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A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model >> RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations., (2008 FRAME.)

Objective: According trend of differentiation cardiomyocyte of embryonic stem cell,the model of EST has been built and the validation of the model is needed to be tested. It is profit to improve the method of safety evaluation., Methods: The embryonic stem cell differentiates into cardiomyocyte in different concentrations of Penicillin G,DPH and 5-FU with hanging and suspending culture conditions. With the results of cytotoxicity, the different embryonic toxicity characteristics of different substants may be detected clearly., Results: The ID50 (D3) concentration of three embryo toxicants were 1099, 47.4 and 0.023 microg/ml. Penicillin G, DPH and 5-FU were discriminated as none-embryotoxicity, weak-embryotoxicity and strong-embryotoxicity., Conclusion: Test compounds between three classes were discriminated correctly .The validation of EST model that established by us is high.

Human-based in vitro hepatic experimental systems are now used routinely in drug development. The rationale for the use of human-based in vitro systems is based on the known species-species differences in drug properties. Human-specific drug properties, by definition, cannot be defined using nonhuman experimental animals, and therefore can only be assessed in the preclinical phase of drug development using in vitro human-based approaches. A widely applied human-based in vitro experimental system for preclinical evaluation of drug properties is human hepatocytes. Our laboratory was one of the first to successfully isolate highly viable and functional hepatocytes from human livers, and we recently developed cryopreservation procedures to retain high viability and high attachment efficiency of the isolated hepatocytes. Successful cryopreservation of human hepatocytes greatly enhances the utility of this valuable in vitro experimental system, allowing storage, transport, convenient scheduling of experimentation and repeat experimentation using hepatocytes isolated from the same donors. Effective assays have been developed with cryopreserved human hepatocytes using multiwell plates for the evaluation of critical drug properties, including metabolic stability, drug-drug interaction potential, and drug toxicity. Human hepatocytes represent an alternative experimental system that plays a significant role in the 3Rs - the reduction, refinement, and replacement of the use of animals in preclinical drug development research.

Albert Schweitzer developed an egalitarian biocentrism which follows the maxim "I am life that wants to live, in the midst of life that wants to live". Following Schweitzer's idea of the Reverence for Life obviously leads to ethical dilemmas - as for example in the case of animal experimentation. In many situations we cannot but kill or harm, even if we don't want to, and must live at the cost of other living beings. How can the Reverence for Life stand up to that? Overcoming ethical dilemmas often means agreeing to compromises, which often leave us behind in discomfort. This discomfort and its meaning for Schweitzer's ethical concept can be illustrated by means of an example. Imagine two biologists, both conducting animal experiments that seem ethically justified and necessary to them. Nevertheless they can hold very different positions concerning their action. In some respect Schweitzer's ideas may be problematic and fairly radical. But they are also interesting and topical in so far as they don't let us get away easily after the decision-making process in an ethical dilemma. His theory opens up for the idea of compensation and development of alternative methods arising from what he calls a unique solidarity between human and non-human animals.

The assumption that animal models are reasonably predictive of human outcomes provides the basis for their widespread use in toxicity testing and in biomedical research aimed at developing cures for human diseases. To investigate the validity of this assumption, the comprehensive Scopus biomedical bibliographic databases were searched for published systematic reviews of the human clinical or toxicological utility of animal experiments. In 20 reviews in which clinical utility was examined, the authors concluded that animal models were either significantly useful in contributing to the development of clinical interventions, or were substantially consistent with clinical outcomes, in only two cases, one of which was contentious. These included reviews of the clinical utility of experiments expected by ethics committees to lead to medical advances, of highly-cited experiments published in major journals, and of chimpanzee experiments--those involving the species considered most likely to be predictive of human outcomes. Seven additional reviews failed to clearly demonstrate utility in predicting human toxicological outcomes, such as carcinogenicity and teratogenicity. Consequently, animal data may not generally be assumed to be substantially useful for these purposes. Possible causes include interspecies differences, the distortion of outcomes arising from experimental environments and protocols, and the poor methodological quality of many animal experiments, which was evident in at least 11 reviews. No reviews existed in which the majority of animal experiments were of good methodological quality. Whilst the effects of some of these problems might be minimised with concerted effort (given their widespread prevalence), the limitations resulting from interspecies differences are likely to be technically and theoretically impossible to overcome. Non-animal models are generally required to pass formal scientific validation prior to their regulatory acceptance. In contrast, animal models are simply assumed to be predictive of human outcomes. These results demonstrate the invalidity of such assumptions. The consistent application of formal validation studies to all test models is clearly warranted, regardless of their animal, non-animal, historical, contemporary or possible future status. Likely benefits would include, the greater selection of models truly predictive of human outcomes, increased safety of people exposed to chemicals that have passed toxicity tests, increased efficiency during the development of human pharmaceuticals and other therapeutic interventions, and decreased wastage of animal, personnel and financial resources. The poor human clinical and toxicological utility of most animal models for which data exists, in conjunction with their generally substantial animal welfare and economic costs, justify a ban on animal models lacking scientific data clearly establishing their human predictivity or utility.

