bluuebunny/crossref_metadata_2025 huggingface dataset:
The Impact of the Introduction of the Ethical Review process for Research using Animals in the UK: Attitudes to Alternatives among those Working with Experimental Animals
Authors: Purchase Iain F.H. , Nedeva Maria
Published: April 2019

A postal questionnaire survey was carried out in late 1999 of those involved in working under the Animals (Scientific Procedures) Act 1986, namely the Certificate Holders, Project Licence Holders, Personal Licence Holders, Named Veterinary Surgeons, and Named Animal Care and Welfare Officers. The aim of the survey was to elicit views on the effectiveness of the introduction of the Ethical Review Process (ERP), introduced in April 1999. This report covers issues related to the use of alternatives, which were incorporated into the questionnaires. The number of returned questionnaires (45% of 1636 questionnaires) was sufficient for a meaningful analysis to be made of attitudes to the use of alternatives. In response to questions about the reason for the use of alternatives, more than 80% answered that alternatives should be used on moral or ethical grounds. Only about 50% of Certificate Holders and Licence Holders answered that alternatives were used because of legal requirements. Most respondents believed that replacement alternatives did not provide scientific information of equivalent value to that obtained from animal experiments. However, the majority also believed that it was possible to carry out valid scientific experiments by using replacement alternatives. The majority of Named Animal Care and Welfare Officers believed that the ERP had improved many aspects of refinement alternatives. In particular the “culture of care” had improved. Most establishments had a formal mechanism for discussing alternatives, although it was noteworthy that relatively few Personal Licence Holders believed this to be the case. In general, the majority of those working under the 1986 Act and most establishments seem to have a positive approach to the use of alternatives.
Selection of Test Chemicals for the ECVAM International Validation Study on In Vitro Embryotoxicity Tests
Authors: Brown Nigel A.
Published: April 2019

The European Centre for the Validation of Alternative Methods (ECVAM) has sponsored a large international prevalidation and validation study of three embryotoxicity tests, involving embryonic stem cells, limb bud micromass cultures, and postimplantation whole-embryo cultures. The main objective of the study was to assess the performance of these in vitro tests in discriminating between non-embryo-toxic, weakly embryotoxic and strongly embryotoxic compounds. An initial part of the study was to select 20 test substances for the formal validation trial, conducted under blind conditions. A database of in vivo and in vitro developmental toxicity test results was compiled on 309 chemicals that had been used in previous validation studies, or suggested for such use, or that had good quality “segment II”-type in vivo data, or for which there were human data. From this database, a shortlist of about 30 candidates was constructed. Because the ECVAM study would not include metabolic activation, chemicals known to require activation for their developmental effects were excluded as candidates, although some known stable metabolites were included. Attempts were made: to include substances of diverse mechanism; to avoid overemphasis on pharmaceuticals; to avoid biologically inert substances as non-embryotoxicants; and to make the list different from those used previously. The candidates were of three categories: Class 3, strongly embryotoxic, was defined as developmentally toxic in all species tested, inducing multiple developmental effects, and with a high A/D ratio. Class 1, non-embryotoxic, was defined as not developmentally toxic at maternally toxic exposures, but which may show some minor embryo/fetal toxicity, which cannot be separated from maternal toxicity. Class 2, weakly embryotoxic, were chemicals of intermediate activity. From this candidate list, chemicals of known receptor (androgen, oestrogen, glucocorticoid, aryl hydrocarbon) mechanisms were excluded, on the basis that simple tests for such activity are already available. In addition, chemicals not freely available were excluded, and an emphasis on human data was applied. The final list of 20 chemicals was: Class 3 — 6-aminonicotinamide, 5-bromo-2′-deoxyuridine, hydroxyurea, methylmercury chloride, methotrexate, all-trans-retinoic acid; Class 2 — boric acid, dimethadione, lithium chloride, methoxyacetic acid, valproic acid (VPA), 2-propyl-4-pentynoic acid (4-yn-VPA), salicylic acid sodium salt; and Class 1 — acrylamide, D-(+)-camphor, dimethyl phthalate, diphenhydramine hydrochloride, 2-ethyl-4-methylpentanoic acid (isobutyl-ethyl-VPA), Penicillin G sodium salt, saccharin sodium hydrate.
Possibilities of Replacing and Reducing Animal Tests in the Pharmacopoeias: An Evaluation of Unused Methods and Proposals for Change
Authors: Miethe Gundel
Published: April 2019

