bluuebunny/crossref_metadata_2025 huggingface dataset:
The Comet Assay with MCL-5 Cells as an Indicator of Genotoxic Treatment with Chemicals and Cigarette Smoke Condensate
Authors: Wolz Lucie , Krause Günter , Scherer Gerhard
Published: April 2019

The metabolically competent human lymphoblastoid cell line MCL-5 was treated with a panel of mutagens to assess the induction of DNA damage. Treatment effects were observed by monitoring cell proliferation and by single-cell gel electrophoresis (SCGE). The direct-acting mutagens benzo[ a]pyrene-7,8-diol 9, 10-epoxide (BPDE) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as pro-mutagens requiring metabolic activation, i.e. benzo[a]pyrene (BaP), 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyri-dine (PhIP), 4- N-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and cigarette-smoke condensate (CSC), were assayed by SCGE. Assay schemes were adapted for the MCL-5 cell line and for low levels of strand break induction, by inclusion of the DNA synthesis inhibitors cytosine arabinoside and hydroxyurea, and by extending the electrophoresis time. For all mutagens tested, dose-dependent increases of median and average tail moment values among 50 nucleoids per slide were observed. The determining factors for selecting the treatment doses for mutation-induction experiments were the solubility of BaP and PhIP in the exposure medium, and the cytotoxicity exhibited by BPDE, MNNG and CSC. Induction of DNA strand breaks was obtained at mutagen concentrations permitting sufficient cell proliferation, except in the case of MNNG.
Microarray Assessment of Fibronectin, Collagen and Integrin Expression and the Role of Fibronectin–Collagen Coating in the Growth of Normal, SV40 T-antigen-immortalised and Malignant Human Oral Keratinocytes
Authors: Sarang Zsolt , Haig Ylva , Hansson Annette , Vondracek Martin , Wärngård Lars , Grafström Roland C.
Published: April 2019

Extracellular matrix proteins affect the growth and survival of epithelial tissues. Accordingly, surface coating with fibronectin and collagen is a common practice for promoting keratinocyte culture. In this study, the expression of fibronectin and collagen-related factors, including integrins, by normal (NOK), SV40 T-antigen-immortalised (SVpgC2a) and malignant (SqCC/Y1) human oral keratinocytes, under standardised, serum-free conditions, was investigated by using microarray analysis. Cell growth was also studied in the presence and absence of a matrix consisting of human fibronectin and bovine collagen type I (FN–COL). Fibronectin transcripts were abundant in all cells, whereas 16 of 29 collagen chains and 14 of 24 integrin subunits were variably detected. With regard to both the expression level and the number of transcripts, higher collagen and lower integrin expression was observed in SVpgC2a cells than in NOKs and SqCC/Y1 cells. The cell types differed with regard to colony-forming efficiency and the rate and kinetics of growth at high cell density. For all cell types, FN–COL coating consistently stimulated cell migration, without influencing growth in mass culture or clonal density. The results demonstrate the transcription of genes associated with the formation and function of fibronectin and collagen in oral epithelium, and variably altered expression patterns in transformed states, and show that keratinocyte lines can be successfully transferred without the stimulus from extracellular FN–COL.
Organotypic Brain Slice Cultures: An Efficient and Reliable Method for Neurotoxicological Screening and Mechanistic Studies
Authors: Noraberg Jens
Published: April 2019

This paper reviews the current state of the use of organotypic brain slice cultures for neurotoxicological and neuropharmacological screening and mechanistic studies, as exemplified by excitotoxin application. At present, no in vitro systems have been approved by the regulatory authorities for neurotoxicity testing. For the evaluation of the slice culture method, organotypic hippocampal slice cultures were exposed to toxic doses of the excitotoxins, glutamate, N-methyl-D-aspartate (NMDA), kainic acid and 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and the glial toxin, DL-α-aminoadipic acid (DL-AAA). Neuronal cell death was quantified by propidium iodide (PI) uptake, and visualised by Fluoro-Jade (FJ) staining. General cell death was monitored by lactate dehydrogenase (LDH) release into the culture medium. EC50 values for the different compounds, based on PI uptake after exposure for 48 hours in entire cultures, were: glutamate, 3.5mM; DL-AAA, 2.3mM; kainic acid, 13μM; NMDA, 11μM; and AMPA, 3.7μM. In the slice cultures, the hippocampal subfields displayed the same differences in vulnerability as those observed in vivo. When subfield analysis was performed on the cultures, the CA1 subfield was most susceptible to glutamate, NMDA and AMPA, while CA3 was most susceptible to kainic acid. The amount of LDH release for DL-AAA was about four times that of L-glutamate, in accordance with the additional toxic effect on glial cells, which was also found by confocal microscopy to stain for FJ. In conclusion, it was found that organotypic brain slice culture, combined with standardised protocols and quantifiable markers, such as PI and FJ staining, is a relevant and feasible in vitro system for neurotoxicity testing. Considering the amount and quality of the available published data, it is recommended that the brain slice culture method could be subjected to pre-validation and formal validation for inclusion in a tiered in vitro neurotoxicity testing scheme to supplement and replace conventional animal tests.
Assessment of Ocular Irritation by Image Processed Quantification of Cell Injury in Human Corneal Cell Cultures and in Corneal Constructs
Authors: Engelke Maria , Patzke Jens , Tykhonova Svitlana , Zorn-Kruppa Michaela
Published: April 2019

