Thursday, 10 May 2012 11:43

Working group – a Portrait: Center for Alternatives to Animal Testing (CAAT) Featured

InvitroJobs presents scientists and their innovative research in a regular feature called “Working Group – a Portrait”. We will focus on newly developed methods, their evaluation and their potential for reducing and where possible replacing animal experimentation according to the 3R principles of Russel & Burch (reduce, refine, replace). In this issue we introduce the European Center for Alternatives to Animal Testing (CAAT).

Working group – a Portrait:

Center for Alternatives to Animal Testing (CAAT)

The Center for Alternatives to Animal Testing (CAAT) is coordinating the research in the field of replacement of animal testing which is proceed in toxicity testing. It bundles international research teams, brings them together to develop on common research objectives thus promoting the research progress. Symposia, workshops, forums and information days serve as panels in which all participating groups from industry, university research, authorities and animal welfare are invited to contribute substantially to the topic. In order to achieve a better research  coordination, CAAT-Europe was founded, based on the model of the CAAT Center in Baltimore, USA in 2009, headquartered at the University of Constance. Thus CAAT has become a driving force in the enforcement of alternative methods to animal testing. CAAT brings together different groups in order to achieve a common goal.

Two cities – a common interest: Constance and Baltimore, Headquaters of CAAT-US and CAAT-Europe.
Images: Left side: Matthias Lohse, Pixelio, right side: Aimin Tang, iStockphoto.

CAAT´s tasks are more extensive. CAAT accompagnies research to alternative methods by grants and funding. At universities, CAAT trains students and young researchers in alternative methods. In addition to the symposia and workshops, the Center´s websites, such as AltWeb, and their magazine, ALTEX, give detailed information on the subject.

CAAT-Europe: Bridging the Gap Between the Old the New Old Worlds
CAAT US and CAAT EU work together to form a transatlantic bridge for the development and application of new 3R methods1 and the implementation of the new approach to toxicity testing in the 21st Century (Tox21c2 see below). This new approach is aiming to change the entire procedure for toxicity research: from being a test and endpoint-oriented procedure to being a metabolic function-oriented analysis and evaluation (so-called "functional pathways"3).

CAAT is led by Prof. Dr. Marcel Leist, together with Prof. Dr. Thomas Hartung, who chairs the toxicology department in Constance and also has a professorship in Baltimore. Prof. Leist holds the Doerenkamp-Zbinden Chair for in vitro toxicology and biomedicine at the Department of Biology at the University of Constance. At Constance the scientists focus is on the development of in vitro4 models to study the human nervous system.

Left side: Prof. Dr. Dr. Thomas Hartung, director of the Center for Alternatives to Animal Testing (CAAT).
Right side: Prof. Dr. Marcel Leist, leader of CAAT-Europe.

The center organize information days, symposia and workshops. CAAT-Europe brings together industry and science with the aim of developing tests whose results are relevant to humans. In the field of education, it is intended that the connection between the departments at Johns Hopkins University and the University of Constance be developed. This is of special importance because the European Commission will shortly decide whether or not to uphold the sales ban on cosmetics tested on animals.

CAAT-Workshop in Constance October 2011. Image: Cristeta Brause

The Center for Alternatives to Animal Testing (CAAT) was founded in Baltimore in 1981 by the toxicologist Prof. Dr. Alan Goldberg under the name Johns Hopkins Center for Alternatives to Animal Testing. CAAT was initially funded by the U.S. CTFA (Cosmetic Toiletry and Fragrance Association) with the aim of furthering a better understanding of the possibilities for replacement methods to whole animal experiments in developing and testing commercial and therapeutic products.
In 1982 CAAT hosted the first symposium on the theme "Product Safety Evaluation: Development of New Methodological Approaches." An initial allocation of research funding through research awards was made possible. In 1985 the Center established its own in vitro toxicology laboratory. In subsequent years CAAT was supported by industry, which had a strong interest in the development of alternative methods to test their products. Amongst other things potential alternatives to the Draize test - an extremely painful eye irritation test on living rabbits – were evaluated.
In 1988, Avon Group financed the first program for the replacement of animal testing in the field of contact allergy. Since 1989 CAAT has also received governmental support and since 1992 has also been funded by the EPA (Environmental Protection Agency).

InVitrojobs asked the CEO of CAAT-EU, Dr. Mardas Daneshian, about tasks and current developments of the European Centre for Alternatives to Animal Testing.