The animal tests currently carried out according to the German and European pharmacopoeias are evaluated. The routine testing of biological medicines, excluding vaccines and pyrogen tests, is examined. The opportunities for replacing obligatory bioassays and safety tests are assessed, taking into account both pharmaceutical quality and animal welfare aspects. The aim is to encourage institutions, public authorities, and the pharmaceutical industry to investigate the importance and the necessity of the remaining tests. In addition, all parties should be encouraged to initiate integrated projects to develop, validate and establish alternative methods.
Stable Biocompatible Adjuvants — a New Type of Adjuvant Based on Solid Lipid Nanoparticles: A Study on Cytotoxicity, Compatibility and Efficacy in Chicken
Authors: Olbrich Carsten , Müller Rainer Helmut , Tabatt Kerstin , Kayser Oliver , Schulze Christoph , Schade Rüdiger
Published: April 2019

A new type of adjuvant was tested for its ability to initiate antibody production in chickens, and its cellular and tissue compatibility were assessed. The stable biocompatible adjuvants tested are based on surface-modified solid lipid nanoparticles (SLNs), made from paraffin or biodegradable glyc-erides, and are simply admixed to the antigens before administration. The tissue-damaging potency of four formulations of the new adjuvants (H1, H2, H3 and H4) were first tested in vitro by using human foreskin fibroblasts and RAW 264.7 macrophages. The adjuvants were well tolerated by both cell types. Immunisation studies in chickens were performed by using a Mycoplasma bovis antigen and mouse immunoglobulin G (IgG). The resulting antibodies were non-invasively extracted from egg yolk. The use of the various adjuvant formulations resulted in a significant production of specific antibodies after the first and second booster immunisations. Freund's complete adjuvant (FCA), considered until now to be the “gold standard” among the adjuvants, revealed the highest antibody titre against mouse IgG. SLNs with a particle size of more than 100nm exhibited a clear adjuvant activity, whereas SLNs with a particle size below 100nm, in various concentrations, revealed a lower adjuvant activity. Immunisation of chickens with the mouse IgG alone, dissolved in phosphate-buffered saline, resulted in a slow development of antibody titre. At the end of the experiment, the chickens were examined for vaccination-associated tissue damage. In contrast to FCA, the SLN formulations caused only minor tissue irritation at the injection sites. In conclusion, SLNs seem to be a promising alternative to FCA for antibody production in chickens, and potentially in other animals.
Arsenic Toxicity and HSP70 Expression in Xenopus laevis Embryos
Authors: Gornati Rosalba , Monetti Claudio , Vigetti Davide , Bosisio Stefano , Fortaner Salvador , Sabbioni Enrico , Bernardini Giovanni , Prati Mariangela
Published: April 2019

The evaluation of the effect of trace metals on health can be difficult, because of their presence in the environment in various chemical forms. Exposure to arsenic compounds is an example of this complexity, as it can be present in the environment in inorganic and organic forms. The effects of arsenic in vertebrates are complicated by several variables, such as speciation of the element, the exposure route, and the susceptibility of the particular animal species. The embryotoxicity and teratogenicity of three arsenic species — sodium arsenite (NaAsO2), disodium hydrogen arsenate (Na2HAsO4) and dimethylarsinic acid [(CH3)2AsOOH] — were evaluated by the modified frog embryo teratogenic assay on Xenopus (FETAX). We also show how the classical FETAX endpoints, based on morphological and morphometrical analysis, can conveniently be integrated with the study of molecular markers. Possible changes in the expression of the mRNA for the heat-shock protein HSP70, following exposure to NaAsO2, were examined by using the reverse transcriptase polymerase chain reaction. HSP70 mRNA is strongly induced by arsenic.
Validation Successes: Chemicals
Authors: Spielmann Horst , Liebsch Manfred
Published: April 2019

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, by the Federal regulatory agencies of the USA, and by 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 to be scientifically valid and appropriate for routine use in laboratories of the European pharmaceutical and chemicals industries.
ECVAM's Research and Validation Activities in the Fields of Topical Toxicity and Human Studies
Authors: Zuang Valérie
Published: April 2019