Currently, there are no accepted alternative tests for the replacement of animals in ocular irritation testing. This study focused on the quantification of cellular viability as a measure of toxic events in immortalised human corneal cell cultures and a three-dimensional corneal construct. Simultaneous vital dye staining by calcein AM and ethidium homodimer-1 was used to provide “live” and “dead” probes, respectively. For further quantification, we have developed image processing tools to evaluate digital images obtained from confocal fluorescence scanning microscopy measurements. Based on the finding that ocular irritation can be related to the extent of cell injury at the various cell layers of the cornea, we extended our studies from corneal cell cultures to an in vitro human corneal equivalent system comprising epithelial, stromal keratocyte and endothelial layers. Our results showed that the microscopic measurement of cellular injury by using either cell cultures or in vitro corneal constructs, combined with image processed quantification, can provide insight into the extent of the toxic effects.
A New Human Breast Carcinoma Cell Line Resistant to DNA-damaging Drugs
Authors: Levina Vera V. , Drobchenko Eugeny A. , Shabalina Elena V.
Published: April 2019

To investigate the phenomenon of active dissociation of the vital dye, Hoechst 33342 (Ho342), from DNA (DNA clearing), a new MCF7HoeR-7 human breast carcinoma cell line was isolated from parent MCF7 cells by step-wise selection with increasing concentrations of Ho342. This cell line possesses an enhanced ability for DNA clearing. The MCF7HoeR-7 line is characterised in detail and compared with the parental MCF7 line and a typical P-glycoprotein-mediated multidrug resistant (MDR) cell line, MCF7/Adr. MCF7HoeR-7 cells have an increased population growth rate, a lower DNA content and a reduced number of chromosomes. Enhanced DNA clearing in MCF7HoeR-7 cells is associated with the high resistance of the cells to the toxic effects of Ho342 and cross-resistance to etoposide, a topoisomerase II inhibitor in clinical use. The MCF7HoeR-7 and parent MCF7 cell lines have similar expression levels of transport proteins. The results obtained confirm that DNA clearing is an atypical MDR mechanism in tumour cells.
Structure–Activity Relationships and Prediction of the Phototoxicity and Phototoxic Potential of New Drugs
Authors: Barratt Martin D.
Published: April 2019

Relationships between the structure and properties of chemicals can be programmed into knowledge-based systems such as DEREK for Windows (DEREK is an acronym for “Deductive Estimation of Risk from Existing Knowledge”). The DEREK for Windows computer system contains a subset of over 60 rules describing chemical substructures (toxophores) responsible for skin sensitisation. As part of the European Phototox Project, the rule base was supplemented by a number of rules for the prospective identification of photoallergens, either by extension of the scope of existing rules or by the generation of new rules where a sound mechanistic rationale for the biological activity could be established. The scope of the rules for photoallergenicity was then further refined by assessment against a list of chemicals identified as photosensitisers by the Centro de Farmacovigilancia de la Comunidad Valenciana, Valencia, Spain. This paper contains an analysis of the mechanistic bases of activity for eight important groups of photoallergens and phototoxins, together with rules for the prospective identification of the photobiological activity of new or untested chemicals belonging to those classes. The mechanism of action of one additional chemical, nitrofurantoin, is well established; however, it was deemed inappropriate to write a rule on the basis of a single chemical structure.
Validation Activities in Japan: A Report
Authors: Ono Hiroshi
Published: April 2019

Japanese activities in the validation of alternative test methods are reviewed. The validation of the Limulus endotoxin test as an alternative method to the rabbit pyrogen test was initiated by the Japanese Pharmacopoeia Society, and was successfully conducted under the direction of the National Institute of Health Sciences (NIHS), with the participation of many pharmaceutical company laboratories. The validation of in vitro alternatives to the Draize eye test for cosmetics were carried out by the NIHS and cosmetic company laboratories. Research on alternatives in Japan has been promoted by the Japanese Society of Alternatives to Animal Experiments (JSAAE) since 1989. Validation studies on alternatives were also attempted by the JSAAE. Interlaboratory reproducibility studies were carried out by the JSAAE on five different cytotoxicity tests. The JSAAE is planning the validation of in vitro alternatives to a local irritation test for intramuscular injection. Cell transformation assays have been carried out in Japan as adjuncts to the in vivo carcinogenicity test.
Are Animal Tests Inherently Valid?
Authors: Balls Michael
Published: April 2019

