Future activities: ECVAM and the quality control of biologicals

ECVAM's activities in the field of biologicals have contributed in many ways to the successful incorporation of Three Rs methods, as summarised elsewhere in these proceedings. The progress achieved is impressive, but large numbers of animals are still needed in order to meet the requirements stipulated by various regulations. ECVAM's activities in this area should therefore be continued and extended. Besides the well-established organisation of ECVAM workshops and contributions to conferences, further prevalidation and validation studies should be funded. In addition, studies on refinement, and training courses on validated and well-established Three Rs methods, could be initiated. There is a need for more communication and information exchange, especially between regulators and industry concerning the Three Rs. ECVAM could provide a suitable forum for such activities. An ECVAM Biologicals Task Force should be established in order to define a list of priorities.     

The ECVAM workshops: a critical assessment of their impact on the development,validation and acceptance of alternative methods

ECVAM initiated its workshop programme in 1994, to enable it to become well informed about the state of the art of non-animal test development and validation, and about the possible incorporation of alternatives into regulatory requirements for safety testing. Fifty-one such workshops have been held on specific topics, up to 2002. In these workshops, the current status of in vitro tests and their potential uses were reviewed and recommendations were made as to the best ways forward to progress and enhance the utilisation of in vitro methods. Reports for 46 of these workshops have been published in ATLA. Most of the workshops focused on in vitro replacement methods, although an increasing number have dealt with reduction and refinement. The recommendations in the ECVAM workshops have been progressed further by: a) the formation of ECVAM task forces; b) the organisation of further workshops; c) the activities of scientific committees; d) the provision of earmarked research funding; and e) the conduct of validation studies. Examples of each of these activities are discussed. Some individual workshops are covered in more detail and several recommendations that have so far not been acted on are also considered. The workshops and their reports have had a substantial effect on the development and implementation of alternative methods, and have been a major factor in contributing to the success of the first nine years of ECVAM's existence. It is strongly recommended that ECVAM continues to organise workshops and to publish their findings, and several suggestions are made for topics of future workshops.     

ECVAM-ICCVAM: prospects for future collaboration

The level and complexity of testing for hazard and risk assessment of marketed products and environmental agents has increased substantially over time, resulting in the use of greater numbers of both animals and humans for testing. Today, industry and regulatory bodies worldwide face increasing pressures to demonstrate responsible utilisation of laboratory animals, to limit their use, and to employ alternative non-animal tests. Institutions have also been established to identify, encourage development of, conduct research on, and validate new, improved, and surrogate test methods that will reduce and replace animal use. Two such organisations are ECVAM and the Interagency Coordinating Committee for the Validation of Alternative Methods (ICCVAM). As the evolutionary changes occurring in the field of toxicology result in an unprecedented increase in the introduction of alternative methodologies, these will strain the capacities of such alternative methods institutions. That realisation is causing a shift in thinking and creating an impetus to seek approaches by which to collaborate and develop more-efficient operational procedures for the validation and regulatory acceptance of alternative methods. Similarities in objectives, functions, scientific standards, and commitment to the principles of validation and animal welfare support the value of a cooperative arrangement between ECVAM and ICCVAM, to minimise duplication of effort, maximise productivity, and influence the international adoption of alternative tests. Opportunities for ECVAM-ICCVAM collaboration are discussed, which illustrate the feasibility and potential benefits of such a partnership.     

ECVAM's contributions to the implementation of the Three Rs in the production and quality control of biologicals