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's Activities on Biologicals
Authors: Halder Marlies , Balls Michael , Hendriksen Coenraad , Cussler Klaus
Published: April 2019

This paper summarises key activities initiated and the progress achieved between April 1993 and June 2002 in implementing the Three Rs in one of ECVAM's priority areas — the production and quality control of biologicals. These have included: organising nine key workshops; financially supporting and/or participating in a number of prevalidation and/or validation studies; financial contributions and sponsorship to relevant international workshops, symposia and conferences; and financial support for the compilation of manuals and expert reports, and training in test methods. The paper complements the papers of Hendriksen et al. and Cussler et al. included in these proceedings.
The ECVAM Scientific Information Service (SIS)
Authors: Roi Annett Janusch
Published: April 2019

The aim of this ECVAM Status Seminar was to critically review the contributions made by ECVAM in relation to its four main tasks. The establishment and maintenance of the ECVAM Scientific Information Service (SIS) is a precise means of fulfilling one of these four principal duties of ECVAM. The major achievements of the SIS, and the efforts required to achieve them, are discussed, together with the immediate future for the SIS.
ECVAM: Desperately Needed or Superfluous? An Animal Welfare Perspective
Authors: Kolar Roman
Published: April 2019

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 they 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.
Dialogue and Collaboration with ECVAM: The View of the EFPIA
Authors: Garthoff Bernward
Published: April 2019

An evaluation is presented of past experience of dialogue and collaboration between ECVAM and the European Federation of Pharmaceutical Industries and Associations (EFPIA) over the last nine years. Lessons learnt from the viewpoint of EFPIA company representatives are given. Also, proposals for the future ECVAM approach are made, such as support for other research areas for new methods to be validated, giving realistic statements to ECVAM's European Union and external customers, and being open to any new technology development that might help in opening and establishing new alternative avenues. Finally, the need for proper publications on the implementation of alternatives is recommended, for example, through the existing national platforms and their umbrella organisation, ecopa.
Ecopa: A Powerful Concept in the Way Forward for Alternative Methods
Authors: Rogiers Vera
Published: April 2019

ecopa, the European Consensus-Platform on Alternatives, is an international not-for-profit organisation, based in Belgium and complying with Belgian Law. It is the only quadripartite organisation that promotes the Three Rs at the European level. Ecopa brings together national consensus platforms on alternative methods. Consensus means that all parties concerned are represented, including animal welfare, industry, academia and government. Ecopa currently includes the National Platforms of 14 EU Member States (or future Member States; eight full members, namely, Austria, Belgium, Finland, Germany, The Netherlands, Spain, Switzerland and the UK, and six associate members, being the Czech Republic, Denmark, Italy, Norway, Poland and Sweden). Ecopa also has three working groups, concerned with: a) the 6th Framework Programme of the EC for Research, Technological Development and Demonstration Activities; b) the EC White Paper Strategy for a Future EU Chemicals Policy; and c) the formation of educational programmes on alternative methods within the EU. Ecopa is thus uniquely placed and has huge expertise to offer to the debate around political topics, including the White Paper, the 6th Framework Programme, and the 7th Amendment of the EU Cosmetics Directive. Ecopa should be considered a key stakeholder by the European Commission and Parliament, and it is essential that the views of ecopa are fully incorporated into future legislation. Recently, the ecopa working groups made a strong common statement on the Chemicals Policy White Paper and made a number of recommendations to the Commission based on scientific, practical and realistic grounds. These are to be found on the ecopa Web site ( http://ecopa.tsx.org/ or http://ecopa.vub.ac.be ).
The Seventh Amendment to the Cosmetics Directive: What does DG Enterprise want from ECVAM?
Authors: Schumann Regina
Published: April 2019

A short overview is given of the current situation regarding the draft seventh amendment to the EU cosmetics directive, Council Directive 76/768/EEC. Future perspectives are discussed.
ECVAM and Pharmaceuticals
Authors: Vanparys Philippe
Published: April 2019