Dr. Mardas Daneshian has  been working  at the Department of Biochemical  Pharmacology at the University of Constance since 2009 and is the managing director of CAAT Europe  (Center for Alternatives  to  Animal  Testing - Europe). He is  also a board member of the scientific journal ALTEX (Alternatives to Animal Experimentation). When he is  not  involved in the organization of international workshops and symposia, he conducts research on the effects of toxic mould spores on the immune system.

Dr. Mardas Daneshian, CEO of CAAT-EU (Image: CAAT-EU).

InVitroJobs: Dr. Daneshian, you are the manager of CAAT-Europe, which  was established nearly two years ago. Can you briefly tell us about the core activities of the CAAT Centre?

Dr. Daneshian: CAAT-Europe would like to bring together various stakeholders, including industry, academia and regulators to support the  development  of  animal-free,  human-relevant  methods. This means that we have to be up-to-date with the current status of relevant developments but also must be aware of the needs and concerns of the various industries involved in the area. To promote the development of new methods and concepts, we organize regular conferences and symposia. In addition, we also organize workshops to try to develop different approaches. For these workshops, experts from academia, industry and from state regulatory bodies  are invited to participate in multi-day sessions to develop and optimize their ideas.

The results of these activities are published in scientific journals such as ALTEX.  In the near future we will start another program that will allow us to offer courses to industry partners, teachers and students on many of the facets of the field of alternative methods.

InVitroJobs: How are these activities financed?

Dr. Daneshian: CAAT-Europe is supported in part by industry partners and in part by funding from the Doerenkamp-Zbinden Society.

InVitroJobs: Can you give us a overview of what CAAT Europe has already achieved?

Dr. Daneshian: Since the inauguration on 30th March 2010 we have organized six symposia and 12 workshops. On several occasions, we have been invited to speak at conferences about various topics that arose at these symposia and workshops. Furthermore, we can look back on 18 publications in scientific journals. Another achievement is the CAAT-Europe Policy Program, whereby the center will be present in Brussels in 2012 to promote the implementation of 3R-related thoughts and human-relevant alternative methods in legal texts and guidelines. For this purpose, we talk directly with parliamentarians and draw their attention to this issue. This also includes the center´s participation in all relevant events of the Parliament as the Environment, Public Health and Food Safety (ENVI), Industry, Research and Energy (ITRE), Internal Market and Consumer Protection (IMCO), and to remain up-to-date on the current state of of political decision.The achievements of CAAT Europe which exists since two years only are also emphasized by the fact that the public authorities and relevant industry federations from both sides of the Atlantic appreciate our activities and respond always positive to our invitations.

We are very proud that we can welcome experts of the Food and Drug Administration (FDA), the U.S. Environmental Protection Agency (U.S. EPA), the European Food Safety Association (EFSA), European Medicines Agency (EMEA), European Federation of Pharmaceutical Industries and Associations (EFPIA), the European Chemicals Agency (ECHA) as workshop participants and symposium speakers at our events.

InVitroJobs: There are a number of institutions in the field of alternatives to animal testing and the 3Rs. How does you can describe the interactions between these institutions?

Dr. Daneshian: All organizations in this area do have the same goal, namely the implementation of the 3R-thoughts; however, they work on various aspects of this topic. So it is only natural that cooperation leads to synergies. CAAT Europe cooperates very closely with various institutions working in this field such as Eurogroup for Animals, the European Society for Alternatives to Animal Testing (ESAAT), with the German Animal Protection Academy and the German Animal Welfare Association and the set Foundation. We also have strong interactions with the European Society Of Toxicology In Vitro (estiv) and the In Vitro Testing Industrial Platform (ivtip). Since 2011, the European Consensus-Platform for Alternatives (ecopa) - the umbrella organization of the national platforms for alternative methods in Europe - and CAAT-Europe have pooled their resources and cooperate harmonized with each other. Based on this collaboration in Europe there is currently a very productive atmosphere which which benefits both humans and animals.

InVitroJobs: Thank you very much for the interview.

The new Approach in Toxicology

Tox 21c
Tox 21c means “Toxicity Testing in the 21st Century - A Vision and a Strategy”. This concept has been developed by the National Research Council (NRC) of the National Academy of Sciences and published in 2007. The goal is to shift regulatory toxicology to a new basis. The mechanism of the toxicity mode should be clarified (the so-called Mechanism of Action, MoA5). Progressive accumulation of scientific knowledge in toxicogenomics6 and epigenetics7, innovative measurements and analyses methods, and the fact that whole animal experiments are time consuming, expensive and their results are only partially transferrable to humans, have led to a demand for a paradigm shift.

