The usefulness of integrated strategy approaches in replacing animal experimentation.

It is currently well accepted that in general, more than one method is necessary to allow the full replacement of an animal experimentation. These so called partial replacement methods can be used within integrated strategy approaches that combine different methods and information sources. A number of integrated strategy approaches were implemented within recent years in different areas of safety and regulatory toxicology. Moreover, latest advances in biomedical research and bioengineering provide a major opportunity to make use of in vitro human-based and/or three-dimensional complex models that can contribute to achieve more physiologically-relevant models. Examples herein describe currently existing integrated strategy frameworks aiming at full or partial replacement purposes and/or at gaining mechanistic insights. Furthermore, a general concept is provided on how 3R methods might be integrated in a strategy approach in order to ensure that animal experimentation is conducted only as a last resort.

"Watching the Detectives" report of the general assembly of the EU project DETECTIVE Brussels, 24-25 November 2015.

SEURAT-1 is a joint research initiative between the European Commission and Cosmetics Europe aiming to develop in vitro- and in silico-based methods to replace the in vivo repeated dose systemic toxicity test used for the assessment of human safety. As one of the building blocks of SEURAT-1, the DETECTIVE project focused on a key element on which in vitro toxicity testing relies: the development of robust and reliable, sensitive and specific in vitro biomarkers and surrogate endpoints that can be used for safety assessments of chronically acting toxicants, relevant for humans. The work conducted by the DETECTIVE consortium partners has established a screening pipeline of functional and "-omics" technologies, including high-content and high-throughput screening platforms, to develop and investigate human biomarkers for repeated dose toxicity in cellular in vitro models. Identification and statistical selection of highly predictive biomarkers in a pathway- and evidence-based approach constitute a major step in an integrated approach towards the replacement of animal testing in human safety assessment. To discuss the final outcomes and achievements of the consortium, a meeting was organized in Brussels. This meeting brought together data-producing and supporting consortium partners. The presentations focused on the current state of ongoing and concluding projects and the strategies employed to identify new relevant biomarkers of toxicity. The outcomes and deliverables, including the dissemination of results in data-rich "-omics" databases, were discussed as were the future perspectives of the work completed under the DETECTIVE project. Although some projects were still in progress and required continued data analysis, this report summarizes the presentations, discussions and the outcomes of the project.

Los métodos alternativos: un reto para las administraciones y para los científicos / Alternative methods: a challenge for governments and scientists

La normativa actual que regula en el ámbito europeo la utilización de los animales con fines científicos establece con absoluta rotundidad que su prioridad absoluta es el fomento y la implantación de los enfoques alternativos a los métodos tradicionales de utilización de los animales. Insistimos sobre dos aspectos que consideramos relevantes. En primer lugar es necesario aclarar que los métodos alternativos no son solo aquellos en los que se alcanza el Reemplazo total de los animales, sino que también comprenden aquellos otros métodos y estrategias en las que se reduce el número de animales utilizados (Reducción) o se refinan las condiciones en los que éstos se utilizan y mantienen (Refinamiento). En segundo lugar quisiéramos animar a la comunidad científica a dar los pasos que sean necesarios para poder implementar también en el ámbito de las 3 erres el cambio de paradigma que los avances y el desarrollo que en el campo de la biología molecular y de sistemas están haciendo posible en toxicología. En el futuro es posible que algunas pruebas puedan consistir ya no tanto en analizar los efectos que determinadas sustancias tóxicas tienen sobre los animales, sino más bien en evaluar los cambios metabólicos que a nivel molecular son los que realmente causan los mencionados efectos sobre los animales (y lógicamente el ser humano) (AU)

Current European regulations on the use of animals for scientific purposes categorically states that the first priority is the development and implementation of alternative approaches to traditional methods using animals. We insist on two aspects that we consider relevant. First able it is necessary to clarify that alternative methods are not only those in which the total replacement of animals is reached, but also include those other methods and strategies in which the number of animals used decreases (reduction) or refine the conditions under which they are used and maintained (Refinement). Secondly, we would like to encourage the scientific community to take the steps necessary to also implement in the field of the 3Rs principle that progress and development in the field of molecular biology and systems are possible in toxicology. In the future it is possible that some studies may consist not so much as to analyze the effects that certain toxic substances have on the animals, but rather to evaluate metabolic changes at the molecular level that actually cause the effects on animals (and logically on humans) (AU)