In the pharmaceutical industry, toxicology testing is normally done by preclinical scientists during the Development phase. In the last decade, the implementation of high-throughput screens during the Discovery phase has resulted in an ever-increasing number of lead candidates to be selected for drug development. The low throughput of the conventional safety tests is a bottleneck in the drug-development process. The pharmaceutical industry needs new techniques, down-scaled tests and in vitro alternative test models to determine the absorption, distribution, metabolism, and excretion (ADME) and toxicology profiles of compounds in the late-Discovery phase and/or early in the Development phase. Medium-throughput ADME and toxicity tests will enhance the selection of safer new chemical entities for animals and/or humans. Consequently, this testing strategy will not only reduce the use of resources and the overall development time, but will also result in a substantial decrease in animal use.
Biokinetic and Toxicodynamic Modelling and its Role in Toxicological Research and Risk Assessment
Authors: Blaauboer Bas J.
Published: April 2019

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 Critical Assessment of the European Commission's Proposals for the Risk Assessment and Registration of Chemical Substances in the European Union
Authors: Combes Robert , Dandrea Jennifer , Balls Michael
Published: April 2019

In May, 2003, the European Commission published detailed proposals relating to its 2001 White Paper – Strategy for a Future Chemicals Policy. The White Paper described a new registration system called the REACH (Registration, Evaluation and Authorisation of Chemicals) system, for both new and existing chemicals. Subsequently, these detailed proposals were available for an eight-week consultation period for stakeholders to voice their views and concerns. In this paper, we describe our reactions to the Commission's more-detailed proposals. These include the creation of a European Chemicals Agency to implement the REACH system in conjunction with Competent Authorities (CAs) in Member States and the Commission itself. Unfortunately, many of our concerns and suggestions, previously voiced and shared with several other key stakeholders, remain unanswered, but are as relevant as when the White Paper was published. In particular, we are concerned about the lack of a clear and coherent strategy. There is no guidance for registrants on intelligent testing to maximise the use of non-animal approaches to safety testing, based on a combination of factors for estimating exposure levels, rather than mainly on production volumes. We are also concerned about the absence of a clear programme for the development, improvement and validation of new alternative methods, in conjunction with the Commission's own unit, the European Centre for the Validation of Alternative Methods, as well as other organisations with relevant expertise and experience, including FRAME. Finally, we explain why such measures should be introduced, together with clearer guidelines for the respective roles of the Agency, the CAs and the Commission in implementing and harmonising the REACH system at the European Union and Member State levels. A series of recommendations are made, to improve the situation and to improve the risk assessment process.
ECVAM's Response to the Changing Political Environment for Alternatives: Consequences of the European Union Chemicals and Cosmetics Policies
Authors: Hartung Thomas , Bremer Susanne , Casati Silvia , Coecke Sandra , Corvi Raffaella , Fortaner Salvador , Gribaldo Laura , Halder Marlies , Roi Annett Janusch , Prieto Pilar , Sabbioni Enrico , Worth Andrew , Zuang Valerie
Published: April 2019

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.
A Survey of Stakeholder Organisations on the Proposed New European Chemicals Policy
Authors: Dandrea Jennifer , Combes Robert D.
Published: April 2019

In February 2001, the European Commission published a White Paper proposing that a single new system of chemical regulation should be applied throughout the Member States of the European Union. The proposed Registration, Evaluation and Authorisation of Chemicals (REACH) system was to include both new and existing chemicals, with the aim of ensuring that sufficient pertinent data were made available to enable human health and the environment to be protected. The policy was founded on the principle of sustainable industrial development, and ambitiously attempted to incorporate the needs and views of key stakeholder organisations, such as industry, trade associations, consumer groups, environmentalists, animal welfarists and Member State governments. During the period between the publication of the White Paper and the on-line publication of consultation documents, as part of a public consultation exercise, in May 2003, many of these key stakeholder organisations produced material in support of or critical of the White Paper, either in part or as a whole. In this paper, we have attempted to review this extensive material and to present it in the context of the current chemical regulatory system that the REACH system will replace. Emphasis is placed on the impact of the new policy on the number of animals used in the testing regimes within the REACH system and the inclusion of alternative methods into the legislation. Although supportive of the overriding aims of the new policy, FRAME believes that the fundamental concept of a risk-free environment is flawed, and that the new REACH system will involve the unjustifiable use of millions of laboratory animals. The new policy does include alternative methods, particularly for baseset substances. Nevertheless, alternative testing methods that are already available have been excluded and replaced with outdated in vivo versions. There is also insufficient detail with regard to the further development and validation of alternative methods, particularly for substances of high concern, such as endocrine disrupters or reproductive toxins, for which no alternative testing methods currently exist.
FRAME and the Royal Commission on Environmental Pollution: Common Recommendations for Assessing Risks Posed by Chemicals under the EU REACH System
Authors: Combes Robert D. , Dandrea Jennifer , Balls Michael
Published: April 2019

