bluuebunny/crossref_metadata_2025 huggingface dataset:
Reflections on Dialogue Processes, FRAME and the Three Rs
Authors: Smith Jane A.
Published: April 2019

My contribution to FRAME's 40th Anniversary meeting started by looking back: offering some reflections on the benefits and difficulties of engaging in wide-ranging dialogue on laboratory animal issues, largely based on experience with two forums — both of which have involved FRAME. Drawing on this discussion, I then looked forward: arguing that such dialogue now has an especially important role to play in developing strategies to replace (and reduce or avoid) the use of animals in research.
Collaboration between ZEBET, FRAME and ECVAM: FRAME's Contribution to Establishing the Three Rs in Europe
Authors: Spielmann Horst
Published: April 2019

FRAME has more than any other institution contributed to the implementation of the Three Rs concept in Europe. The first step was achieved by establishing the European Research Group on Alternatives to Toxicity Testing (ERGATT), in which individual scientists in several European countries began to collaborate to promote the development and validation of in vitro toxicity tests in order to refine, reduce and replace the use of animals in toxicity testing. The first successful project was the start of the INVITOX in vitro toxicology database, which is today managed by ECVAM. Major milestones were the establishment in 1989 of ZEBET, the first national centre for alternatives, in Germany, and of ECVAM in 1991. In 1990, ERGATT and CAAT organised a workshop in Amden, Switzerland, at which European and US scientists developed the concept of experimental validation of toxicity tests, which today remains the basis for the independent, scientific validation process. ECVAM has applied this validation concept in all of its successful validation studies, which have provided in vitro toxicity tests that have been accepted for regulatory purposes by the EU Commission and by the OECD. By re-launching the ATLA journal in 1983, FRAME provided another powerful scientific tool for promoting the Three Rs in Europe and around the world. The close cooperation of scientists from FRAME, ZEBET and ECVAM has most effectively promoted and established the Three Rs as the basic scientific, ethical and legal concept for refining, reducing and replacing the use of experimental animals.
Personal Reflections on Russell and Burch, FRAME, and the HSUS
Authors: Stephens Martin L.
Published: April 2019

The coincidence of anniversaries associated with the publication of William Russell and Rex Burch's The Principles of Humane Experimental Technique, the founding of the Fund for the Replacement of Animals in Medical Experiments (FRAME), and the establishment of the collaboration between FRAME and the University of Nottingham, provides an opportunity to reflect on Russell and Burch's legacy and how it was carried forward by FRAME. The Principles, published in 1959, was the pioneering work in what later became the alternatives or Three Rs field of replacement, reduction, and refinement of animal use. Such was the book's initial and undeserved obscurity, however, that FRAME, following its founding in 1969, pioneered a similar approach independently of Russell and Burch's work. The Humane Society of the United States (HSUS) was also an early champion of the alternatives framework, and through the establishment of the Russell and Burch Award, helped unite Russell and Burch with what had emerged as the alternatives community. Thanks largely to FRAME, Russell and Burch were able to participate in Three Rs activity before their deaths. They lived long enough to see their ideas take hold, but not long enough to see the emerging revolution currently under way in toxicity testing, toward the use of non-animal methods.
Laboratory Animal Science in China: Current Status and Potential for the Adoption of Three R Alternatives
Authors: Kong Qi , Qin Chuan
Published: April 2019

This paper aims to describe the development of laboratory animal science in China on the basis of historical evidence and recent national survey data, and to identify the problems facing the adoption of Three R alternatives. The authors undertook a national survey in 2006, by means of a questionnaire sent to 31 provinces, municipalities and autonomous regions, and also compared data from a variety of sources, including several national surveys and published papers. Laboratory animal science in China has developed rapidly over the past 30 years, as a result of a combination of economic, governmental and societal forces. More than 100,000 people work in the field of laboratory animal science, in 2,000 laboratory animal centres, institutes, universities, organisations, and companies. During the year of our survey, more than 19 million laboratory animals were produced from 320 licensed production facilities. Approximately 16 million laboratory animals were used in animal experiments, in 1530 facilities licensed for their use. The scale of the market for the supply and use of laboratory animals is huge, and thus it is very important to improve the level of adoption of these alternatives, in education, research and testing. For China, this presents a significant economic and technological opportunity in the field of biosciences research. The concept of the Three Rs first appeared in China in the 1980s, when the scale of laboratory animal sciences was starting to increase. In the 1990s, the Three Rs concept became commonly accepted among laboratory animal scientists, and began to appear in government documents. In the first decade of the 21st century, the Three Rs principles have become increasingly applied in our day-to-day work. But further time is still needed to achieve the full application of the Three Rs principles, especially the adoption of Three R alternatives. This paper describes the achievements in China relating to laboratory animal science, the use of Three R alternatives, and animal welfare, and shows that there is currently great potential for the adoption of alternatives. The information will help scientists and organisations around the world to gain better insight into the current state of laboratory animal science in China, and hopefully, will enable them to give advice on how we can improve the adoption of Three R alternatives in our country.
The use of Organotypic Hippocampal Slice Cultures to Evaluate Protection by Non-competitive NMDA Receptor Antagonists against Excitotoxicity
Authors: Ring Avi , Tanso Rita , Noraberg Jens
Published: April 2019