A summary is presented of the activities initiated, and the progress achieved, between April 1993 and December 2001 in implementing the Three Rs in one of the main priority areas of the European Centre for the Validation of Alternative Methods (ECVAM) - the production and quality control of biologicals. These have included organising eight key workshops, and financial contributions to, and sponsorship of, relevant international workshops, symposia and conferences. Noteworthy activities include financial support and/or participation in a number of prevalidation and validation studies. These involved alternative methods for the batch potency testing of: human tetanus vaccines; human and veterinary tetanus antisera and immunoglobulin; rabies vaccines; Leptospira hardjo vaccines; Clostridium perfringens vaccines; and erysipelas vaccines. They also involved a cell culture test for specific toxicity testing of diphtheria toxoid vaccines. In addition, ECVAM funded a study on the use of humane endpoints for vaccine quality control tests involving severe suffering, such as the potency testing of erysipelas, rabies and pertussis vaccines. ECVAM has also contributed financially to the compilation of manuals and expert reports, and to training in test methods. Following the report of an ECVAM Task Force, ECVAM financially supported the prevalidation of some in vitro methods for the potency testing of a recombinant hormone. A proposal is presented for promotion of regulatory acceptance, and suggestions are made for possible future activities.     

The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM): a review of the ICCVAM test method evaluation process and current international collaborations with the European Centre for the Validation of Alternative Methods (ECVAM)

Over the last decade, national authorities in the USA and Europe have launched initiatives to validate new and improved toxicological test methods. In the USA, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and its supporting National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEATM) were established by the Federal Government to work with test developers and Federal agencies to facilitate the validation, review, and adoption of new scientifically sound test methods, including alternatives that can refine, reduce, and replace animal use. In Europe, the European Centre for the Validation of Alternative Methods (ECVAM) was established to conduct validation studies on alternative test methods. Despite differences in organisational structure and processes, both organisations seek to achieve the adoption and use of alternative test methods. Accordingly, both have adopted similar validation and regulatory acceptance criteria. Collaborations and processes have also evolved to facilitate the international adoption of new test methods recommended by ECVAM and ICCVAM. These collaborations involve the sharing of expertise and data for test-method workshops and independent scientific peer reviews, and the adoption of processes to expedite the consideration of test methods already reviewed by the other organisation. More recently, NICEATM and ECVAM initiated a joint international validation study on in vitro methods for assessing acute systemic toxicity. These collaborations are expected to contribute to accelerated international adoption of harmonised new test methods that will support improved public health and provide for reduced and more-humane use of laboratory animals.     

The contributions of the European cosmetics industry to the development of alternatives to animal testing: dialogue with ECVAM and future challenges

COLIPA (the European Federation of the Cosmetics Industry) represents 24 international companies and 2000 small and medium-sized enterprises. Together with ECVAM, COLIPA has been involved in the development and validation of alternative methods since the beginning of the validation efforts. The work of the Steering Committee on Alternatives to Animal Testing (SCAAT) is based on collaboration between companies, but also with academia, trade associations, the Scientific Committee on Cosmetics and Non-Food Products (SCCNFP), European Commission Directorates General, and ECVAM. Some success has been achieved, but some validation efforts have failed. One lesson is that the search for alternatives requires a lot of humility.     

ECVAM's research and validation activities in the fields of topical toxicity and human studies

This paper outlines the research, prevalidation and validation activities that ECVAM has undertaken in collaboration with its partners in the field of topical toxicity testing and human volunteer studies, from its creation until now (1994-2002).     

ECVAM,ECETOC and the EU chemicals policy

ECVAM's initiatives in validation have received significant support from the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), especially through the provision of reference chemical data banks, which contain peer-reviewed, high-quality in vivo data on commercially available chemical substances. Chemicals have been selected from these ECETOC data banks for validation studies on alternative methods for skin corrosion and irritation and for eye irritation and, in addition, an ECETOC task force peer-reviewed the selection and classification, on the basis of in vivo data, of chemicals used in the validation of three alternative methods for developmental toxicity. More recently, ECVAM and ECETOC have been pursuing parallel initiatives on the proposed new EU chemicals policy, with the common goals of ensuring that industry and European Commission resources are used to investigate only those chemicals that pose a significant risk to human health and the environment, and that the Policy requires that any testing which is required follows the Three Rs principles of reduction, refinement and replacement.     