The principle item of this new vision is a network of cells responding to environmental influences. Interconnected pathways (so-called "pathways") consist of complex biochemical interactions of genes, proteins and small molecules which sustain the normal cell function and communication and control the cells, allowing them to adapt to environmental changes. It has to be clarified, how and to what extent the substances can disturb these pathways, which leads to toxic effects. It is supposed that from a certain disturbance intensity adverse (damaging) effects occur to the cells. This process is called toxicity pathway. This view is the basis for a new toxicity test system which identifies and evaluates the biologically significant disruptions in key (metabolism) pathways. Modern methods of physical and chemical characterization of substances, the extensive application of in vitro methods for the mapping of toxicity pathways, micro array8 and high throughput methods9, as well as the functional genomics (in which, for example with the help of small interfering RNAs (siRNA10) the gene expression of certain proteins can be prevented) have to be used for studying the functioning of the pathway without that protein.

The use of new methods of computational biology means computational techniques and mathematical models that play a role in predicting the toxicity of a substance that has not yet been tested. The concept is far more extensive.

Prof. Dr. Dr. Thomas Hartung
Since 2009 Prof. Hartung is leading the chair of evidence-based toxicology at the Bloomberg School of Public Health, Johns Hopkins University, in Baltimore. He is also director of the Center for Alternatives to Animal Testing (CAAT). There he established a laboratory for developmental neurotoxicology where the methods of genomics and metabolomics are used. From 2002 to 2008 he was the leader of the European Center for the Validation of Alternative Methods (ECVAM) at Joint Research Center in Ispra, Italy. Additionally since 2003 he has a professorship at Constance University, and he is the inventor of the in vitro pyrogen test which has been implemented in the European Pharmacopeia under the name monocyte activation test in 2010. It is a replacement method to a very painful pyrogen test on rabbits. Prof. Hartung has a comprehensive expertise in clinical and experimental pharmacology and toxicology, and he received numerous awards and distinctions, for example 2002 the RIVM Award of the World Conference on Animal Use and its Alternatives as well as in 2006 the US Society of Toxicology Enhancement of Animal Welfare Award.

The Human Toxome Project
In its entirety the Human Toxome is all the significant "Pathways of Toxicology (PoT)11". It is the basis of the new test approach. With the approach, substances, but also substance concentrations which do not cause a pathway of toxicology, can get identified and listed. This is not possible with a conventional animal approach, as animal experiments are a kind of "black box" and if there is no result (toxic effects) it is not known whether a successful defense process was held in the animal at that moment. It is also not known whether a defense pathway has occurred successfully or not. Also it is not known whether there are differences between humans and animals in the biochemical defense mechanisms and also among the people (e. g. between babies and adults, different ethnic groups, men and women). For risk assessment in the evaluation of environmental chemicals under REACH the so-called in vitro no-effect level (IVNOEL) are also of great importance.

As the scientists assume that pathways of toxicology operate in networks, scientists want to examine first which pathway combinations generate a toxic effect after cell exposure to a substance and, whether there is a pathway of defense using human-specific primary cells. The measuring methods are described as omics (genomics, transcriptomics, proteomics and metabolomics). The omics measurement results can be compared with the measurements of cells which were not exposed to any substances. Defense and stress responses of the cells must be considered and also have to be excluded. The knowledge of the interruption of a normal metabolic pathway can be used as a basis for developing so-called biomarkers.  A biomarker is a measurable product of an organism which serves as an indicator for an environmental stress or disease (for example a blood glucose level, Wikipedia) - in this case for a toxic effect. It can be a pathway (e. g. the Wnt / beta-catenin signaling pathway, Uibel et al. (2010)).

The new concept is connected to a new view of the entire validation process: while test systems are being validated until now, the mechanism of metabolic pathways (Mechanism of Action), which play a role in the assessment of toxicity, will be validated in the future.

Evidence-based Toxicology (EBT)
The concept of evidence-based toxicology is borrowed from the evidence-based medicine (EBM, the equivalent of evidence-based medicine). Goal of the EBM is to evaluate the quality of published medical information (Wikipedia). EBM itself does not deal with the execution of clinical trials, but with the systematic use of its results. In order to evaluate these studies, certain classification systems have been developed. The EBM concept was transferred to the field of toxicology, since traditional medical and scientific research approaches are mixed in a way similar as in medicine. Otherwise for many methods there is no way to assess their quality, and the immense flow of information makes it impossible to filter out relevant information.

In 2009 a Doerenkamp-Zbinden chair at the Bloomberg School of Public Health, Johns Hopkins University in Baltimore, led by Prof. Thomas Hartung, has been established to work especially on the evidence-based toxicology. Several workshops have been held on this subject and a tool for the development of quality factors have been developed for toxicological studies. The concept is still in development; however, governments, companies and CAAT are already working together to transfer the principles and approaches of the Toxicology EBM. Here CAAT serves as secretariat for the founded consortium. For the European CAAT team Dr. Sebastian Hoffmann (seh-consulting) is responsible.

