Non-animal Test Research Articles

FRAME and the Royal Commission on Environmental Pollution: Common Recommendations for Assessing Risks Posed by Chemicals under the EU REACH System

This document discusses recommendations made by FRAME and the Royal Commission on Environmental Pollution (RCEP) with regard to the current European Commission proposals on the Registration, Evaluation and Authorisation of Chemicals (REACH) system for assessing the risks of chemicals to humans, wildlife and the environment. Of several common aims and recommendations, the two most important are: a) the greater use of non-animal testing methods, especially computational prediction methods (for example, [quantitative] structure-activity relationships, expert systems and biokinetic modelling) for prioritising chemicals for hazard assessment; and b) the greater use of intelligent exposure-based targeted risk assessment, with less emphasis being placed on tonnage-triggers. FRAME has produced a decision-tree testing scheme to illustrate the way in which these approaches could be used, together with in vitro test methods. This scheme has been slightly modified to take account of proposals subsequently made by the RCEP. In addition, FRAME points out that new and improved computational methods are needed through more coordinated research, and that these and existing methods need to be validated. The similarities between the independent publications of FRAME and the RCEP add weight to the recommendations that each have made concerning the implementation of the REACH system.

Biokinetic and Toxicodynamic Modelling and its Role in Toxicological Research and Risk Assessment

Toxicological risk assessment for chemicals is still mainly based on highly standardised protocols for animal experimentation and exposure assessment. However, developments in our knowledge of general physiology, in chemicobiological interactions and in (computer-supported) modelling, have resulted in a tremendous change in our understanding of the molecular mechanisms underlying the toxicity of chemicals. This permits the development of biologically based models, in which the biokinetics as well as the toxicodynamics of compounds can be described. In this paper, the possibilities are discussed of developing systems in which the systemic (acute and chronic) toxicities of chemicals can be quantified without the heavy reliance on animal experiments. By integrating data derived from different sources, predictions of toxicity can be made. Key elements in this integrated approach are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (for example, tissue-blood partition coefficients, in vitro biotransformation parameters), tests or batteries of tests for determining basal cytotoxicity, and more-specific tests for evaluating tissue or organ toxicity. It is concluded that this approach is a useful tool for various steps in toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitro and other non-animal tests have already been developed.

A prevalidation study on the in vitro skin irritation function test (SIFT) for prediction of acute skin irritation in vivo: results and evaluation of ECVAM Phase III

A prevalidation study sponsored by the European Centre for the Validation of Alternative Methods (ECVAM) on in vitro tests for acute skin irritation is aimed at identifying non-animal tests capable of discriminating irritants (I) from non-irritants (NI), as defined according to European Union and OECD. This paper reports on Phase III for one of the methods, the skin integrity function test (SIFT), assessing the protocol performance of the SIFT, in terms of reproducibility and predictive ability, in three laboratories. The barrier function properties of excised mouse skin were determined using a set of 20 coded chemicals (10 I, 10 NI), using the endpoints of trans-epidermal water loss (TEWL) and electrical resistance (ER). The basis of the SIFT prediction model is if the ratios of the pre- and post-application values for either TEWL or ER are greater than five-fold, then the test chemical is deemed irritant (I). If the ratio of both parameters is less than five-fold then the chemical is deemed non-irritant (NI). Analysis of variance (ANOVA) indicated that the intra-lab reproducibility was acceptable but that the inter-lab reproducibility was not. Overall, the SIFT test under-predicted the irritancy of the test chemicals chosen for Phase III with an overall accuracy of only 55%. The sensitivity value (ability to correctly predict I) was only 30%. The specificity (ability to predict NI) of the test was better at 80%. A retrospective examination of the SIFT results was undertaken using Student's t-test and a significance level of P<0.05 to predict an irritant based on changes in the TEWL ratio values. This improved the predictivity of the SIFT test, giving a specificity of 60%, a sensitivity of 80% and an overall accuracy of 70%. Appropriate modifications to the prediction model have now been made and the SIFT will be re-examined in a new validation exercise to investigate the potential of this non-animal method to predict acute skin irritation potential.