The proposition that animal tests are inherently valid, merely because they are animal tests, is discussed and is rejected. It is concluded that there is no justifiable reason for subjecting new or substantially modified animal test procedures or testing strategies to a validation process that is any less stringent than that applied to non-animal tests and testing strategies.
Use of a Perfusion Co-culture System Consisting of Caco-2 and Hep G2 Cell Compartments for the Kinetic Analysis of Benzo[a]pyrene Toxicity
Authors: Sakai Yasuyuki , Fukuda Osamu , Choi Sue-Hyung , Sakoda Akiyoshi
Published: April 2019

Conventional cytotoxicity tests cannot usually include various metabolic processes in humans. We therefore developed a physiologically based, multi-compartment perfusion co-culture system, using a Caco-2 cell monolayer on a semi-permeable membrane and a microcarrier-based, three-dimensional culture of Hep G2 cells to mimic permeation across the small intestine and biotransformation of the small intestine and the liver. Stable operations allowed us to maintain various activities of both cells for at least 4 days. Cocultivation improved the growth of Hep G2 cells and enhanced the cytochrome P450 1A1/2 capacities of both Hep G2 and Caco-2 cells. When benzo[ a]pyrene (BaP) was loaded on the apical side of the Caco-2 cell layer, the enhanced P450 capacities produced a higher amount of BaP-7,8-hydrodiol, a precursor of the ultimate carcinogen of BaP, BaP-7,8-dihydrodiol-9,10-epoxide (BPDE). These phenomena led to the initially retarded, but later stronger, expression of BaP toxicity in the co-culture system than in pure cultures, which agreed with the actual load of BaP-7.8-hydrodiol to the Hep G2 cells. Because this kind of system can reproduce such complicated phenomena, including those influenced by organ–organ interactions, it is useful as a new in vitro experimental system, for understanding the unknown mechanisms involved in final toxicity in humans and thereby improving physiologically based pharmacokinetic (PBPK) simulation models.
Taming and Training of Pregnant Sheep and Goats and of Newborn Lambs, Kids and Calves before Experimentation
Authors: Mellor David J.
Published: April 2019

Familiarity with handling and routine procedures reduces the stress experienced by pregnant ewes and nanny goats and newborn lambs, kids and calves during experimentation. The factors that need to be considered when taming and training pregnant ewes and goats include: the behavioural and physiological effects of daily handling for six to eight weeks; the impact of the progesterone/oestrogen status of ewes on their responses to taming and training; the attainment of physiological stability after surgery; and comparison of tamed and trained ewes with non-handled ewes. The factors that need to be considered for newborn lambs, kids and calves include: beneficial effects of the experimenter being part of the birth environment of lambs, kids and calves; the impact of handling on lamb responses to feeding and painful procedures; and remote sampling effects on physiological responses. The relative value of taming and training for the humane management of animals and for producing meaningful results is assessed with reference to absence of the confounding effects of stress, and, conversely, the possible confounding effects of taming and training, on the animals’ responses.
Revision of Council of Europe Convention ETS 123 Guidelines for the Accommodation and Care of Animals Used for Experimental and Other Scientific Purposes
Authors: Anderson David
Published: April 2019

Within the 43 Member States of the Council of Europe, Appendix A of Convention ETS 123 provides guidance on the standards of accommodation and care to be provided for animals used for experimental and other scientific purposes. At a meeting of the Parties to the Convention in 1997, a resolution providing additional guidance on accommodation and care was adopted. At the same meeting, it was agreed that, as scientific knowledge and experience had progressed since adoption of ETS 123 in 1986, a Working Party should be convened to consider the revision of Appendix A. The Working Party first met in January 1999, and since then, there have been a number of Working Party meetings, and a number of meetings of Expert Groups, who are tasked with producing guidelines incorporating species-specific needs. The Expert Groups have drafted proposals for the accommodation and care of rodents, rabbits, dogs, cats, ferrets, farm animals, birds, amphibia, reptiles and fish. These proposals will complement the general principles of accommodation and care included in the general section of Appendix A. This comprehensive review has provided an opportunity to incorporate new ideas on housing and care based on scientific data and on best contemporary practises. It is expected that these revisions will be used throughout the scientific community to inform accommodation and care practises for animals used for scientific purposes.
Research on Vertebrate Pesticides and Traps: Do Wild Animals Benefit?
Authors: Warburton Bruce , O'Connor Cheryl
Published: April 2019

New Zealand has a range of carnivorous and herbivorous mammals that were originally introduced for recreational, financial, aesthetic and biocontrol reasons, but which now pose significant threats to conservation and animal health values. Research is undertaken to develop new tools and strategies to manage these pests. Captive trials that are carried out include those to determine the toxicity of poisons, the efficacy of fertility control agents, and welfare impacts of poisons and traps. Field trials are undertaken to test the efficacy of poisons and traps, and large-scale management trials carried out to optimise control strategies. Although this research is aimed at managing animals, including killing them, we believe that there are benefits to both individual animals and animal populations that far exceed any cost of harm to individuals that are managed (often killed). We suggest that there are three levels of beneficiaries: the individuals of the pest species that are killed, the individuals in the surviving population, and the individuals and populations of other species. We provide examples of how wild animals can benefit from vertebrate pesticide and trap research.
Ethics for Research Using Animals in Italy
Authors: Passantino Annamaria , Panichi Marzio , Russo Maria Rachele , Passantino Michele
Published: April 2019

