The establishment of ECVAM and its progress since 1993

The background to the establishment of ECVAM in 1991 is summarised, and progress made since 1993 is briefly reviewed in relation to 12 recommendations made at the ECVAM Opening Symposium in 1994 and to statements on tests for chemicals and biologicals endorsed by the ECVAM Scientific Advisory Committee since 1997.     

The role of ECVAM in promoting the regulatory acceptance of alternative methods in the European Union. European Centre for the Validation of Alternative Methods

The roles played by the European Centre for the Validation of Alternative Methods (ECVAM) and its advisory committee, the ECVAM Scientific Advisory Committee (ESAC), in the evolution of alternative methods are described. Particular emphasis is given to the process by which ECVAM and the ESAC assess the scientific validities of alternative methods, and, in appropriate cases, initiate the progression of scientifically validated methods toward regulatory acceptance.     

ECVAM's response to the changing political environment for alternatives: consequences of the European Union chemicals and cosmetics policies

The European Centre for the Validation of Alternative Methods (ECVAM) has restructured its services by directly targeting the animal tests that need to be replaced. In view of the short time-lines for making available and implementing validated methods, ECVAM is offering to steer the process by bringing together the inputs of stakeholders and encouraging the early involvement of regulators. In essence, steering groups formed by ECVAM senior staff, and complemented with external experts, will carry out the project management and will coordinate the various inputs.     

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.     

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.     

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.     

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.     

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.     

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.     

Prediction of maize hybrid performance using similarity in state and similarity by descent information

We evaluated the efficiency of the best linear unbiased predictor (BLUP) and the influence of the use of similarity in state (SIS) and similarity by descent (SBD) in the prediction of untested maize hybrids. Nine inbred lines of maize were crossed using a randomized complete diallel method. These materials were genotyped with 48 microsatellite markers (SSR) associated with the QTL regions for grain yield. Estimates of four coefficients of SIS and four coefficients of SBD were used to construct the additive genetic and dominance matrices, which were later used in combination with the BLUP for predicting genotypic values and specific combining ability (SCA) in unanalyzed hybrids under simulated unbalance. The values of correlations between the genotypic values predicted and the means observed, depending on the degree of unbalance, ranged from 0.48 to 0.99 for SIS and 0.40 to 0.99 using information from SBD. The results obtained for the SCA ranged from 0.26 to 0.98 using the SIS and 0.001 to 0.990 using the SBD information. It was also observed that the predictions using SBD showed less biased than SIS predictions demonstrating that the predictions obtained by these coefficients (SBD) were closer to the observed value, but were less efficient in the ranking of genotypes. Although the SIS showed a bias due to overestimation of relatedness, this type of coefficient may be used where low values are detected in the SBD in the group of parents because of its greater efficiency in ranking the candidates hybrids.     

ECVAM's role in the implementation of the Three Rs concept in the field of biologicals

Biologicals can be defined as products that are derived from living organisms or are produced by them. They include vaccines, hormones, monoclonal and polyclonal antibodies, blood products and rDNA products. The production of conventionally produced biologicals requires an extensive batch-related quality control, to ensure that these products are both safe and potent. As several of the control tests rely on animal models, it is inevitable that the large numbers of animals are used. Many initiatives have been undertaken in the last few decades to reduce and refine the use of animals in this area. ECVAM has been involved in many activities to support the development, validation and implementation of these Three Rs methods. The role that ECVAM has played in a number of validation studies is summarised. It is concluded that ECVAM should continue to support the activities that have been shown to be successful, preferably in collaboration with the regulatory bodies.     

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.     

Follow-up to the ECVAM prevalidation study on in vitro tests for acute skin irritation. The European Centre for the Validation of Alternative Methods Skin Irritation Task Force report 2

The European Centre for the Validation of Alternative Methods (ECVAM) Skin Irritation Task Force was established in 1996, to review the status of the development and validation of alternative tests for skin irritation and corrosion, and to identify appropriate non-animal tests for predicting human skin irritation that were sufficiently well-developed to be prevalidated and validated by ECVAM. The EpiDerm method, based on a reconstituted human skin model, was proposed as being sufficiently well advanced to enter a prevalidation (PV) study. Based on a review of test protocols, prediction models (PMs), and data submitted by test developers on ten specified chemicals, with 20% sodium lauryl sulphate as a reference standard, the task force recommended the inclusion of four other tests: EPISKIN and PREDISKIN, based on reconstituted human epidermis or on human skin; the non-perfused pig-ear test, based on pig skin; and the skin integrity function test (SIFT), with ex vivo mouse skin. The prevalidation study on these methods was funded by ECVAM, and took place during 1999-2000. The outcome of the PV study was that none of the methods was ready to enter a formal validation study, and that the protocols and PMs of the methods had to be improved in order to increase their predictive abilities. Improved protocols and PMs for the EpiDerm and EPISKIN methods, the pig ear test, and the SIFT were presented at an extended Task Force meeting held in May 2001. It was agreed that, in the short term, the performance of the revised and harmonised EpiDerm and EPISKIN methods, as well as the modified SIFT, should be evaluated in a further study with a new set of 20 test chemicals. In addition, it was decided that the SIFT and the pig ear test would be compared to see if common endpoints (transepidermal water loss, methyl green-pyronine stain) could be identified.     

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.     

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.     

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.     

Selective activation of intra-muscular compartments within the trapezius muscle in subjects with Subacromial Impingement Syndrome. A case-control study

Neuromuscular control of the scapular muscles is important in the etiology of shoulder pain. Electromyographical (EMG) biofeedback in healthy people has been shown to support a selective activation of the lower compartment of the trapezius muscle, specifically. The aim of the present paper was to investigate whether patients with Subacromial Impingement Syndrome (SIS) were able to selectively activate the individual compartments within the trapezius muscle, with and without EMG biofeedback to the same extent as healthy controls (No-SIS).Fifteen SIS and 15 No-SIS participated in the study. Sessions with and without visual biofeedback were conducted. Surface EMG was recorded from four compartments of the trapezius muscle. Selective activation was defined as activation above 12% with other muscle parts below 1.5% or activation ratio at or above 95% of the total activation. Without biofeedback significantly fewer SIS subjects than No-SIS achieved selective activation (p = 0.02–0.03).The findings of the study show that without biofeedback No-SIS had a superior scapular muscle control. However, when provided with visual EMG feedback the SIS group performed equally well as the No-SIS group. This indicated that individuals with SIS may benefit from biofeedback training to gain control of the neuromuscular function of the scapular muscle.     

Preparation of porcine small intestinal submucosa sponge and their application as a wound dressing in full-thickness skin defect of rat

Small intestinal submucosa (SIS) sponge was prepared by crosslinking with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). The prepared SIS sponges exhibited elastic and soft property on touch and were ease to handle. The SIS sponges have the pore diameter of 100-200 microm and an interconnective porous structure. The SIS sponges exhibited high water absorption ability over 8000%. The water uptake of SIS sponges decreased as SIS concentration used to manufacture SIS sponge increased. In wound healing test, SIS sponge attained uniform adherence to the wound surface. The SIS sponges absorbed higher extent of exudation for wound than that covered with Tegaderm as control. Wound area contracted above 80% at the 21st postoperative day. The SIS sponge treated wound was almost completely covered with a thin layer of epidermis at 4 weeks. In addition, the dermal collagen in the wound regenerated at only SIS sponges treated wounds. The progress of granulous tissue formation was faster in SIS sponges as wound dressing than in Tegaderm. In conclusion, we found that the SIS sponges might be a potential material as a wound dressing.