The principles of validation and the ECVAM validation process

The validation of a test method is the process by which the relevance and reliability of the method are assessed for a particular purpose. It is an essential stage in the evolution of the method from its development to its acceptance and application for regulatory purposes. The principles according to which alternative tests should be validated have been agreed at an international level, although the actual process by which the validation process is conducted varies between different validation authorities. This paper summarises the principles of alternative test development and validation, and describes how the principles have been applied to the validation of in vitro tests by ECVAM.     

Peer review of validation studies: an assessment of the role of the OECD by reference to the validation of the uterotrophic assay for endocrine disruptors

The involvement of the OECD in managing the validation of the rat uterotrophic assay for endocrine disruptors, and in organising the peer review of the results of this study, has been assessed and compared with the many conclusions and recommendations in several published reports of international workshops on validation, and information in guidance documents, produced by the European Centre for the Validation of Alternative Methods (ECVAM), the US Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the OECD itself. It is concluded that the OECD has not followed the recommendations for full transparency and independence of the peer-review process. This is based on the fact that it has published a draft guidance document that differs from the report of a recent OECD workshop on validation, in such a way as to give the OECD the flexibility to fully control the peer-review process and, in so doing, to avoid full transparency. Comparison of the timing of the organisation of workshops by the OECD and the progression of the validation study, together with the fact that a draft test guideline for the assay was written before completion of the peer review, suggest that the OECD has given a higher priority to the expedition of the validation and regulatory acceptance of the uterotrophic assay than it has to good scientific and logistical practice. This severely undermines its credibility in the validation process, so, in order for the OECD to be rightly perceived as an honest broker, it is recommended that the OECD should play no role in the validation of new or revised tests, until after they have been successfully validated, peer reviewed, and endorsed by the appropriate authorities, and are ready for test guideline development. With regard to the on-going OECD validation studies of other in vivo assays for endocrine disruptors, the OECD should take immediate steps to ensure full independence and transparency of their peer review.     

ECVAM: desperately needed or superfluous? An animal welfare perspective

Eurogroup for Animal Welfare is the umbrella organisation of the major animal welfare organisations in Europe. Whereas its long-term goal is the complete replacement of animal experiments by methods that do not involve pain, suffering or distress in animals, it is also committed to any reasonable effort to reduce and refine animal experiments, as long as these continue to be carried out. Eurogroup therefore supports the activities of ECVAM, and it acknowledges the contributions to animal protection in various areas of animal use for scientific purposes made by ECVAM to date. Eurogroup is not satisfied with the number of alternative methods accepted in the past, but it sees the main responsibility for the slow progress as being outside ECVAM. The insufficient involvement of ECVAM by the EU Commission in various issues that would require its competence is also a matter of concern to Eurogroup.     

Report of the EPAA-ECVAM workshop on the validation of Integrated Testing Strategies (ITS)

The use of Integrated Testing Strategies (ITS) permits the combination of diverse types of chemical and toxicological data for the purposes of hazard identification and characterisation. In November 2008, the European Partnership for Alternative Approaches to Animal Testing (EPAA), together with the European Centre for the Validation of Alternative Methods (ECVAM), held a workshop on Overcoming Barriers to Validation of Non-animal Partial Replacement Methods/Integrated Testing Strategies, in Ispra, Italy, to discuss the extent to which current ECVAM approaches to validation can be used to evaluate partial replacement in vitro test methods (i.e. as potential ITS components) and ITS themselves. The main conclusions of these discussions were that formal validation was only considered necessary for regulatory purposes (e.g. the replacement of a test guideline), and that current ECVAM approaches to validation should be adapted to accommodate such test methods. With these conclusions in mind, a follow-up EPAA-ECVAM workshop was held in October 2009, to discuss the extent to which existing validation principles are applicable to the validation of ITS test methods, and to develop a draft approach for the validation of such test methods and/or overall ITS for regulatory purposes. This report summarises the workshop discussions that started with a review of the current validation methodologies and the presentation of two case studies (skin sensitisation and acute toxicity), before covering the definition of ITS and their components, including their validation and regulatory acceptance. The following main conclusions/recommendations were made: that the validation of a partial replacement test method (for application as part of a testing strategy) should be differentiated from the validation of an in vitro test method for application as a stand-alone replacement, especially with regard to its predictive capacity; that, in the former case, the predictive capacity of the whole testing strategy (rather than of the individual test methods) would be more important, especially if the individual test methods had a high biological relevance; that ITS allowing for flexible and ad hoc approaches cannot be validated, whereas the validation of clearly defined ITS would be feasible, although practically quite difficult; and that test method developers should be encouraged to develop and submit to ECVAM not only full replacement test methods, but also partial replacement methods to be placed as parts of testing strategies. The added value from the formal validation of testing strategies, and the requirements needed in view of regulatory acceptance of the data, require further informed discussion within the EPAA forum on the basis of case studies provided by industry.     