In vitro toxicology generally refers to the study of toxicological phenomena in non-whole-animal models. This broader connotation includes studies utilizing isolated organs, tissue slices, explants cultures, and isolated primary cells, as well as cell lines and subcellular fractions (e.g., microsomes). These model systems have made major contributions to toxicological sciences, particularly in our understanding of the mechanisms of toxicity, xenobiotic metabolism, and species differences in expressions of toxicity. This unit reviews the use of in vitro models and their value in toxicology testing., (© 2007 by John Wiley & Sons, Inc.)

The contribution of animal experimentation to biomedical research is of undoubted value, nevertheless the real usefulness of animal models is still being hotly debated. Laboratory Animal Science is a multidisciplinary approach to humane animal experimentation that allows the choice of the correct animal model and the collection of unbiased data. Refinement, Reduction and Replacement, the "3Rs rule", are now widely accepted and have a major influence on animal experimentation procedures. Refinement, namely any decrease in the incidence or severity of inhumane procedures applied to animals, has been today extended to the entire lives of the experimental animals. Reduction of the number of animals used to obtain statistically significant data may be achieved by improving experimental design and statistical analysis of data. Replacement refers to the development of validated alternative methods. A Laboratory Animal Science training program in biomedical degrees can promote the 3Rs and improve the welfare of laboratory animals as well as the quality of science with ethical, scientific and economic advantages complying with the European requirement that "persons who carry out, take part in, or supervise procedures on animals, or take care of animals used in procedures, shall have had appropriate education and training".

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.

Public support is a strong impetus for the adoption of alternatives to laboratory animals. It is therefore important to find out what a society thinks about ethical animal use. In the case of China, a useful line of enquiry was to survey Chinese people's as their country is renowned for the deplorable conditions under which animals are kept. This report concerns an investigation into the attitudes of Chinese university students toward the use of animals in laboratory research. The survey revealed a moderate concern amongst students; for example, they agreed that the use of animals for testing cosmetics and household products is unnecessary and should be stopped, and disagreed that humans have the right to use animals as they see fit. This finding is very encouraging. Further research is needed, in order to understand Chinese views about the justification of using animals in research.

This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.

The final text of the REACH legislation, that has recently been published by the European Parliament, and which will come into force in the middle of 2007, implies that results obtained from so-called 'suitable' in vitro methods could be used for testing. It is also stated that a 'suitable' test is one that has been deemed to be ready to enter a validation study - for example, according to criteria specified by ECVAM. It is argued that, because the wording of the legal text is too vague and imprecise, it is totally unsatisfactory, mainly because it seems to endorse the application of non-validated methods for regulatory risk assessment. While their use might be suitable for providing information for classification in a weight-of-evidence approach, setting safe dose levels should only ever be based on the use of data from a properly validated test method. This concern, and other problems raised by the final legislation, is discussed, and recommendations are made which could avoid the premature use of potentially unsuitable methods for risk assessment, whilst ensuring that potentially suitable tests can be used for this purpose, once they have been formally validated.

