of possible downstream effects, such as 'gross' phenotypic cellular outcomes.

Selecting reference chemicals using a mode-of-action approach also has implications for how they are actually chosen for research purposes. Essentially, SEURAT-1 investigators should first decide on which mode-of-action is of relevance to their particular study or test system, and then select the associated reference chemicals. Thus mode-of-action thinking is brought to the forefront, with the design, optimisation, and evaluation of in vitro test systems being driven by the aim to capture one or more specific modes-of-action with high sensitivity and selectivity. As a consequence, the specifications of the biological model, the exposure protocol, the biomarkers to be measured, and the reference chemicals to be used as positive controls, all depend on the mode-of-action chosen.

Mode-of-Action Selection. The selection of modes-of-action will be performed in the context of the development of AOP descriptions. The OECD AOP template (OECD, 2012a) clearly indicates which information should be provided, both to describe the toxicological process itself and the evidence that supports the postulation of the associated key events (KE), including the molecular initiating event (MIE). What is lacking however is a practical guide on how to go about the actual task of developing an AOP. The SEURAT-1 Research Initiative therefore adopted a learning-by-doing approach, which resulted in the emergence of a generic AOP development process, as outlined in Figure 3.

Figure 3. A generic stepwise process to develop an AOP in line with the OECD template and guidance from the OECD (2012a).

It is very likely that there are common MIEs for many pathways, and a threshold value must be reached to significantly disturb a certain pathway. It is, however, a priori not clear whether the MIEs relevant for repeated dose systemic toxicity differ from those relevant for acute toxicity, but it is reasonable to assume that there is at least some mechanistic overlap between both exposure scenarios. Most current knowledge of toxicity mechanisms stems from the acute exposure scenario and thus it will be important to decide whether the same mechanisms also hold true for repeated dose exposures, or whether different or additional mechanisms come into play under the latter condition. Mechanisms to be considered include: (i) repeated hits on the same molecular target; (ii) overload of defence/repair mechanisms through accumulation of a chemical at certain initiation sites; (iii) progressive change in the epigenome; (iv) effects on the immune system, such as proliferation of memory cells and progressive activation and transformation of hepatic stellate cells; and finally (v) induction of a sequence of adverse reactions involving different cell types (and organs).

The development process outlined in Figure 3 relies heavily on a systematic review of the literature to mine out the mechanistic knowledge applicable to the AOP in question. Considering the wealth of information already available, an AOP can be typically brought to a relatively mature state of development by studying relevant review papers and reported studies. However, at some point the process plateaus since the finer mechanistic detail specific to the AOP is often lacking in the description of third-party investigations. This is due to the fact that these investigations were usually conducted with different research objectives in mind. Thus, more extensive development of an AOP can require the undertaking of prospective experimental (in vitro) investigations that are specifically designed to shed light on the less understood aspects of the AOP. Such 'knowledge discovery' research is necessary, for example, to develop more precise descriptions of the dynamic relationships between key events and to be explicit in quantitative terms about the conditions that dictate the transitioning from one event to another in the pathway.

Although experimental investigation to elucidate mechanisms is an important activity within SEURAT-1, there is widespread acknowledgement that mining publicly available databases reporting both in vitro and in vivo toxicity studies (e.g., the 'Open TG-GATEs' database established by the Toxicogenomics Project in Japan, or the DrugMatrix database of the US National Toxicology Program) can provide an invaluable source of mode-of-action information to support AOP development. In particular, there is an enormous amount of toxicogenomics data that, with the appropriate analysis, could uncover hidden mechanisms and key events and provide supporting evidence for an AOP during its development and evaluation.

Given the composition of the SEURAT-1 Research Initiative it was reasonable to start with modes-of-action that are of relevance to the liver (as all of the projects are addressing liver toxicity in their work programme) and to try to define related pathways based on the identification of interactions of a chemical with known targets. A first report presenting the definition and detailed documentation of chosen toxicological modes-of-action associated with repeated dose target organ toxicity was prepared as an initial step in building a 'prototype' safety assessment framework. In addition to providing a detailed description of two chosen modes-of-action related to chronic liver toxicity, namely 'Mode-of-Action from Protein Alkylation to Liver Fibrosis' and 'Mode-of-Action from Liver X Receptor Activation to Liver Steatosis', the working process leading to this result including the problems that have been encountered, such as scarcity of quantitative data and the difficulty in capturing and describing complex non-linear processes in a narrative manner, was described (Landesmann et al., 2012).

Biokinetics. An important factor, too frequently ignored, relates to the biokinetics of the chemical in question. This can be very different in an in vitro system when compared to in vivo because of, for example: (i) accumulation of a chemical in a target organ due to slow metabolism; (ii) inhibition of an inactivating enzyme; (iii) lowering of metabolic clearance (damage to liver); or (iv) induction of a bioactivating enzyme. It is obvious, therefore, that the apparent toxicodynamic behaviour of a given compound will be strongly influenced by its biokinetics. As a consequence, a central issue for the SEURAT-1 Research Initiative is how to relate treatment concentrations used in the various in vitro test systems to in vivo serum and target organ concentrations, and vice versa.