The purpose of this report is to describe the legislative and ethical context in Italy regarding technology and the need for innovative research versus responsibility toward the living world. Questions regarding responsible behaviour in the use of animals for research are examined, and special attention is focused on the implementation of the Three Rs concept ( reduction, refinement and replacement) and on considerations of Catholic dogma.
A Demographic Analysis of Primate Research in the United States
Authors: Conlee Kathleen M. , Hoffeld Erika H. , Stephens Martin L.
Published: April 2019

An analysis of primate research in the USA, including the number and species of non-human primates used, types of research, levels of invasiveness, housing conditions and funding, is an important step in addressing various concerns (ethical and scientific) surrounding primate research. An analysis of monkey and chimpanzee research, conducted by The Humane Society of the United States (HSUS), demonstrated that the USA uses more non-human primates (including great apes) in research per year, than any other country in the world. The US government devotes approximately $575–800 million per year to primate research and care. Chimpanzees are most commonly used for hepatitis research; monkeys are most commonly used for HIV research, and other research areas include vaccine and drug testing, cognition, human pathologies/diseases, drug abuse and xenotransplantation. Legislation (including great ape research bans), media attention and proposed increased primate use also contribute to the overall picture of current and future non-human primate research in the USA and throughout the world. The HSUS proposes that cost–benefit analyses of non-human primate research in the USA be conducted to properly assess “value added” to relevant fields of research and whether the use of non-human primates is the only, or most effective, strategy for biomedical progress. Finally, The HSUS proposes a ban on the use of apes in research in the USA and worldwide.
Refinement and Reduction in the Production of Genetically Modified Mice
Authors: Robinson Vicky , Jennings Maggy
Published: April 2019

This paper introduces the sixth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement (JWGR), which addresses reduction and refinement in the generation, management and care of genetically modified (GM) mice. The report provides a comprehensive review of the potential for reduction and refinement at all stages of the generation and maintenance of GM animals. The issues covered include scientific, procedural and practical issues. The need for good training in all of these areas is emphasised. The report concludes with a section on the need to develop comprehensive criteria for welfare assessments for GM mice and a preliminary list of criteria is provided. The full report has been publisheded in Laboratory Animals.
Effective Policies in the Animal Genomics Era: How to Best Involve Ethics, Expertise and the Public
Authors: Paula Lino Enrique
Published: April 2019

Genomics will have a significant impact on animal use in life science research. Together with related developments such as animal biotechnology, it also raises ethical issues that have to be addressed by society. Societal demands for regulatory accountability and transparency make it very unlikely that these issues can be addressed solely via current Institutional Animal Care and Use Committee (IACUC) reviews. New, more open ways of public policymaking are needed to effectively deal with these issues. Policy science can offer guidelines on how to structure such new policies. In particular, the so-called theory of policy problems sheds light on how to approach such a new development. Taking the Dutch animal biotechnology policy as an example, and keeping this theory in mind, some of the do's and don'ts with regard to involving experts and the public in public policy will be discussed.
Animal Experimentation and Ethics in India: The CPCSEA Makes a Difference
Authors: Pereira Shiranee , Veeraraghavan Prema , Ghosh Sonya , Gandhi Maneka
Published: April 2019

The Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) is a statutory body formed by the Act of the Indian Parliament under the Prevention of Cruelty to Animals Act 1960. Formed in 1964, it was revived in 1998, under the committed chairpersonship of Maneka Gandhi. In the last two years, the CPCSEA has bettered the life of the animals in laboratories across India. This committee is composed of members of the scientific community, regulatory authorities and animal activists. The CPCSEA functions with a brilliant network of volunteers who liaise with the laboratories. For the first time in India: over 665 laboratories are registered with the CPCSEA; Institutional Animal Ethics Committees (IAECs) are constituted in every laboratory, which are only empowered to approve research project proposals that use rats, mice, guinea-pigs or rabbits; every project that uses canines, ovines, bovines or non-human primates can only be conducted if approved by the panel of scientific experts constituted for this purpose; guidelines on laboratory animal care and practice have been formulated and enforced; a protocol for the production of immunobiologicals from equines has been formulated and ratified by the Supreme Court of India; the CPCSEA has been deliberating on alternatives and working out modalities to introduce alternatives in basic/regulatory research and education, in keeping with the international arena; the CPCSEA, to date, has rehabilitated and homed over 300 dogs, 150 equines, 200 non-human primates and several cattle, cats, birds, rabbits and mice; the CPCSEA proactively trains and guides scientific and non-scientific personnel on issues of alternatives and laboratory animal welfare; and the CPCSEA has fought legal issues on laboratory animal care and use and have had verdicts that favoured alternatives and animal welfare.