Validation and regulatory acceptance of new carcinogenicity tests.

The 3 Rs concept, which was developed by Russell and Burch in 1959, was implemented into the legal framework in the European Union(EU) for the protection of vertebrate animals used for experimental and other scientific purposes, when Directive 86/609/EEC was adopted in 1986. To reduce or replace animal testing for regulatory purposes, nonanimal tests must be independently validated to prove that they can provide information that is relevant and reliable for hazard prediction in vivo. At the end of the 1980s, no scientific concept existed for the formal validation ofin vitro toxicity tests, so a small group of European and American scientists developed a set of principles for experimental validation, which was accepted internationally by the OECD in 1996. A major breakthrough was the acceptance in the EU of the scientifically validated in vitro toxicity test for phototoxic potential in 2000, which was accepted by the OECD at the worldwide level in 2002. Taking the progress in the development and validation of alternative toxicity tests during the past decade into account, the current concepts of developing alternatives to the standard 2-year rodent bioassay for carcinogenicity testing are discussed.

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.

Metabolism-mediated neurotoxicity: the significance of genetically engineered cell lines and new three-dimensional cell cultures.

Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process. Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.

The applicability of in vitro-derived data in hazard identification and characterisation of chemicals

Toxicological hazard and risk assessments for chemicals presently are mainly based on highly standardised protocols for animal experimentation and exposure assessment. In this paper the possibilities are being discussed of developing systems in which the systemic (acute and chronic) toxicity of chemicals can be quantified, without the heavy reliance on animal experiments. On the basis of a chemical's structure, in vitro data on its toxicity, and biokinetic modelling a decision/flow scheme is presented. Key elements are the evaluation of chemical functionalities representing structural alerts for toxic actions, the construction of biokinetic models on the basis of non-animal data (e.g. tissue–blood partition coefficients (PCs), in vitro biotransformation parameters), tests or batteries of tests for determining basal cytotoxicity and more specific tests for evaluating tissue- or organ toxicity. It is concluded that such a flow chart is a useful tool for different steps in the toxicological hazard and risk assessment, especially for those forms of toxicity for which validated in vitro and other non-animal tests have already been developed.

Non-animal testing strategies for assessment of the skin corrosion and skin irritation potential of ingredients and finished products

The dermatotoxicologist today is faced with a dilemma. Protection of workers and consumers from skin toxicities (irritation and allergy) associated with exposure to products, and the ingredients they contain, requires toxicological skin testing prior to manufacture, transport, or marketing. Testing for skin corrosion or irritation has traditionally been conducted in animals, particularly in rabbits via the long established Draize test method. However, this procedure, among others, has been subject to criticism, both for its limited predictive capacity for human toxicity, as well as for its use of animals. In fact, legislation is pending in the European Union which would ban the sale of cosmetic products, the ingredients of which have been tested in animals. These considerations, and advancements in both in vitro skin biology and clinical testing, have helped drive an intensive effort among skin scientists to develop alternative test methods based either on in vitro test systems (e.g. using rat, pig or human skin ex vivo, or reconstructed human skin models) or ethical clinical approaches (human volunteer studies). Tools are now in place today to enable a thorough skin corrosion and irritation assessment of new ingredients and products without the need to test in animals. Herein, we describe general testing strategies and new test methods for the assessment of skin corrosion and irritation. The methods described, and utilized within industry today, provide a framework for the practicing toxicologist to support new product development initiatives through the use of reliable skin safety testing and risk assessment tools and strategies.

Alternatives to animal testing in the safety evaluation of products.

The conventional method for assessing the safety of products, ranging from pharmaceuticals to agrochemicals, biocides and industrial and household chemicals - including cosmetics - involves determining their toxicological properties by using experimental animals. The aim is to identify any possible adverse effects in humans by using these animal models. Providing safe products is undoubtedly of the utmost importance but, over the last decade or so, this aim has come into conflict with strong public opinion, especially in Europe, against animal testing. Industry, academia and the regulators have worked in partnership to find other ways of evaluating the safety of products, by non-animal testing, or at least by reducing the numbers of animals required and the severity of the tests in which they are used. There is a long way to go before products can be evaluated without any animal studies, and it may be that this laudable aim is an impossible dream. Nevertheless, considerable progress has been made by using a combination of in vitro tests and the prediction of properties based on chemical structure. The aim of this review is to describe these important and worthwhile developments in various areas of toxicological testing, with a focus on the European regulatory framework for general industrial and household chemicals.