Relevance of clonogenic assays in hematotoxicology

Clonogenic assays have been established in hematology for 30 years. They have been widely used in fundamental studies on hematopoiesis and they are also routinely used in clinical hematology to confirm diagnosis or to predict time to recovery in cases of bone marrow failure. Their use in toxicological studies is more recent. Adverse effects of xenobiotics can induce hematological problems and pathologies such as neutropenia, thrombocytopenia, anemia, and aplastic anemia. Three clonogenic assays are proposed for granulopoiesis, megakaryopoieisis and erythropoieisis. Hematopoietic progenitors from murine or human origin can be cultured in the presence of xenobiotics using validated protocols to complete standard animal toxicological studies. These clonogenic assays can help to predict adverse effects of drugs or toxicants. Clonogenic assays using white blood cell progenitors (CFU-GM culture) have recently been validated by ECVAM and can be used routinely. Megakaryocyte progenitor (CFU-MK) culture is under development and prevalidation in toxicological studies supported by ECVAM. Red blood cells progenitor culture (BFU-E) has been proposed but needs international validation to be recognized. This revised version was published online in July 2006 with corrections to the Cover Date.     

Validation successes: chemicals

The ECVAM validation concept, which was defined at two validation workshops held in Amden (Switzerland) in 1990 and 1994, and which takes into account the essential elements of prevalidation and biostatistically defined prediction models, has been officially accepted by European Union (EU) Member States and by the Federal regulatory agencies of the USA and the OECD. The ECVAM validation concept was introduced into the ongoing ECVAM/COLIPA validation study of in vitro phototoxicity tests, which ended successfully in 1998. The 3T3 neutral red uptake in vitro phototoxicity test was the first experimentally validated in vitro toxicity test recommended for regulatory purposes by the ECVAM Scientific Advisory Committee (ESAC). It was accepted by the EU into the legislation for chemicals in the year 2000. From 1996 to 1998, two in vitro skin corrosivity tests were successfully validated by ECVAM, and they were also officially accepted into the EU regulations for chemicals in the year 2000. Meanwhile, in 2002, the OECD Test Guidelines Programme is considering the worldwide acceptance of the validated in vitro phototoxicity and corrosivity tests. Finally, from 1997 to 2000, an ECVAM validation study on three in vitro embryotoxicity tests was successfully completed. Therefore, the three in vitro embryotoxicity tests, the whole embryo culture (WEC) test on rat embryos, the micromass (MM) test on limb bud cells of mouse embryos, and the embryonic stem cell test (EST) including a permanent embryonic mouse stem cell line, are considered for routine use in laboratories of the European pharmaceutical and chemicals industries.     

Mapping urban growth probability in South Korea: comparison of frequency ratio, analytic hierarchy process, and logistic regression models and use of the environmental conservation value assessment