The greatness of a nation and its moral progress can be judged by the way its animals are treated. Mahatma Gandhi
The Use of Animals in National High School Student Science Fair Projects in the United States
Authors: Miller-Spiegel Crystal
Published: April 2019

Science fair projects can provide a sound opportunity to teach students the value of scientific methodology without relying on the routine and unnecessary use of animals. Unfortunately, students are often encouraged to use animals in an expendable manner that both duplicates previous experiments and neglects the opportunity to “think outside the box” in order to generate new hypotheses/theories about human health, physiological processes or basic biological concepts. Although at least one national science fair sponsor has changed its policy regarding students’ utilisation of vertebrate animals, others continue to encourage the more traditional in vivo experimental projects. This paper will review the guidelines of two major national science fairs in the USA; types of projects conducted that involve animals; numbers of animals involved; interview responses by some student finalists who used vertebrates in their projects; successful initiatives by animal advocates in the USA to eliminate the use of animals in science fairs; and potential areas of outreach to science educators, science fair sponsors, high schools and students.
Technological Advances that Enhance Teaching Using Animals, and the Application of the Three Rs
Authors: Davies Alexander S.
Published: April 2019

The inventions that have progressively contributed to education have never offered opportunities as vast as in the digital era, even though this is still scarcely 25 years old. In this time, digital handling of data led first to text processing, then to bitmap and vector graphics, and now, to digital sound and movies. As these advanced, the storage methods became larger, faster, easier and cheaper. The advancement of technology has been so rapid that the standard of most teaching aids now available is well below what can currently be achieved. We are confronted with an unprecedented opportunity for applying the principles of reduction, refinement and replacement of animals in education. Not only are the visual teaching aids improved by digitising, but all aspects of their development, including the ease, higher speed, and low cost of creation, editing, copying, distribution and access, are improved, as well. Several examples will be given, including access to interactive panoramic movies, animated sequences to explain difficult concepts, on-line tutorials and image databases using digital photography, radiography and other diagnostic methods, as well as the production of desktop movies. The speed of technical advance brings its own problems, but the challenges and possibilities for developing viable alternatives to the use of animals in teaching are vast.
How Can We Encourage Teachers to Use Computer-based Alternatives: The UK Higher Education Experience
Authors: Dewhurst David
Published: April 2019

Computer-assisted learning (CAL) programs, which are now widely available, have the potential to make a significant contribution to reducing the use of animals in teaching, particularly in pharmacology and physiology. A number of databases exist to support teachers in finding information about potential alternatives. The evidence from a number of evaluative studies is that computer programs that provide a virtual simulation of an animal lab can achieve many of the teaching and learning objectives for many students at least as effectively as the live experience. However, the key to reducing animal use is persuading teachers to integrate the alternatives into mainstream practice. Proven strategies to facilitate this process are described.
A Proposed Higher Education Institution-based Three Rs Advisory Service
Authors: Berdoy Manuel , Repp Catherine R.
Published: April 2019

The University of Oxford's Ethical Review Process (ERP) is promoting a pilot scheme for a collaborative UK higher education institution-based Three Rs advisory service. It is believed that there is scope for UK academia to make a significant contribution, through the spread of its science base and the availability of high-quality library and IT services. A collaborative approach based on the concept of the critically appraised topic is envisaged, to minimise duplication and to ensure that limited resources are used to good effect. The initial objective is to identify, research, and validate refinement and replacement alternatives, drawing on both the skills of the information professional and the analogy of evidence-based health care. The results would be disseminated among member institutions by way of web-based systems, which could also offer on-line training in search strategies. The service would provide assistance to university-based project licence applicants, and would contribute to achieving the aims of the ERP, as an initiative “leading to the widest possible application of the Three Rs”. It is hoped to develop the scheme in partnership with research council initiatives now under way, as the councils play a key role as funders, not only of research, but also of research student training. Universities are uniquely positioned, as centres of scientific education and, through continuing education, of continuing professional development, to provide a base for training in good practice. A successful pilot project could provide a foundation for a similar approach to reduction strategies and experimental design.
A Pharmacological Approach for the Selection of Potential Anticancer Agents
Authors: Double John A.
Published: April 2019

Historically, the process of developing new anticancer agents was largely empirical. Today, because of improvements in our knowledge of the molecular processes involved in the development of cancer, the process of developing new agents is becoming more rational. Researchers from Cancer Research UK, the European Organisation for Research and Treatment of Cancer and the National Cancer Institute have shown that, by undertaking a pharmacological approach to the selection of potential anticancer agents, both meaningful antitumour data and an 80% reduction in animal usage can be obtained. It has also been demonstrated that a new pharmacological tool, the “hollow fibre system”, in which tumour cells are grown in biocompatible fibres which are implanted into mice, can be used to produce meaningful antitumour data with pharmacodynamic endpoints. By increasing the amount of data that can be obtained from a single animal and opening up the possibility of eliminating the need for untreated control animals, the hollow fibre system has the potential to make a significant contribution to both reduction and refinement.
Modern Concepts of Socialisation for Dogs: Implications for their Behaviour, Welfare and Use in Scientific Procedures
Authors: Boxall Jackie , Heath Sarah , Bate Simon , Brautigam John
Published: April 2019