Tiered testing strategies--acute local toxicity.

More work has been done to develop alternatives to animal use in the areas of eye and skin irritation than in any area other than carcinogenicity. There has long been a belief both in the scientific community and among the public that the development of nonanimal tests in these areas should be simple and straightforward. After more than 20 yr of research, we can identify materials corrosive to the skin without using animals, but the assessment of irritation using in vitro methods alone is still an illusive goal. This review of current recommendations and industry practices that reduce the number of animals needed for these two tests concludes that animal use for skin irritation testing is not necessary today, with currently available and accepted methodology, except for regulatory reasons. Scientifically sound improvements in current eye irritation methods are also available. Advances in the understanding of the mechanisms of eye irritation that have been made in the last 5 yr should lead to improved in vitro methods for this endpoint. In the meantime, changes should be made in the current animal protocol to reduce pain and distress. This paper provides an overview of the progress that has been made toward discontinuing the use of animals in tests to determine the potential of materials to cause skin or eye damage after a single acute exposure. It also discusses some additional changes that could be made now to reduce animal use further or to reduce pain and distress in the testing that must still be done until such time as we can meet the ultimate goal--validated and accepted nonanimal methods for these endpoints.

FRAME Annual Lecture. International co-operation: an essential requirement for replacing animal toxicity tests.

The Three Rs concept, which was developed by Russell and Burch in 1959, was implemented into the legal framework in the European Union (EU) for the protection of vertebrate animals used for experimental and other scientific purposes, when Directive 86/609/EEC was adopted in 1986. One focus of activity under this Directive is the use of animals and alternative methods in regulatory testing. To reduce or replace animal testing for regulatory purposes, non-animal tests must be independently validated to prove that they can provide information that is relevant and reliable for hazard prediction in relation to specific types of toxicity in vivo. At the end of the 1980s, no scientific concept existed for the formal validation of in vitro toxicity tests, so a small group of European and American scientists met to develop a set of principles for experimental validation, which were first adopted by ECVAM in Europe in 1995, and, after harmonisation with experts from the USA and Japan, accepted internationally by the OECD in 1996. ECVAM has directly funded a number of validation studies, and a major breakthrough in the year 2000 was the acceptance for regulatory purposes in the EU of scientifically validated in vitro toxicity tests for phototoxic potential and for skin corrosivity. These, and other examples which are discussed, confirm that the internationally harmonised ECVAM/ICCVAM/OECD validation concept is a practical and effective way of making possible the replacement of regulatory testing in animals.

Novel advanced in vitro methods for long-term toxicity testing: the report and recommendations of ECVAM workshop 45. European Centre for the Validation of Alternative Methods.

This is the report of the forty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become wellinformed about the state-of-the-art of nonanimal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of in vitro tests and their potential uses, and make recommendations about the best ways forward. In addition, other topics relevant to the Three Rs concept of alternatives to animal experiments have been considered in several ECVAM workshops.

Approaches for the development of cell-based in vitro methods for contact sensitization

Allergic contact dermatitis (ACD) is a cell-mediated immune response to small molecular weight chemicals that contact and penetrate the skin. There are a variety of characteristics that determine whether a chemical can function as a contact sensitizer (or allergen) including the ability to penetrate into the skin, react with protein and be recognized as antigenic by immune cells. The ultimate challenge for developing non-animal test methods for skin sensitization testing will be applying our mechanistic understanding of ACD to the design of predictive in vitro alternative test methods. Specifically, the in vitro approach should be designed so that a chemical's potential to penetrate the skin, react with protein/peptide (biotransformation may be required) and initiate an antigen-specific immune response is incorporated in the test methods developed. In this review, we have focused on cellular-based assays that have been developed or proposed for assessing a chemical's skin sensitization potential in vitro. All of the promising leads to date are based on observations made from in vivo studies conducted in animals and humans, and therefore have a strong mechanistic foundation. However, it remains to be demonstrated whether a single in vitro test, or several in vitro tests in combination, which model the critical steps in sensitization, can replace animal experiments for predicting contact allergic reactions in humans. Regardless, the future looks promising with continued development of our understanding of the chemical and biological aspects of allergic contact dermatitis, and most importantly, with the application of genomics/proteomics to this field on the immediate horizon.