A critical analysis is presented of the reports produced by four Technical Expert Working Group Sub-groups (SGs) on Ethical Review, Cost-Benefit, Authorisation and Scope, which were published on the EC website (http://ec.europa.eu/environment/chemicals/lab_animals/ia_info_en.htm), as part of the European Commission (EC)s review of EU Directive 86/609 EEC. This is in addition to our official response to the internet consultation questionnaire, submitted to the Commission on behalf of FRAME. Whilst the respective SG reports were extensive and detailed, we have identified several shortcomings, and in particular, a frequent lack of consensus among the SG members, resulting in a lack of clear guidance for the EC on what the revised Directive should contain, with reference to a number of crucial issues. Such indecisiveness could lead to wide discrepancies in the approaches of the EC, the European Parliament and the EU Member States concerning many issues of importance to animal welfare and the implementation of alternatives to animal experiments. These concerns range from logistical issues, such as requirements for named officers in authorised establishments, and the recording and publishing of statistics on animal use, to ethical and scientific problems, including the use of non-human primates, local ethical review, and education and training on the essential link between the Three Rs concept and best scientific practice. In each case, the basis for our concerns is explained, and suggestions for improvements to be incorporated into the revised Directive are made, in the hope that the harmonisation of approaches to laboratory animal experimentation and the use of alternative methods in the Member States can be maximised.

The European Chemicals Bureau and the Organisation for Economic Cooperation and Development are currently compiling a sanctioned list of quantitative structure-activity relationship (QSAR) risk assessment models and data sets to predict the physiological properties, environmental fate, ecological effects and human health effects of new and existing chemicals in commerce in the European Union. This action implements the technical requirements of the European Commission's Registration, Evaluation and Authorisation of Chemicals legislation. The goal is to identify a battery of QSARs that can furnish rapid, reliable and cost-effective decision support information for regulatory decisions that can substitute for results from animal studies. This report discusses issues and concerns that need to be addressed when selecting QSARs to predict human health effect end points.

When addressing the use of fish for the environmental safety of chemicals and effluents, there are many opportunities for applying the principles of the 3Rs: Reduce, Refine, and Replace. The current environmental regulatory testing strategy for bioconcentration and secondary poisoning has been reviewed, and alternative approaches that provide useful information are described. Several approaches can be used to reduce the number of fish used in the Organization for Economic Cooperation and Development (OECD) Test Guideline 305, including alternative in vivo test methods such as the dietary accumulation test and the static exposure approach. The best replacement approach would seem to use read-across, chemical grouping, and quantitative structure-activity relationships with an assessment of the key processes in bioconcentration: Adsorption, distribution, metabolism, and excretion. Biomimetic extraction has particular usefulness in addressing bioavailable chemicals and is in some circumstances capable of predicting uptake. Use of alternative organisms such as invertebrates should also be considered. A single cut-off value for molecular weight and size beyond which no absorption will take place cannot be identified. Recommendations for their use in bioaccumulative (B) categorization schemes are provided. Assessment of biotransformation with in vitro assays and in silico approaches holds significant promise. Further research is needed to identify their variability and confidence limits and the ways to use this as a basis to estimate bioconcentration factors. A tiered bioconcentration testing strategy has been developed taking account of the alternatives discussed.

The European Partnership for Alternative Approaches to Animal Testing (EPAA) pointed out the need to involve authorities throughout the process of validation and legal acceptance of alternatives to animal experiments. The Paul-Ehrlich-Institute (PEI), Federal Agency for Sera and Vaccines, is the national competent authority in Germany which is responsible for the quality and safety of biologicals including blood and cell-based products. This paper is intended to contribute to the discussion concerning the use of alternative methods in safety testing of medicinal products and considers the scientific work of the PEI in this field. From a regulator's perspective, adequate demonstration of safety and quality of medicinal products are of major interest. Additionally, the availability of the products to the patient has to be taken into consideration. It has to be carefully explored whether the respective in vitro method for demonstration of non-clinical safety as part of the non-clinical development programme is able to guarantee safety level comparable to the corresponding experiment in animals. The topics cited above shall be discussed in this paper using the example of the Alternative Pyrogen Test or also called Monocyte Activation Test. The Alternative Pyrogen Test could serve as paradigm to exemplify how an alternative test can provide at least a comparable level of safety estimation in comparison with a conventional animal test. Furthermore, this alternative test creates additional information which cannot be obtained from the animal experiment, and might also open further scientific insight into the mechanisms of pyrogenicity and acute pro-inflammatory reactions in patients. This test method allows the definition of pyrogen limits for medicinal products. Due to its use of relevant cell systems this in vitro test might contribute significantly to safety assessments of advanced medicinal products during the pre-clinical phase.