The provision of a socialisation and training programme for dogs can lead to an improvement in the quality of the data that are produced from scientific procedures, and also to an improvement in animal welfare. A reduction in the number of animals needed to produce statistically significant data can result from decreasing the variability of the experimental data. The effects of a dog's behaviour can be a major source of random variability. A well socialised, habituated and trained dog should be calm during experimental procedures and, for example, during an ECG measurement, thus providing good quality data. A fearful, poorly socialised dog may also appear to be well-behaved on an examination table, and during an ECG measurement, because of the freezing response. However, there is likely to be a difference in the level of stress that these two individuals experience during the procedures. The stress response can have an impact on vital physiological parameters, such as heart rate. The variability in these parameters and the behaviour exhibited within a group of socialised, habituated and trained dogs that have been well prepared for experimental procedures, should be less than the variability present within a similar group of dogs that have not been prepared for these procedures. This paper describes two socialisation programmes, which were designed in order to compare the heart rates and behaviour of dogs which had received different degrees of socialisation, habituation and training. The behaviour of small groups of dogs from this study was compared with that of dogs on a standard socialisation programme, by using a simple, reproducible behavioural score scheme. The heart rate of the dogs was also measured. The results showed that there was little difference in heart rate between the groups, but that there were significant differences in the scores for key behaviours. There was evidence of a decrease in the variability of the behavioural scores for the groups of dogs that had undergone an intensive socialisation programme. Therefore, a socialisation programme can have a significant effect on behaviour and welfare, and has the potential to improve the quality of the data that are recorded.
An Analysis of the Home Office Statistics of Scientific Procedures on Living Animals, Great Britain 2004
Authors: Hudson Michelle , Bhogal Nirmala
Published: April 2019

The 2004 Statistics of Scientific Procedures on Living Animals were released by the Home Office in December 2005. They indicate that, for the third year running, there has been a significant increase in the number of laboratory animal procedures undertaken in Great Britain, and that increasing numbers of animals are involved. The overall trends in the use of toxicological and non-toxicological procedures involving animals are described. Particular emphasis is placed on the production and use of genetically modified animals, the production of biological materials, and acute toxicity testing. The use of non-human primates and dogs is also discussed. The implications of these latest statistics are considered with reference to the implementation of the Three Rs and their consequences for animal welfare.
The EU Physical Agents (EMF) Directive and its Impact on MRI Imaging in Animal Experiments: A Submission by FRAME to the HSE
Authors: Hudson Michelle
Published: April 2019

The EU Physical Agents (EMF) Directive, Directive 2004/40/EC, which threatens to greatly restrict the use of magnetic resonance imaging (MRI) in both clinical and research situations, will come into force on 30 April 2008. This could severely affect experimental animal welfare and scientific progress, as well as patient care. FRAME made a submission to a Health and Safety Executive round-table discussion about the Directive, held in January 2006, detailing concerns about the implications that the legislation would have on implementing the Three Rs in animal-based research and testing, and the subsequent consequences for animal welfare and the quality of scientific output. The submission is reproduced here, with additional comments on the outcome of the meeting and recommendations for further research into the consequences of the Directive.
The Comparative Effects of Metals on the Hatching of Earthworm Cocoons
Authors: Gupta Shrawan K. , Srivastava Richa , Mathur Neeraj , Saxena Prem N.
Published: April 2019

To establish the use of Metaphire posthuma as a sensitive model for ecotoxicological studies, the comparative effects of five metals on the hatching profiles of the cocoons of the earthworms, Metaphire posthuma, Eisenia foetida and Perionyx excavatus, were studied. The cocoons of the three species of earthworms were exposed to copper, chromium (III), chromium (VI), lead and zinc at 1.25, 2.5, 5.0 and 10.0ppm. Viable cocoons were incubated at 20 ± 1°C by using the immersion method. The results indicated that the inhibition of cocoon hatching was concentration dependent. The normal hatching, delayed hatching and non-viability of cocoons were recorded. At a concentration of 1.25ppm, there was almost no effect on the hatching of the cocoons of all three species of earthworms, except when exposed to chromium (VI), but higher concentrations (2.5 and 5.0ppm) caused severe effects. It was concluded that M. posthuma was more sensitive than the other two species, and that it is a suitable model for use in eco-toxicity testing.
Why do the Numbers of Laboratory Animal Procedures Conducted Continue to Rise? An Analysis of the Home Office Statistics of Scientific Procedures on Living Animals: Great Britain 2005
Authors: Hudson Michelle
Published: April 2019