Estrategias de identificación de planteamientos alternativos a la experimentación animal / Strategies for the identification of alternative approaches to animal experimentation

Los investigadores deben asegurarse de que la información que podrían obtener con su experimentación no está ya disponible, que no existe otro procedimiento para llevarlo a cabo sin emplear animales y que el protocolo se ha diseñado teniendo en cuenta consideraciones de protección animal. Sin embargo, la identificación de procedimientos alternativos empleados por otros científicos sigue siendo un proceso muy complejo, debido, sobre todo, a la deficiente indexación de las publicaciones en las bases de datos bibliográficas. Una búsqueda eficiente debe basarse en emplear siempre varias bases de datos, en revisar al menos los documentos de los últimos 5-10 años. En primer lugar debe evitarse la duplicación inútil de investigaciones, es decir, asegurarse de que la información que pudiera obtenerse en el estudio no está ya disponible. A continuación se realiza la búsqueda de posibles alternativas de reemplazo. Si ésta no fuera productiva, se identificarían alternativas de reducción y refinamiento, tratando de mejorar, en lo posible, cada una de las fases de la experimentación animal. Los estudios toxicológicos de finalidad reguladora presentan la exigencia de utilizar protocolos oficiales, por lo que deben localizarse en sus directorios específicos. Las alternativas en la enseñanza y entrenamiento, como los modelos mecánicos, audiovisuales y de simulación, se encuentran recogidas en bases de datos específicas. Finalmente, cuando no se encuentran opciones válidas en otras fuentes, es posible recurrir a expertos, tanto directamente como en foros especializados. Todo ello se facilita con el buscador Buscaalternativas.com (http://buscaalternativas.com) (AU)

The researchers should be sure that the information obtainable with the experiments is not yet available, that there is no other possible procedure without the use of animals or that the protocol was designed taking into account animal protection considerations. However, the identification of alternative procedures employed by other scientists is a very complex process, mainly due to the deficient indexation of the articles. An efficient search must be based on the use of several data bases and the review of documents of the last 5-10 years. The search strategy presents several phases. Firstable, the unnecessary duplication of studies should be avoided, assuring the information obtainable in the study is not yet available. A search for replacement alternatives is then carried out. If it is not productive, reduction and refinement alternatives are identified to improve every phase of animal research. Toxicological regulatory studies must use official protocols, which should be localized in specific directories. Alternatives in education and training, including mechanical models, audiovisuals and simulations are included in specific databases. Finally, when no valid options are found in other sources, it is possible to ask experts, directly or through specialized debate lists. The procedure is facilitated thanks to the Web Buscaalternativas.com (http://buscaalternativas.com) (AU)

Actividades de promoción y difusión de alternativas en España / Advocacy and outr each on alternatives in Spain

Se revisan las actividades de promoción y desarrollo de los métodos alternativos y complementarios a la experimentación animal en España. Se muestran las reuniones o jornadas que se han celebrado y organizado, incluyendo la constitución de la Red Española de Métodos Alternativos. Como foro de discusión para lograr un menor y más racional uso de los animales de experimentación, fomentando el desarrollo, validación y utilización de los métodos in vitro, tiene como fin conseguir el eficaz desarrollo de los procedimientos alternativos mediante el uso de los principios de reducción, refinamiento y reemplazo de los animales en la experimentación, lo que constituye la aplicación de las 3Rs. Se destaca el papel de la citada Red detallando información sobre la misma, sus miembros, la participación en numerosos cursos, seminarios y jornadas que se organizan por Universidades españolas y otras entidades, y la estrecha colaboración con el Ministerio de Sanidad, Servicios Sociales e Igualdad y el Ministerio de Agricultura Alimentación y Medio Ambiente, contando con observadores en su Junta Directiva (AU)

A revision of advocacy and development of alternative and complementary methods to animal testing in Spain was done. Meetings and conferences that have been organized, including the constitution of the Spanish Network for Alternative Methods, are shown. As a discussion forum for a lower and more rational use of experimental animals, promoting the development, validation and use of in vitro methods. The aims were the effective development of alternative procedures using the principles of reduction, refinement and replacement of animal use in experiments, which is the application of the 3Rs. The role of the aforementioned net is detailed, with the information about the network and their members. It has participated in many courses, seminars and conferences in the Spanish universities and other entities, maintaining a close collaboration with the Ministry of Health and the Ministry of Agriculture, Food and Environment, with observers included in the Board (AU)

Report on the international workshop on alternative methods for human and veterinary rabies vaccine testing: state of the science and planning the way forward.