Toxicity Testing: The Many Arts of Persuasion

Last October, Vice President Al Gore announced a 6-year program to rapidly collect health and safety data on 2800 major industrial chemicals. But animal rights groups quickly denounced the so-called High Production Volume Challenge because they estimated it would require the destruction of more than a million animals. The groups charged that the killing of so many animals was needless because much of the information already existed, more could be derived from nonanimal tests, and some simply wasn9t worth collecting.

The use of human keratinocytes and human skin models for predicting skin irritation.

This is the report of the thirty-eighth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). ATLA 27, 723–743, 1999 723

The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

Non-animal Tests for Evaluating the Toxicity of Solid Xenobiotics

This is the report of the thirtieth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM’s main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well-informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and Non-animal Tests for Evaluating the Toxicity of Solid Xenobiotics

In Vitro Alternatives for Ocular Irritation

The necessity of using animals to test whether new chemicals and products are eye irritants has been questioned with increasing frequency and fervor over the last 20 years. During this time many new nonanimal methods have been proposed as reliable alternatives to the traditional rabbit (Draize) test. To date, however, none of these nonanimal (in vitro) tests have become universally accepted as a complete replacement for the Draize test. To understand why a complete replacement has not been found, one has to first understand the reasonably complex structure of the eye, the standard Draize scoring scale--which is based on a qualitative evaluation of three different tissues--the differences between human and rabbit eyes, the intrinsic variability of the animal test, and the details of the different in vitro tests that have been proposed as replacements. The in vitro tests vary from relatively simple assays using single cells to more sophisticated assays that use discarded animal tissue or artificially constructed human tissue. It is clear that appropriately designed in vitro tests will eventually give more useful mechanistic information about ocular injury from which we can more comfortably predict the risk of human eye irritation from new products and ingredients.

Mechanistic approaches and the development of alternative toxicity test methods.

A mechanism can be defined as an explanation of an observed phenomenon that explains the processes underlying the phenomenon in terms of events at lower levels of organization. A prerequisite for new, more mechanistic, approaches, which would use in vitro systems rather than conventional animal analogy models, is a strengthening of the underlying scientific basis of toxicity testing. This will require greater recognition of the differences between fidelity and discrimination models and between analogy and correlation models. The development of high-fidelity, high-discrimination tests with a sound mechanistic basis will also require greater appreciation of the interdependence of all the components of test systems and the development of new alternative (i.e., nonanimal) testing strategies that can provide the specific knowledge needed for making relevant and reliable predictions about the potential effects of chemicals and products in human beings. The optimal use of this new knowledge will require fundamental changes to current practices in risk assessment.

In vitro alternatives for ocular irritation.

The necessity of using animals to test whether new chemicals and products are eye irritants has been questioned with increasing frequency and fervor over the last 20 years. During this time many new nonanimal methods have been proposed as reliable alternatives to the traditional rabbit (Draize) test. To date, however, none of these nonanimal (in vitro) tests have become universally accepted as a complete replacement for the Draize test. To understand why a complete replacement has not been found, one has to first understand the reasonably complex structure of the eye, the standard Draize scoring scale--which is based on a qualitative evaluation of three different tissues--the differences between human and rabbit eyes, the intrinsic variability of the animal test, and the details of the different in vitro tests that have been proposed as replacements. The in vitro tests vary from relatively simple assays using single cells to more sophisticated assays that use discarded animal tissue or artificially constructed human tissue. It is clear that appropriately designed in vitro tests will eventually give more useful mechanistic information about ocular injury from which we can more comfortably predict the risk of human eye irritation from new products and ingredients.