The publication of the Statistics of Scientific Procedures on Living Animals: Great Britain 2005 once again provides evidence that the levels of animal experimentation in Great Britain are rising, the underlying reason for this being the continued and increasing reliance on genetically modified animals as model systems. There has been a gradual increase in fundamental research, as applied toxicological studies have declined. Of particular concern is the impact that the forthcoming REACH legislation will have and the apparent lack of urgency in facing up to this challenge. The major issues arising from the Statistics are discussed, including the increases in rabbit and primate procedures. The potential of newly validated and emerging techniques to counteract these worrying trends are also considered.
An In Vitro Study of the Toxic Effects of Stachybotrys chartarum Metabolites on Lung Cells
Authors: Kováčiková Zuzana , Tátrai Erzsébet , Piecková Elena , Tulinská Jana , Pivovarová Zuzana , Mataušic-Pišl Mirjana , Kuricová Miroslava , Wsolová Ladislava
Published: April 2019

During a study of indoor fungal colonisation, several isolates of Stachybotrys chartarum were recovered, and the effects of metabolites from four isolates on lung epithelial Type II cells and alveolar macrophages were studied in vitro. All the isolates showed toxic effects on both cell types, and they differed only in the extent of the changes induced. In Type II cells, the number of alkaline phosphatase positive cells was reduced, the pattern of Maclura pomifera agglutinin (MPA) binding was changed, and acid phosphatase activity in alveolar macrophages was diminished. In both cell types, the production of monocyte chemotactic protein-1 (MCP-1) and tumour necrosis factor-alpha (TNF-alpha) was changed, and parameters related to antioxidant status (superoxide dismutase, glutathione peroxidase, glutathione) were decreased.
The Effects of Heavy Metals on Common Carp White Blood Cells In Vitro
Authors: Witeska Małgorzata , Wakulska Marta
Published: April 2019

The in vitro effects of cadmium, copper, lead and zinc, and various cadmium compounds (chloride, sulphate and nitrate) on common carp (Cyprinus carpio) lymphocyte viability and phagocyte activity, were evaluated. The percentage of dead lymphocytes was determined after Trypan blue staining, and phagocyte activity was measured by using the nitroblue tetrazolium (NBT) reduction test. Lead was the most toxic to lymphocytes — the maximum mortality exceeded 30%, and was significantly higher at 1μM of lead, compared to the control. The maximum mortality caused by cadmium was below 10%, but was significantly elevated with 5μM or more of cadmium. Zinc induced lymphocyte mortality from 10μM, whilst no effect was observed with copper. The incubation of full blood with the three cadmium compounds (at 5mg/l of cadmium) showed that cadmium nitrate and cadmium sulphate were more toxic (over 35% and 25% mortality, respectively) than cadmium chloride (about 15% mortality). This was confirmed by the results of tests on isolated cells —1mg/l of cadmium as nitrate and sulphate increased lymphocyte mortality compared to the control and cadmium chloride. Phagocytic activity was less sensitive to heavy metals than was lymphocyte viability. It was significantly reduced following exposure to 50μM and 100μM cadmium, and 100μM zinc, but no effects were observed with either lead or copper.
Proposed Integrated Decision-tree Testing Strategies for Mutagenicity and Carcinogenicity in Relation to the EU REACH Legislation
Authors: Combes Robert , Grindon Christina , Cronin Mark T.D. , Roberts David W. , Garrod John
Published: April 2019

Liverpool John Moores University and FRAME recently conducted a research project sponsored by Defra, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This paper focuses on the prospects for using alternative methods (both in vitro and in silico) for mutagenicity (genotoxicity) and carcinogenicity testing — two toxicity endpoints, which, together with reproductive toxicity, are of pivotal importance for the REACH system. The manuscript critically discusses well-established testing approaches, and in particular, the requirement for short-term in vivo tests for confirming positive mutagenicity, and the need for the rodent bioassay for detecting non-genotoxic carcinogens. Recently-proposed testing strategies focusing on non-animal approaches are also considered, and our own testing scheme is presented and supported with background information. This scheme makes maximum use of pre-existing data, computer ( in silico) and in vitro methods, with weight-of-evidence assessments at each major stage. The need for the improvement of in vitro methods, to reduce the generation of false-positive results, is also discussed. Lastly, ways in which reduction and refinement measures can be used are also considered, and some recommendations are made for future research to facilitate the implementation of the proposed testing scheme.
Why ‘Suitable’ In Vitro Methods, as Defined in the Final EU REACH Legislation, are an Inappropriate Basis for Risk Assessment
Authors: Combes Robert D.
Published: April 2019

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.
Richard Clothier: An Appreciation
Authors: Balls Michael
Published: April 2019