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.
Two-stage Testing of Safety: A Statistical View
Authors: Hauschke Dieter , Hothorn Ludwig A.
Published: April 2019

Sample sizes given in regulatory guidelines are not based on statistical reasoning. However, from an ethical, scientific, and regulatory point of view, a mutagenicity experiment must have a reasonable chance of supporting the decision as to whether a result is negative or positive. Consequently, the sample size should be based on type I and type II errors, the underlying variability, and the specific size of a treatment effect. A two-stage adaptive interim analysis is presented, which permits an adaptive choice of sample size after an interim analysis of the data from the first stage. Because the sample size of the first stage is considered to be a minimum requirement, this stage can also be regarded as a pilot study.
Human Research Tissue Banks: The ATRA Project for Establishing a Human Research Tissue Bank in Switzerland
Authors: Tettamanti Massimo , Tralamazza Sara , Berati Marina , Molteni Max , Gamba Natascia
Published: April 2019

A large number of experiments in biomedical research are carried out on tissues, but, even though the results should be applicable to humans, these tissues are mainly of animal origin. The difficulty encountered in obtaining human organs and tissues is an acknowledged problem: not enough human tissues are available to meet research needs. We are introducing the ATRA Project, with the purpose of supporting progress in biomedical research in Switzerland through the establishment of one or more human tissue banks, which will be able to find, treat, preserve and supply human material. Where similar projects have already been launched, concerns have been expressed that donation for research purposes might compete with donation for transplantation, but most organs and tissues are in any case non-transplantable. Surplus surgical tissue is considered “sanitary waste”, and must be treated according to specific regulations for collection, packaging, transport, treatment and disposal. A human tissue bank would not only abate the costs of treating sanitary waste, but would actually turn what is now considered waste into a resource which could be used to save human and animal lives.
A Human Corneal Equivalent Constructed from SV40-immortalised Corneal Cell Lines
Authors: Zorn-Kruppa Michaela , Tykhonova Svitlana , Belge Gazanfer , Bednarz Jürgen , Diehl Horst A. , Engelke Maria
Published: April 2019

Within the last decade, extensive research in the field of tissue and organ engineering has focused on the development of in vitro models of the cornea. The use of organotypic, three-dimensional corneal equivalents has several advantages over simple monolayer cultures. The aim of this study was to develop a corneal equivalent model composed of the same cell types as in the natural human tissue, but by using immortalised cell lines to ensure reproducibility and to minimise product variation. We report our success in the establishment of an SV40-immortalised human corneal keratocyte cell line (designated HCK). A collagen matrix, built up with these cells, displayed the morphological characteristics of the human stromal tissue and served as a biomatrix for the immortalised human corneal epithelial and endothelial cells. Histological cross-sections of the whole-cornea equivalents resemble human corneas in tissue structure. This organotypic in vitro model may serve as a research tool for the ophthalmic science community, as well as a model system for testing for eye irritancy and drug efficacy.
The Fourth EC Report on the Statistics of Laboratory Animal Use: Trends, Recommendations and Future Prospects
Authors: Grindon Christina , Bhogal Nirmala
Published: April 2019

At the beginning of 2005, the European Commission published its fourth report on the statistics of the number of animals used for experimental and other scientific purposes. A total of 10.7 million animals were used within the Member States of the European Union (EU) in 2002, an increase of almost a million animals since the 1999 report. France, Germany and the UK continue to be the largest users of animals for scientific purposes, and mice, rats, fish and birds remain the most commonly-used animals. For the first time, all 15 Member States used the standardised “EU tables”, as had been agreed in 1998. This has made it easier to identify areas on which Three Rs initiatives should be focused. Nevertheless, the reporting system still has a number of serious deficiencies. In particular, there are insufficient data on the numbers of animals that are kept or bred for research purposes, the numbers of transgenic animals, and the severity of procedures that are applied.
The In Vitro Acute Skin Irritation of Chemicals: Optimisation of the EPISKIN Prediction Model within the Framework of the ECVAM Validation Process
Authors: Cotovio José , Grandidier Marie-Hélène , Portes Pascal , Roguet Roland , Rubinstenn Gilles
Published: April 2019