Potency testing of most human and veterinary rabies vaccines requires vaccination of mice followed by a challenge test using an intracerebral injection of live rabies virus. NICEATM, ICCVAM, and their international partners organized a workshop to review the availability and validation status of alternative methods that might reduce, refine, or replace the use of animals for rabies vaccine potency testing, and to identify research and development efforts to further advance alternative methods. Workshop participants agreed that general anesthesia should be used for intracerebral virus injections and that humane endpoints should be used routinely as the basis for euthanizing animals when conducting the mouse rabies challenge test. Workshop participants recommended as a near-term priority replacement of the mouse challenge with a test validated to ensure potency, such as the mouse antibody serum neutralization test for adjuvanted veterinary rabies vaccines for which an international collaborative study was recently completed. The workshop recommended that an in vitro antigen quantification test should be a high priority for product-specific validation of human and non-adjuvanted veterinary rabies vaccines. Finally, workshop participants recommended greater international cooperation to expedite development, validation, regulatory acceptance, and implementation of alternative test methods for rabies vaccine potency testing.

The Dr Hadwen Trust for Humane Research: 39 years of replacement science.

The year 2009 will not be remembered mainly because it is the Chinese year of the ox, but probably, for many, because it is Charles Darwin's bicentenary, the 150th anniversary of the publication of his masterpiece, The Origin of Species, the 50th anniversary of the publication by Russell and Burch of The Principles of Humane Experimental Technique, where they introduced the concept of Three Rs, and also the 40th anniversary of the foundation of the Fund for the Replacement of Animals in Medical Experiments (FRAME). FRAME will always remain our senior, since the Dr Hadwen Trust for Humane Research is only celebrating its 39th anniversary this year! After four decades of learning, challenging, contesting, creating, promoting and advancing the Three Rs concept, the idea of the replacement of the use of animals in research has itself become a science, giving the old sterile debate between pro- and anti-vivisectionists a new dimension. Although scientific progress has been the main reason for such changes, it is important to remember that, without the people and organisations whose aim has been devoted to this cause, these changes might never have happened. Still, as illustrated by the 2008 statistics on animal procedures, many more changes will have to be made, and it is by considering the progress achieved during the last 40 years that we can find the strength and motivation to pursue our goal of scientific research and testing that no longer relies on animal-based techniques.

The importance of anniversaries.

This year marks a number of important anniversaries for the Three Rs and those organisations involved in their advancement. These include the 50th anniversary of the publication of Russell and Burch's book, The Principles of Humane Experimental Technique, the 40th anniversary of FRAME, and the 5th anniversary for the UK's National Centre for the 3Rs. These anniversaries provide an important opportunity to reflect on past achievements and future challenges.

Establishing a Three Rs programme at the Canadian Council on Animal Care.

The anniversary of the Fund for the Replacement of Animals in Medical Experiments (FRAME) provides an opportunity to celebrate the Charity's achievements over the past 40 years, and in particular, its contribution to the international acceptance of the Three Rs as the basis of an ethic for animal experimentation, and its role as a leader in the implementation of the Three Rs in the life sciences. The Canadian Council on Animal Care (CCAC) has based its work on the Three Rs, especially since the establishment of its fundamental CCAC Policy Statement on the Ethics of Animal Investigation (1989). Following recommendations from the evaluation of its programmes by external committees established by the national granting agencies, the CCAC recently launched a Three Rs Program. This programme will build on the work of FRAME and other similar Three Rs organisations, to further the promotion and implementation of the Three Rs in Canadian science.

The Institute for Laboratory Animal Research - an international resource for promoting the Three Rs.