The career of Richard Clothier is reviewed in the light of his long-standing collaboration with Michael Balls and Laurens Ruben at the University of East Anglia (UEA), the University of Nottingham, and Reed College, Portland, Oregon, USA. It began with work at UEA on the aetiology of the lymphosarcoma of Xenopus laevis, followed by studies on the effects of exposure to N-nitroso- N-methylurea on T-cell functions, which led to many contributions to comparative immunology. This was followed by the establishment of the FRAME Research Programme, which led to participation in extensive studies on the development of in vitro cytotoxicity tests and their application in acute and topical toxicity testing. A FRAME Trustee since 1983, Richard Clothier was a co-founder, and subsequently Director, of the FRAME Alternatives Laboratory in the University of Nottingham Medical School, where he led successful collaborations with a number of industrial partners and, in particular, with the European Centre for the Validation of Alternative Methods (ECVAM).
The FRAME Research Programme under the Direction of Dr Richard Clothier
Authors: Combes Robert D.
Published: April 2019

The work of Dr Richard Clothier (Reader in Cellular Toxicology and Director of the FRAME Alternatives Laboratory in the University of Nottingham Medical School) in relation to the FRAME Research Programme, is reviewed. He made a very substantial contribution to FRAME's laboratory research work over the last 20 years, by publishing many research papers, mainly with respect to method development and the application of new replacement toxicity test methods, particularly those involving the use of human cells in tissue culture. In addition, he participated in a number of international validation studies that have facilitated the regulatory acceptance of certain new methods. Lastly, he has been closely involved in the logistical aspects of obtaining and maintaining external research funding for FRAME from industrial sponsors.
A Comparative Evaluation of Corneal Epithelial Cell Cultures for Assessing Ocular Permeability
Authors: Becker Ulrich , Ehrhardt Carsten , Schneider Marc , Muys Leon , Gross Dorothea , Eschmann Klaus , Schaefer Ulrich F. , Lehr Claus-Michael
Published: April 2019

The purpose of this study was to evaluate the potential value of different epithelial cell culture systems as in vitro models for studying corneal permeability. Transformed human corneal epithelial (HCE-T) cells and Statens Serum Institut rabbit corneal (SIRC) cells were cultured on permeable filters. SkinEthic human corneal epithelium (S-HCE) and Clonetics human corneal epithelium (C-HCE) were received as ready-to-use systems. Excised rabbit corneas (ERCs) and human corneas (EHCs) were mounted in Ussing chambers, and used as references. Barrier properties were assessed by measuring transepithelial electrical resistance, and by determining the apparent permeability of markers with different physico–chemical properties, namely, fluorescein, sodium salt; propranolol hydrochloride; moxaverine hydrochloride; timolol hydrogenmaleate; and rhodamine 123. SIRC cells and the S-HCE failed to develop epithelial barrier properties, and hence were unable to distinguish between the permeation markers. Barrier function and the power to differentiate compound permeabilities were evident with HCE-T cells, and were even more pronounced in the case of C-HCE, corresponding very well with data from ERCs and EHCs. A net secretion of rhodamine 123 was not observed with any of the models, suggesting that P-glycoprotein or similar efflux systems have no significant effects on corneal permeability. Currently available corneal epithelial cell culture systems show differences in epithelial barrier function. Systems lacking functional cell–cell contacts are of limited value for assessing corneal permeability, and should be critically evaluated for other purposes.
The Intercostal NMJ Assay — A New Alternative to the Conventional LD50 Assay for the Determination of the Therapeutic Potency of Botulinum Toxin Preparations
Authors: Huber Alexander , France Richard M. , Riccalton-Banks Lisa , McLaren Jane , Cox Helen , Quirk Robin A. , Shakesheff Kevin M. , Thompson David , Panjwani Naveed , Shipley Sarah , Pickett Andy
Published: April 2019

Therapeutic botulinum neurotoxin type A preparations have found an increasing number of clinical uses for a large variety of neuromuscular disorders and dermatological conditions. The accurate determination of potency in the clinical application of botulinum toxins is critical to ensuring clinical efficacy and safety, and is currently achieved by using a lethal dose (LD50) assay in mice. Ethical concerns and operational constraints associated with this assay have prompted the development of alternative assay systems that could potentially lead to its replacement. As one such alternative, we describe the development and evaluation of a novel ex vivo assay (the Intercostal Neuromuscular Junction [NMJ] Assay), which uses substantially fewer animals and addresses ethical concerns associated with the LD50 assay. The assay records the decay of force from electrically-stimulated muscle tissue sections in response to the toxin, and thus combines the important mechanisms of receptor binding, translocation, and the enzymatic action of the toxin molecule. Toxin application leads to a time-related and dose-related reduction in contractile force. A regression model describing the relationship between the applied dose and force decay was determined statistically, and was successfully tested as able to correctly predict the potency of an unknown sample. The tissue sections used were found to be highly reproducible, as determined through the innervation pattern and the localisation of NMJs in situ. Furthermore, the efficacy of the assay protocol to successfully deliver the test sample to the cellular target sites, was critically assessed by using molecular tracer molecules.