Approximately 30% of animal use within the European Union (EU) is done to meet regulatory requirements. The tests are often repetitive in nature and may cause severe suffering, due to the procedures used and to rigidly predefined end points. In addition, product evaluation procedures often take long and are very expensive. Over the last decades the heavy reliance on animal experimentation in this area has met serious objections, both ethical and economical in nature. This study describes obstacles and opportunities to implement the 3Rs in regulatory animal testing. The findings are based primarily on interviews with legislators, regulators, industry, science and animal welfare organisations and reflect shared perceptions of these respondents. In order to increase the application of the 3Rs in regulatory testing a number of technical, political and social obstacles must be overcome. This study offers insight into the persistent character of regulatory animal testing and can function as a starting point for further discussion on how to tackle these problems. To this end, several recommendations are made ranging from strategic test approaches and data sharing to strengthening the policy network and improving communication between 3Rs experts and regulators. The study is an initiative of the national project group "Regulatory Animal Testing", which consists of a group of Dutch experts on animal testing working for a variety of organisations in the field.1 They felt the need for cooperation to initiate a discussion at relevant levels and to identify possible solutions in order to implement the objectives of the three R's in testing for regulatory purposes without loss of scrutiny in safety and/or efficacy evaluation needed for product release.

The study is a contribution to the EDQM's efforts to meet some of the expectations of the 3 Rs: Replacement, Reduction and Refinement of animal assays as proposed by Russell and Burch in 1959 and adopted by the European Union in 1986, and specifically to validate alternative assays to replace, for batch-release purposes, the European Pharmacopoeia (Ph. Eur.) in vivo direct challenge procedures for the potency determination of diphtheria toxoid vaccines. The study results may be used in support of the replacement of the multi-dilution direct challenge procedures in different animal models by a single dilution serology test, where appropriate, and to use sera from the same animals for potency testing of several components in combined vaccines. With regard to the latter, the present study explores the possibility of testing both diphtheria and tetanus toxoid potencies using serum from the same animals.

It is claimed that the modular approach to validation, which involves seven independent modules, will make the assessment of test validity more flexible and more efficient. In particular, the aspects of between-laboratory variability and predictive capacity are formally separated. Here, the main advantage of the approach is to offer the opportunity for reduced labour, and thus to allow study designs to be more time efficient and cost effective. The impact of this separation was analysed by taking the ECVAM validation study on in vitro methods for skin corrosivity as an example of a successful validation study - two of its methods triggered new OECD test guidelines. Lean study designs, which reduced the number of tests required by up to 60%, were simulated with the original validation data for the EPISKIN model. By using resampling techniques, we were able to demonstrate the effects of the lean designs on three between-laboratory variability measures and on the predictive capacity in terms of sensitivity and specificity, in comparison with the original study. Overall, the study results, especially the levels of confidence, were only slightly affected by the lean designs that were modelled. It is concluded that the separation of the two modules is a promising way to speed-up prospective validation studies and to substantially reduce costs, without compromising study quality.

Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.

Traditionally, regulatory required vaccine quality control relies on the use of laboratory animals. Both batch testing for safety and testing for potency are based on animal models. Quite often these models include procedures that induce severe pain and suffering. This has urged the development of 3Rs methods. Some examples of recent achievements in the quality control of toxoid vaccines are the replacement of challenge procedures by serological methods, the reduction of numbers of animals required by changing from multi-dose to single dose testing, and developments in the area of in vitro models and physicochemical techniques. Central in the progress towards the elimination of animal use is the acceptance of the so-called consistency approach. The essence of the consistency approach is that a new batch of vaccine is no longer seen as a unique product but as only one of a series of batches produced from the same starting material (seed lot). Consequently, the new batch shares many of the characteristics of the previous batches produced from the same seed lot. This allows for a new strategy of vaccine quality control consisting of demonstrating consistency in production, placing emphasis on aspects such as in-process testing, and implementing Good Manufacturing Practice and Quality Assurance (QA). The new strategy particularly focuses on non-animal test models such as physicochemical methods and in vitro models. Implementation of the consistency approach might significantly contribute towards the elimination of the use of animals in regulatory required vaccine quality control.