In 1995, an international group of scientists met in Sheringham, Norfolk, UK, for a workshop entitled The Three Rs: The Way Forward. There, with the participation of William Russell and Rex Burch, the group discussed each of the Three Rs in detail, and made specific recommendations on how to promote them within the scientific community. The Institute for Laboratory Animal Research (ILAR) was established as part of the US National Academy of Sciences, to develop and disseminate information and guidelines for the care and use of laboratory animals. Over the years, the focus of ILAR has been to improve the health, welfare, and psychological well-being of the research animal, using the Three Rs as a foundation. ILAR's programmes include an international component, through which it reaches out to other countries via translations of its report; the ILAR Journal, a quarterly publication that focuses on animal models, animal welfare and protocol review; communications and outreach through the ILAR website and presence at scientific meetings; and special reports in which expert committees make recommendations to improve science and animal welfare. Through the efforts of ILAR and similar organisations, it is now recognised that high standards of humane care lead to better science. However, continued effort is needed to promote the Three Rs in developing countries.

Proposal for a Brazilian centre on alternative test methods.

Several initiatives have recently taken place in Brazil in order to foster the creation of centers dedicated to alternatives to animal testing. In 2008, Vanessa Sá-Rocha organized a meeting with Brazilian regulatory authorities and the major stakeholders in the field of testing to foster discussions on the process of funding, development, and validation of alternative methods in Brazil. Octavio Presgrave published a scientific article on "The Need for the Establishment of a Brazilian Centre for the Validation of Alternative Methods." Also in 2008, Jadir Nunes, together with Dermeval de Carvalho, prepared and presented a proposal to the Brazilian National Agency of Health Surveillance (ANVISA) for the creation of a Centre for the Validation of Alternative Methods. ECVAM and other European stakeholders have been involved in the initiatives. Furthermore, also in 2008, a new legislation has been adopted in Brazil regarding the use of animals for scientific purposes ("lei Arouca"). The legislation establishes, among other provisions, the task of monitoring and evaluating the introduction of alternative methods. However, the legislation does not provide for promotion of or information about, existing alternative methods to the larger Brazilian scientific community. In order to streamline the different activities, Chantra Eskes acted as a facilitator by establishing a new joint proposal with the current Brazilian stakeholders, aimed at setting up a Brazilian Center on Alternative Test Methods.

The need for the establishment of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM).

Brazil has recently approved legislation for regulating experimental animal use. On the other hand, it is a requirement that the safety of many products on the market is controlled on the basis of animal testing. Some groups at official laboratories, universities and industries are studying alternative methods, but there is no approved mechanism for funding collaborative studies, nor is there an institution responsible for coordinating these studies. These shortcomings obstruct the development of these assays in Brazil. The creation of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM) would facilitate the development and validation of tests in all the institutions working on alternative methods in Brazil, and could also offer support to other Latin American countries.

The FRAME Research Programme under the direction of Dr Richard Clothier.

The work of Dr Richard Clothier (Reader in Cellular Toxicology and Director of the FRAME Alternatives Laboratory in the University of Nottingham Medical School) in relation to the FRAME Research Programme, is reviewed. He made a very substantial contribution to FRAME's laboratory research work over the last 20 years, by publishing many research papers, mainly with respect to method development and the application of new replacement toxicity test methods, particularly those involving the use of human cells in tissue culture. In addition, he participated in a number of international validation studies that have facilitated the regulatory acceptance of certain new methods. Lastly, he has been closely involved in the logistical aspects of obtaining and maintaining external research funding for FRAME from industrial sponsors.

Richard Clothier: an appreciation.

The career of Richard Clothier is reviewed in the light of his long-standing collaboration with Michael Balls and Laurens Ruben at the University of East Anglia (UEA), the University of Nottingham, and Reed College, Portland, Oregon, USA. It began with work at UEA on the aetiology of the lymphosarcoma of Xenopus laevis, followed by studies on the effects of exposure to N-nitroso-N-methylurea on T-cell functions, which led to many contributions to comparative immunology. This was followed by the establishment of the FRAME Research Programme, which led to participation in extensive studies on the development of in vitro cytotoxicity tests and their application in acute and topical toxicity testing. A FRAME Trustee since 1983, Richard Clothier was a co-founder, and subsequently Director, of the FRAME Alternatives Laboratory in the University of Nottingham Medical School, where he led successful collaborations with a number of industrial partners and, in particular, with the European Centre for the Validation of Alternative Methods (ECVAM).