Sources and literature recommendations:

3 R-Prinzipien: Russel, William M.S. / Burch, Rex L. (1959): The Principles of Humane Experimental Technique. London: Methuen.
Andersen, M. (2011): Toxicity Testing and safety Assessment in the 21st Century: Creating a Research Program to Accelerate Change. Http://
Andersen, M. E. & Krewski, D. (2009):  Toxicity Testing in the 21st Century: Bringing the Vision to Life. Forum series, Part 1. Toxicological Sciences 107(2): 324-330.
Daneshian, M. (2010): The Center for Alternatives to Animal Testing – europe (CAAT-EU): a Transatlantic Bridge for the Paradigm Shift in Toxicology. ALTEX 27: 63-69.
ebtc (2011): The Evidence-based Toxicology (EBT) Collaboration: An overview.Draft of December 2011,
Hartung, T. (2010): Evidence-Based Toxicology – the Toolbox of Validation for the 21st Century? ALTEX 27: 253-263.
Hartung, T. & Daston, G. (2009): Forum: Are in vitro Tests Suitable for Regolatory Use? Toxicological Sciences 111 (2): 233-237.
Hartung, T. & McBride, M. (2011): Food for Thought … on Mapping the Human Toxome. ALTEX 28: 83-93.
InvitroJobs (2011): CAAT has received grant from NIH for mapping of toxicity.
Krewski, D. et al. (2009): Toxicity Testing in the 21st Century: Implications for human Health Risk Assessment. Risk Analysis 29/4: 474-479.
Leist, M. (2006): Was kann ein Lehrstuhl für Alternativmethoden zu Tierversuchen bewirken? ALTEX 23: 211-213.
Mülhardt, C. (2010): Molekularbiologie/Genomics. Der Experimentator. Heidelberg.
O´Shea, S. H., et al. (2011): In Vitro Screening for Population Variability in Chemical Toxicity. Toxicilogical Sciences 119 (2): 398-407.
Perkel, J. M. (2010): Animal-Free Toxicology. Sometimes, in Vitro is Better.
Seidle, T. & Stephens, M. L. (2009): Bringing toxicology into the 21st century: A global call to action. Toxicology in Vitro 23: 1576-1 579.
Tox21c: Tocixity Testing in the 21st Century. A Vision and a Strategy. Commitee on Toxicity Testing and Assessment of Environmental Agents, National Research Council of the National Academies.
Uibel, F. et al. (2010): ReProGlo: A new stem cell-based reporter assay aimed to predict embryotoxic potential of drugs and chemicals. Reproductive Toxicology 30: 103-112.


1 3R-Methoden: (Replacement, Reduction und Refinement). The methods are developed and published by R. Burch and W. Russell in their book „the Principles of Humane Experimental Technique“ in 1959. Essentially it deals with experimentals techniques to reduce the number of used animals, with the reduction of animals´ pain as well as with the replacement von animals by other methods. Scientists wordwide relate to this 3R-principles.
2 Tox21c: Tocixity Testing in the 21st Century. A Vision and a Strategy.
3 Functional Pathway: In this case: metabolism pathway which cause a toxic reaction in the cell (PoT).
4 In vitro: Transl. appr. „in a test tube“, experiments and measurements which are done on living tissue outside of a living organism in a artificial environment, e. g. a petri dish.
5 MoA (Mechanism of Action): Pharmacological term: a biochemical reaction caused by a  drug which triggers an effect (Wikipedia).
6 Toxicogenomics: Science with regard to the collection, interpretation and storage of information on gene and their proteine activity whithin a special cell culture oder a tissue responding to a toxic substance (Wikipedia).
7 Epigenetics: Epigenetic deals with the cell properties which are transfered to the daughter cells but are not fixed in the DNA sequence.
8 Mircoarray: Microchips on which scientists can hybridise (bind on a probe) DNA oder proteins in order to detect them. Primarily for protein detection there are several technical opportunities as antigen-antibody binding oder protein-protein interactions. Using the chip technology, lots of samples can be investigated at the same time in the smallest space (Mülhardt 2010).
9 High troughput methods: Contemporary methods (automation an robot methods) allow to investigate various samples at the same time.
10 siRNA molecules: (small interfering ribonuclein acid) do not encode a proteine but play an important role in the gene expression (Wikipedia).
11 PoT: Pathway of Toxicology