In view of the increasing need to identify non-animal tests able to predict acute skin irritation of chemicals, the European Centre for the Validation of Alternative Methods (ECVAM) focused on the evaluation of appropriate in vitro models. In vitro tests should be capable of discriminating between irritant (I) chemicals (EU risk: R38) and non-irritant (NI) chemicals (EU risk: “no classification”). Since major in vivo skin irritation assays rely on visual scoring, it is still a challenge to correlate in vivo clinical signs with in vitro biochemical measurements. Being particularly suited to test raw materials or chemicals with a wide variety of physical properties, in vitro skin models resembling in vivo human skin were involved in prevalidation processes. Among many other factors, cytotoxicity is known to trigger irritation processes, and can therefore be a first common event for irritants. A refined protocol (protocol15min–18hours) for the EPISKIN model had been proposed for inclusion in the ECVAM formal validation study. A further improvement on this protocol, mainly based on a post-treatment incubation period of 42 hours (protocol15min–42hours), the optimised protocol, was applied to a set of 48 chemicals. The sensitivity, specificity and accuracy with the MTT assay-based prediction model (PM) were 85%, 78.6% and 81.3% respectively, with a low rate of false negatives (12%). The improved performance of this optimised protocol was confirmed by a higher robustness (homogeneity of individual responses) and a better discrimination between the I and NI classes. To improve the MTT viability-based PM, the release of a membrane damage marker, adenylate kinase (AK), and of cytokines IL-1α and IL-8 were also investigated. Combining these endpoints, a simple two-tiered strategy (TTS) was developed, with the MTT assay as the first, sort-out, stage. This resulted in a clear increase in sensitivity to 95%, and a fall in the false-positive rate (to 4.3%), thus demonstrating its usefulness as a “decision-making” tool. The optimised protocol proved, both by its higher performances and by its robustness, to be a good candidate for the validation process, as well as a potential alternative method for assessing acute skin irritation.
The Use of Non-Human Primates in Regulatory Toxicology: Comments Submitted by FRAME to the Home Office
Authors: Hudson Michelle , Bhogal Nirmala , Balls Michael
Published: April 2019

The Home Office have circulated a document that summarises the discussions of a Primate Stakeholders Forum. The Forum took place in January 2004, and was convened to address the issues raised and the recommendations made in the Animal Procedures Committee 2002 report on the use of primates under the Animals (Scientific Procedures) Act 1986. The report emphasises the need for more resources focused on alternatives to toxicological testing in primates, including harmonising worldwide regulatory requirements, investigating the relevance of primate models, and improving the retrospective analysis of procedures involving primates. The document called for reasoned comments about the report to be submitted to the Home Office. In response, FRAME submitted a comprehensive paper, which evaluated each of the Animal Procedures Committee's recommendations, along with the Home Office Forum's comments. FRAME believes that, in coming to a decision as to whether primates should be used for regulatory testing, there must be full consideration of all the information available, including whether the ethological needs of any given species can be met prior to, during and following experimental use. Where these needs cannot be met, there must be a concerted effort to develop alternative models for research and testing. However, this should not detract from the ultimate goal of phasing out primate research altogether.
Heterologous Co-Expression of Human Cytochrome P450 1A2 and Polymorphic Forms of N-Acetyltransferase 2 for Studies on Aromatic Amines in V79 Chinese Hamster Cells
Authors: Scheuenpflug Jürgen , Krebsfänger Niels , Doehmer Johannes
Published: April 2019