Proving the strategy. The SEURAT-1 Research Initiative will deliver many important computational and experimental tools, and related knowhow, that will be critical components in predictive toxicology approaches. To demonstrate the potential of these tools and how they can be assembled in an integrated manner, the cluster will undertake a proof-of-concept exercise to demonstrate how a mode-of-action based testing strategy can be used to predict aspects of repeated dose target organ toxicity. The concepts to be proven are stratified into three distinct levels: theoretical, systems, and application.

Proof-of-concept at the theoretical level aims to show how toxicological knowledge concerning modes-of-action can be mined or perhaps generated, and then reconciled, consolidated, and explicitly described in a format that can be managed and communicated in an effective and harmonised manner. Proving this concept will require not only acquiring and managing mode-of-action knowledge, but also the demonstration of how this knowledge has been used in a purposeful manner to drive the more applied research activities.

At the systems level, the intention is to demonstrate how test systems can be produced by integrating various in vitro and in silico tools emanating from the SEURAT-1 projects, in order to assess the toxicological properties of chemicals using modes-of-action as an analytical basis. Such systems may include, for example, a combination of computational chemistry models with a battery of in vitro assays to generate a mixed set of chemical-structure and bioactivity descriptors that can be used to group chemicals into mode-of-action based categories, or the combination of biokinetics (PBBK) models with in vitro concentration-response assays to estimate in vivo no-effect levels in rodents and humans.

At the highest level, proof-of-concept will address the desire to show how the data and information derived from the tools and methods developed within the cluster can actually be used in specific safety assessment frameworks and scenarios. Two different types of case studies were proposed that reflect two typical safety assessment scenarios: The objective of the first case study is to arrive at a point-of-departure or reference value for a particular chemical, that can be used as a basis for a safety decision, by conducting an ab initio assessment using only the methods available within SEURAT-1. This quantitative safety assessment case study will explore how far one can actually go with the tools and methodology that are available by the end of SEURAT-1. Of course, the exercise is also designed to highlight major gaps and shortfalls that hinder a comprehensive and credible quantitative safety assessment, thereby providing a clear indication on where future research and development efforts in the field of safety assessment need to focus. The second case study deals with chemical categories and read-across, which are approaches probably best known for hazard and safety assessment of industrial chemicals under REACH, but which are often employed in other sectors too. The purpose of this case study is to demonstrate how information on a chemical generated using SEURAT-1 methods can be used to associate it with a chemical category and to deduce or predict its hazard properties by 'reading across' from the properties of other chemicals belonging to the same category. The intention is to use both toxicokinetic and toxicodynamic data derived from computational and in vitro studies to establish and support a chemical category and read-across argument. Of particular interest in this case study is to show how traditional approaches based primarily on consideration of chemical structure can be supplemented with mechanistic information derived from computational methods and in vitro '-omics’ experiments to either support or reject a chemical category or risk assessment proposal. This is considered as a realistic target for SEURAT-1 and it is expected that the results will demonstrate how the output of the cluster can find immediate use for supporting chemical safety assessment with the potential of reducing animal testing.

Beyond SEURAT-1

Successful completion of SEURAT-1 will lay the foundation for follow-on efforts that will broaden the toxicological, chemical, and regulatory domains addressed. The mode-of-action framework will have been well established, but will be limited in scope, covering mainly repeated dose toxicity associated with primary organs. Thus the mode-of-action ontology will need to be further expanded by harvesting existing knowledge, and generating new knowledge where gaps exist, to cover other adverse health effects linked, for example, to cancer and reproduction.

Uptake and application of SEURAT methodology for safety assessment will begin modestly on a proof-of-concept level within SEURAT-1, but will need to expand continually in both depth and scope. Possible application areas in the relatively near future include satisfying Classification, Labelling, and Packaging (CLP) requirements, or supporting a weight-of-evidence analysis or read-across in a Chemical Safety Assessment under REACH. It is likely that novel tools and safety assessment frameworks deriving from SEURAT will be implemented and evaluated initially in parallel to more traditional approaches. This will identify any shortfalls, build confidence, and define good practice for better safety evaluation that will ultimately replace animal testing.

Acknowledgement

This paper was written with the support of the SEURAT-1 Scientific Expert Panel.

1In total, a series of six Annual Reports, delivered in the form of books, will provide a comprehensive overview of the SEURAT-1 initiative in replacing animal tests in the field of repeated dose systemic toxicity. The books are made available each year through the Scientific Secretariat of the SEURAT-1 Research Initiative. To receive a paper copy, please send an e-mail to: coach-office@eurtd.com or download the report at http://www.seurat-1.eu/

2Council Directive 76/768/EEC on the approximation of the laws of the Member States relating to cosmetic products.

References

Author Biographies


©2013 Tilman Gocht, Michael Schwarz, Elisabet Berggren, and Maurice Whelan