There is great interest in testing neuroprotectants which inhibit the neurodegeneration that results from excessive activation of N-methyl-D-aspartate (NMDA) receptors. As an alternative to in vivo testing in animal models, we demonstrate here the use of a complex in vitro model to compare the efficacy and toxicity of NMDA receptor inhibitors. Organotypic hippocampal slice cultures were used to compare the effectiveness of the Alzheimer's disease drug, memantine, the Parkinson's disease drug, procyclidine, and the novel neuroprotectant, gacyclidine (GK11), against NMDA-induced toxicity. All three drugs are non-competitive NMDA receptor open-channel blockers that inhibit excitotoxic injury, and their neuroprotective capacities have been extensively investigated in vivo in animal models. They have also been evaluated as potential countermeasure agents against organophosphate poisoning. Quantitative densitometric image analysis of propidium iodide uptake in hippocampal regions CA1, CA3 and DG, showed that, after exposure to 10μM NMDA for 24 hours, GK11 was the most potent of the three drugs, with an IC50 of about 50nM and complete protection at 250nM. When applied at high doses, GK11 was still the more potent neuroprotectant, and also the least cytotoxic. These findings are consistent with those from in vivo tests in rodents. We conclude that the slice culture model provides valuable pre-clinical data, and that applying the model to the screening of neuroprotectants might significantly limit the use of in vivo tests in animals.
Prevalidation of the Rat CFU-GM Assay for In Vitro Toxicology Applications
Authors: Pessina Augusto , Bonomi Arianna , Cavicchini Loredana , Albella Beatriz , Cerrato Laura , Parent-Massin Dominique , Sibiril Yann , Parchment Ralph , Behrsing Holger , Verderio Paolo , Pizzamiglio Sara , Giangreco Manuela , Baglio Carolina , Coccè Valentina , Sisto Francesca , Gribaldo Laura
Published: April 2019

In vitro haematotoxicity assays are thought to have the potential to significantly reduce and refine the use of animals for haematotoxicity testing. These assays are used successfully in all types of studies — however, their use is not so common in human toxicology studies in the preclinical setting, as they are not required for regulatory testing in this case. Furthermore, these assays could play a key role in bridging the gap between preclinical toxicology studies in animal models and clinical investigations. In previous studies, the Colony Forming Unit-Granulocyte Macrophage (CFU-GM) assay has been validated for testing drug haematotoxicity (with both mouse bone-marrow and human cord blood) and for predicting the in vivo human maximal tolerated dose (MTD) by adjusting in vivo data on mouse toxicity. Recently, a Colony Forming Unit-Megakaryocyte (CFU-MK) assay has also been prevalidated for testing drug toxicity toward megakaryocytes. The rat CFU-GM assay has been used by many researchers for its ability to evaluate in vitro haematotoxicity. Although it is not yet available, a standardised procedure for data comparison could be very important, since the rat is the most widely-used species for the in vivo testing of toxicants. This report presents the results of the prevalidation study developed to analyse the intra-laboratory and inter-laboratory variability of a standardised operating procedure for this assay and its performance for the in vitro determination of the inhibitory concentration (IC) values of drugs on rat myeloid progenitors (CFU-GM). The results demonstrate that the CFU-GM assay can be performed with cryopreserved rat bone-marrow cells (rBMC). The assay represents a useful tool for evaluating the toxicity of a compound, in terms of both relative toxicity (when different molecules are compared) and the prediction of the degree of in vivo toxicity. The use of this assay could greatly reduce the number of rats used in experimental procedures, and could also contribute to the accumulation of more toxicity data on compounds to be registered according to the criteria established by the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) programme.
Responses of Human Gingival and Periodontal Fibroblasts to a Low-Zinc Environment
Authors: Rudolf Emil , Červinka Miroslav
Published: April 2019