V79 Chinese hamster cells were genetically engineered for the stable co-expression of human cytochrome P450 1A2 and the polymorphic N-acetyltransferase 2 alleles *4, *5B, *6A and *13, in order to generate an in vitro tool for studying the metabolism-dependent toxicity of aromatic amines. N-acetyltransferase 2*4-encoding cDNA was generated by the polymerase chain reaction (PCR) with defined primers from the genomic DNA of a human liver donor homozygous for *4, and served as a template to generate the *5B, *6A and *13 isoforms by site-directed mutagenesis. Human cytochrome P450 (CYP) 1A2-encoding cDNA was generated by the PCR from genomic DNA of the recombinant V79MZh1A2 cell line. All the cDNAs were inserted into a CMV promotor-containing plasmid in conjunction with the selectable marker genes, neomycin and hydromycin. The recombinant expression plasmids were transfected for stable integration into the genomic DNA of the V79 cells. Several cellular clones were obtained and checked for the genomic integration of intact cDNAs with the PCR on the genomic DNA of the recombinant cells. Stable expression was confirmed by the reverse transcriptase PCR (RT-PCR) on RNA preparations. Metabolic function was tested with ethoxyresorufin as a marker substrate for CYP1A2, and 2-aminofluorene and N-sulphametazine for N-acetyltransferase activity, and compared to data obtained from biological samples. 7-Ethoxyresorufin-O-deethylase activities ranged from 0.2 to 4pmol resorufin/min/mg total protein. The N-acetylation of sulphametazine ranged from 0.07 to 1.7nmol N-acetyl-sulphametazine/mg total protein/min. Selected clones showing activities in the range of physiological activities were submitted to metabolism-dependent mutagenicity studies. In particular, the polymorphism-dependent N-acetylation of 2-aminofluorene and the role of CYP1A2 and N-acetyltransferase in the mutagenicity of 2-aminofluorene, were investigated. Surprisingly, the mutagenicity of 2-aminofluorene is dramatically reduced in V79 cells co-expressing CYP1A2 and N-acetyltransferase, compared to V79 cells expressing CYP1A2 only, pointing to a significant species-dependent difference in the metabolic activation of aromatic amines between rats and humans.
Opportunities to Replace the Use of Animals in Sepsis Research
Authors: Langley Chris , Brock Chris , Brouwer Gerard , Brown Alun , Clapp Lucie , Cohen Jon , Evans Tom , Newman Carol , Orr Samantha , Phillips Barry , Rhodes Andy , Webster Nigel , Wooldridge Karl
Published: April 2019

Sepsis and multiple organ failure are common causes of death in patients admitted to intensive care units. The incidence of sepsis and associated mortalities has been steadily increasing over the past 20 years. Sepsis is a complex inflammatory condition, the precise causes of which are still poorly understood. Animal models of sepsis have the potential to cause substantial suffering, and many of them have been poorly representative of the human syndrome. However, a number of non-animal approaches, including in vitro, in silico and clinical studies, show promise for addressing this situation. This report is based on discussions held at an expert workshop convened by Focus on Alternatives and held in 2004 at the Wellcome Trust, London. It provides an overview of some non-animal approaches to sepsis research, including their strengths and weaknesses, and argues that they should be prioritised for further development.
Assessment of the Skin Irritation Potential of Chemicals by Using the SkinEthic Reconstructed Human Epidermal Model and the Common Skin Irritation Protocol Evaluated in the ECVAM Skin Irritation Validation Study
Authors: Kandárová Helena , Liebsch Manfred , Schmidt Elisabeth , Genschow Elke , Traue Dieter , Spielmann Horst , Meyer Kirstin , Steinhoff Claudia , Tornier Carine , De Wever Bart , Rosdy Martin
Published: April 2019

Currently, two reconstructed human skin models, EpiDerm™ and EPISKIN™ are being evaluated in an ECVAM skin irritation validation study. A common skin irritation protocol has been developed, differing only in minor technical details for the two models. A small-scale study, applying this common skin irritation protocol to the SkinEthic reconstructed human epidermis (RHE), was performed at ZEBET at the BfR, Berlin, Germany, to consider whether this protocol could be successfully transferred to another epidermal model. Twenty substances from Phase III of the ECVAM prevalidation study on skin irritation were tested with the SkinEthic RHE™. After minor, model-specific adaptations for the SkinEthic RHE, almost identical results to those obtained with the EpiDerm and EPISKIN models were achieved. The overall accuracy of the method was more than 80%, indicating a reliable prediction of the skin irritation potential of the tested chemicals when compared to in vivo rabbit data. As a next step, inter-laboratory reproducibility was assessed in a study conducted between ZEBET and the Department of Experimental Toxicology, Schering AG, Berlin, Germany. Six coded substances were tested in both laboratories, with three different batches of the SkinEthic model. The assay results showed good reproducibility and correct predictions of the skin irritation potential for all six test chemicals. The results obtained with the SkinEthic RHE and the common protocol were reproducible in both phases, and the overall outcome is very similar to that of earlier studies with the EPISKIN and EpiDerm models. Therefore, the SkinEthic skin irritation assay test protocol can now be evaluated in a formal “catch-up” validation study.
In Vitro Tests within the REACH Information Strategies
Authors: Louekari Kimmo , Sihvonen Kirsi , Kuittinen Marko , Sømnes Vibeke
Published: April 2019