The Dutch Act on Animal Experimentation (1996) requires that local animal experiments committees (AECs) review animal experiments and balance the scientific and societal benefits of the experiments against the suffering caused to the animals used. Each AEC is composed of at least seven members who provide a balance of expertise in animal experiments, alternatives to laboratory animal experiments, ethics, and animal welfare and protection. This study proposes selection criteria for individuals possessing each of the four AEC required areas of expertise. Criteria were established minding that, on the one hand, sufficient knowledge and expertise can be demonstrated whilst, on the other hand, a sufficient number of people would qualify to participate in the AECs. The results of this study may serve as a starting point for further discussion of selection criteria for members of AECs both in the Netherlands and in other countries where ethical review processes have been or are being implemented.

The necessity to revise Directive 86/609/EWG has been discussed since 1993, growing more urgent since the Protocol to the Treaty of Amsterdam (1997) and the Treaty establishing a Constitution for the European Union (2004), both of which recognise the welfare of animals as sentient beings; a revision also proves necessary because of various changes within the framework of animal experimentation. Within an expert questionnaire launched in the summer of 2006 several shortcomings of Directive 86/609/EWG are pointed out; proposals for revision mainly concern the scope of the directive, authorisation of animal experiments by national bodies, controls on institutional level, minimal requirements for housing and care of laboratory animals as well as for qualification of staff, and instruments for reducing duplication of animal experiments. The introduction of an obligatory ethical review as part of a standardised and project-related authorisation procedure, the up-grading of housing and care standards and the implementation of mechanisms for reducing duplication of animal experiments may enhance full exploitation of the 3R and are therefore crucially important for a directive complying with the requirements of the Treaty of Amsterdam.

Many in vitro and ex vivo methods have been developed or are under development to reduce or replace animal usage in toxicity tests. Consistent with the goal of obtaining scientifically sound test data for hazard and risk assessment of chemicals, changes are being made in current policies and procedures to facilitate the acceptance of data developed using these methods. National and international organizations are developing policies and standards for scientific practice to assure quality in implementation of in vitro methods. Consensus is developing in the scientific community for the quality control measures needed for in vitro methods; including appropriate controls, data reporting elements, and benchmarks to be identified in test guidelines so that the potential risks of chemicals can be reviewed and reliably assessed. Additional guidance to the OECD's Good Laboratory Practice principles [Organization for Economic Cooperation and Development (. Advisory Document of the Working Group on Good Laboratory Practice: The Application of the Principles of GLP to in vitro Studies. OECD Series on Principles of Good Laboratory Practice and Compliance Monitoring Number 14 (ENV/JM/MONO(2004)26). Paris, France] will help to ensure that in vitro tests used for regulatory purposes are reproducible, credible, and acceptable. Generic test guidelines incorporating performance standards are being written to allow acceptance of proprietary test methods by regulatory agencies and to provide assurance that any in vitro system performs over time in a manner that is consistent with the test system as it was originally validated.

Cell lines have wide applications as model systems in the medical and pharmaceutical industry. Much drug and chemical testing is now first carried out exhaustively on in vitro systems, reducing the need for complicated and invasive animal experiments. The basis for any research, development or production program involving cell lines is the choice of an authentic cell line. Microsatellites in the human genome that harbour short tandem repeat (STR) DNA markers allow individualisation of established cell lines at the DNA level. Fluorescence polymerase chain reaction amplification of eight highly polymorphic microsatellite STR loci plus gender determination was found to be the best tool to screen the uniqueness of DNA profiles in a fingerprint database. Our results demonstrate that cross-contamination and misidentification remain chronic problems in the use of human continuous cell lines. The combination of rapidly generated DNA types based on single-locus STR and their authentication or individualisation by screening the fingerprint database constitutes a highly reliable and robust method for the identification and verification of cell lines.