Rapid industrialization and economic growth in South Korea since the 1970s have resulted in severe environmental disturbance and pollution, problems aggravated by the imprudent expansion of urban areas. This paper analyzes and predicts urban growth patterns with the aim of contributing to more efficient urban planning. Urban growth probability index (UGPI) maps were prepared using the frequency ratio (FR), analytic hierarchy process (AHP), and logistic regression (LR) methods, with and without considering development restrictions based on the national environmental conservation value assessment map (ECVAM). Environmental and legal restrictions were associated with an average difference of 41.70% in conservation areas and an 81.32% average difference in agriculture and forest land use–land cover (LULC). Accuracy of the models was examined by area under the curve (AUC) analysis. Accuracies of UGPI maps produced with the ECVAM were higher than UGPI maps produced without the ECVAM. In addition, effectiveness and accuracy tests based on LULC showed that the UGPI maps produced with the ECVAM had a higher rate of accuracy that UGPI maps produces without the ECVAM. Using the ECVAM and assuming that urban and built-up areas will be 1.5 times greater than in 2005 and that environmental restrictions are removed, urban development can be expected to more than double in conservation areas and borderlands, increase by more than 1.5 times in developable areas, and decrease by half in old downtown areas. If legal restrictions are removed, urban development is expected to occur mostly in former conservation areas, followed by borderlands, old downtowns, and developable areas.     

Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests

A detailed report is presented on the performance of the embryonic stem cell test (EST) in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the EST can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.     

ECVAM's activities in validating alternative tests for skin corrosion and irritation

ECVAM has funded and managed validation studies on in vitro tests for skin corrosion, resulting in the validities of four in vitro tests being endorsed by the ECVAM Scientific Advisory Committee: the rat skin transcutaneous electrical resistance (TER) assay, two tests based on the use of commercial reconstituted human skin equivalents, EPISKIN and EpiDerm, and another commercially-produced test, CORROSITEX. In the European Union (EU), a new test method on skin corrosion (B.40), incorporating the rat skin TER and human skin model assays, was included in Annex V of Directive 67/548/EEC in mid-2000, thereby making the use of in vitro alternatives for skin corrosion testing of chemicals mandatory in the EU. At the recommendation of its Skin Irritation Task Force, ECVAM has funded prevalidation studies on five in vitro tests for acute skin irritation: EpiDerm, EPISKIN, PREDISKIN, the pig-ear test, and the mouse-skin integrity function test (SIFT). However, none of the tests met the criteria (set by the Management Team for the studies) for inclusion in a large-scale formal validation study. Thus, to date, there are no validated in vitro tests for predicting the dermal irritancy of chemicals. Following further work on the EPISKIN, EpiDerm and SIFT test protocols and/or prediction models after the completion of the prevalidation studies, it appears that the modified tests could meet the performance criteria defined for progression to a validation study. This will now be assessed independently by the ECVAM Skin Irritation Task Force, with the objective of taking a decision before the end of 2002 on whether to conduct a formal validation study.     

Validation of the postimplantation rat whole-embryo culture test in the international ECVAM validation study on three in vitro embryotoxicity tests

A detailed report is presented on the performance of the postimplantation rat whole-embryo culture (WEC) test in a European Centre for the Validation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects in animals and/or humans, were tested in four laboratories. The outcome showed that the WEC test can be considered to be a scientifically validated test, which is ready for consideration for use in assessing the embryotoxic potentials of chemicals for regulatory purposes.     

Validation of the rat limb bud micromass test in the international ECVAM validation study on three in vitro embryotoxicity tests

A detailed report is presented on the performance of the rat limb bud micromass (MM) test in a European Centre for the Evaluation of Alternative Methods (ECVAM)-sponsored formal validation study on three in vitro tests for embryotoxicity. Twenty coded test chemicals, classified as non-embryotoxic, weakly embryotoxic or strongly embryotoxic on the basis of their in vivo effects on animals and/or humans, were tested in four laboratories. The outcome showed that the MM test is an experimentally validated test, which holds promise for use for identifying strongly embryotoxic chemicals, but which needs to be improved before it can be recommended for use for regulatory purposes.     

ECVAM's approach to intellectual property rights in the validation of alternative methods

In this article, we discuss how intellectual property rights affect the validation of alternative methods at ECVAM. We point out recent cases and summarise relevant EU and OECD documents. Finally, we discuss guidelines for dealing with intellectual property rights during the validation of alternative methods at ECVAM.