Tonnage-based information requirements are specified in the proposal on the regulation on the Registration, Evaluation and Authorisation of Chemicals (REACH) in the European Union. The hazard assessment for toxic endpoints should be performed by using a tiered approach, i.e. as an information strategy (IS), starting with an evaluation of all of the data already available, including animal in vivo and in vitro data, and human evidence and case reports, as well as data from (Quantitative)-Structure Activity Relationships ([Q]SARs) or read-across, before any further testing is suggested. To contribute to the implementation of the REACH system, the Nordic countries launched two projects: 1) a review of currently used testing strategies, including a comparison with the REACH requirements; and 2) the development of detailed ISs for skin and eye irritation/corrosion. The review showed that the ISs and classification criteria for the selected endpoints are inconsistent in many cases. In the classification criteria, human data and in vivo test results are usually the prerequisites. Other types of information, such as data from in vitro studies, can sometimes be used, but usually as supportive evidence only. This differs from the REACH ISs, where QSARs, read-across and in vitro testing are important elements. In the other part of the project, an IS for skin and eye irritation/corrosion was proposed. The strategy was “tested” by using four high production volume (HPV) chemicals: hydrogen peroxide, methyl tertiary-butyl ether (MTBE), trivalent chromium, and diantimony trioxide, but only MTBE and trivalent chromium are dealt with in this paper. The “test” revealed that in vivo data, human case reports and physical-chemical data were available and could be used in the evaluation. Classification could be based on the proposed IS and the existing data in all cases, except for the eye irritation/corrosion of trivalent chromium. Weight-of-evidence analysis appeared to be a useful step in the ISs proposed, and including it in the REACH strategies should be considered. For these chemicals, few in vitro and (Q)SAR data were available — more of these data would be generated, if the relevant guidance and legislation on classification were updated.
Management of an Integrated Project (Sens-it-iv) to Develop In Vitro Tests to Assess Sensitisation
Authors: Rovida Costanza , Basketter David , Casati Silvia , de Silva Odile , Hermans Helma , Kimber Ian , Manou Irene , Weltzien Hans Ulrich , Roggen Erwin
Published: April 2019

Sens-it-iv is an integrated project, funded by European Commission Framework Programme 6, the overall objective of which is to develop in vitro tests and test strategies to be used by the chemical, cosmetic and pharmaceutical industries to assess the risk for potential contact and respiratory sensitisers. Such tests, once formally validated and accepted, will permit the evaluation of the sensitising potential of existing and new chemical entities and the products of the European industries for classification and labelling, as required by the new EU REACH legislation on chemicals, or for the purpose of risk assessment as required by the 7th Amendment to the EU Cosmetics Directive. Sens-it-iv involves 28 partners, representing industries, universities and regulatory bodies, including various institutes in the EU Member States and different competencies, all with the common aim of achieving a final deliverable — increasing the safety of consumer products, whilst reducing animal experimentation. This paper provides an overview of the structure of the project and a detailed description of the organisation of its management.
Systematic Reviews of Animal Experiments Demonstrate Poor Human Clinical and Toxicological Utility
Authors: Knight Andrew
Published: April 2019

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.
Toward a New Toxicology — Evolution or Revolution?
Authors: Hartung Thomas
Published: April 2019

This essay summarises the author's thoughts on the current paradigm change in toxicology. The driving factors and mechanisms of this change, and obstacles to it, are discussed. Current developments are discussed on the basis of some key assumptions in Thomas Kuhn's famous book, The Structure of Scientific Revolutions. The author's personal view is that there is clear evidence that revolutionary changes in regulatory toxicology are emerging.