Morphology, motility, proliferation rate and markers of oxidative stress in primary human gingival fibroblasts (GF) and periodontal ligamental fibroblasts (PDL-F) grown in zinc-deficient cultivation medium (ZDM), were studied over a 5-week culture period. A low-zinc environment effectively reduced the total, as well as the free, intracellular zinc content in both cell types, over the course of the experiment. Decreased intracellular zinc content resulted in altered cellular morphology, reduced motility, and rearrangement of actin and tubulin in the cytoskeleton. In addition, fibroblasts with low zinc content exhibited decreased proliferation, accompanied by changes in cell cycle distribution, expression of specific biochemical markers, increased oxidative stress and the activation of caspase-3. Supplementation of ZDM with exogenous zinc prevented the loss of intracellular zinc, while also restoring the morphology, cell proliferation and mitogenic signalling of the cultured cells. Moreover, such supplemented cells were protected against oxidative stress and cell death. Of the two primary cell cultures examined, GF were more sensitive to decreased intracellular zinc content, when compared to PDL-F. The results obtained suggest that the human primary cell cultures can be useful for the longer-term evaluation of the effects of nutritional factors originating from the environment.
An Exploratory Study of Two Caco-2 Cell Models for Oral Absorption: A Report on Their Within-laboratory and Between-laboratory Variability, and Their Predictive Capacity
Authors: Prieto Pilar , Hoffmann Sebastian , Tirelli Valentina , Tancredi Francesco , González Isabel , Bermejo Marival , De Angelis Isabella
Published: April 2019

In 2005, the European Centre for the Validation of Alternative Methods (ECVAM) sponsored a study aimed at evaluating the reproducibility (between-laboratory and within-laboratory variability) and the predictive capacity of two in vitro cellular systems — the Caco-2/ATCC parental cell line and the Caco-2/TC7 clone — for estimating the oral fraction absorbed (Fa) in humans. Two laboratories, both of which had experience with Caco-2 cultures, participated in the study. Ten test chemicals with documented in vivo oral absorption data were selected. Atenolol, cimetidine and propranolol were included as reference compounds for low, medium and high intestinal absorption, respectively. Transport experiments were independently carried out in the two laboratories, according to an agreed protocol. The apparent permeability coefficient ( Papp) was calculated in either the apical to basolateral (absorption) or the basolateral to apical (efflux) direction. To investigate the involvement of possible active transport processes, experiments were also performed in the presence of sodium azide plus 2-deoxy-D-glucose in the donor compartment. Before performing the permeability experiments, the highest concentration that did not impair barrier integrity was identified for each test chemical in both cell models, by applying the chemicals together with a marker of the paracellular pathway. In addition, barrier integrity was assessed by measuring the trans-epithelial electrical resistance. All the permeability data obtained were independently analysed. Reproducibility was assessed for the seven substances for which sufficient data were available. Within-laboratory variability was based on coefficient of variation (CV) values. Median CV values of 10.4% and 14.7% were found for the two laboratories. Concerning between-laboratory reproducibility, comparable response levels were obtained for the three reference compounds and for paracetamol, while, for the other chemicals, the results were less reproducible — in particular, for compounds known to be actively transported. The Papp values obtained for both cell lines were comparable for identical experimental conditions. Despite the limited number of substances tested, the predictive capacity was investigated by using two mathematical models available in the literature. Good estimations of the human Fa were obtained for five well-absorbed compounds, while moderately and poorly absorbed compounds were overestimated. It is proposed that a confirmatory study addressing the main results, including power considerations, would now be useful.
European Experimental Animal Use Declines Ever So Slightly
Authors: Hudson Michelle , Balls Michael
Published: April 2019

The latest report on experimental animal use in the EU indicates a slight decrease. However, the figures, which are for 2008, show that around 12 million animals are still used in laboratories throughout Europe. To date, only a summary of the data has been published, and so this report is limited to giving a comparison of trends and significant changes. The impact on the figures of the revision of Directive 86/609/EEC and the 7th Amendment to the Cosmetics Directive 76/768/EEC is discussed. In addition, consideration is given to which areas of research and regulation will need to be closely monitored, and the importance of continuing to push for the implementation of Three Rs initiatives is highlighted.
A Social Science View on the FRAME Symposium: Identities and Networks
Authors: Harrington Jean , Stephens Neil
Published: April 2019

This paper, written by two social scientists, presents a social science perspective on the issues raised at the FRAME symposium on Human Alternatives to Animal Studies. Drawing upon the authors’ experience of conducting research with stem cell scientists, issues around access to human tissue for in vitro uses are considered. The paper concludes by raising questions pertinent to both interested social scientists and the Three Rs agenda.
Development and Application of Human Virtual Excitable Tissues and Organs: From Premature Birth to Sudden Cardiac Death
Authors: Holden Arun V.
Published: April 2019

The electrical activity of cardiac and uterine tissues has been reconstructed by detailed computer models in the form of virtual tissues. Virtual tissues are biophysically and anatomically detailed, and represent quantitatively predictive models of the physiological and pathophysiological behaviours of tissue within an isolated organ. The cell excitation properties are quantitatively reproduced by equations that describe the kinetics of a few dozen proteins. These equations are derived from experimental measurements of membrane potentials, ionic currents, fluxes, and concentrations. Some of the measurements were taken from human cells and human ion channel proteins expressed in non-human cells, but they were mostly taken from cells of other animal species. Data on tissue geometry and architecture are obtained from the diffusion tensor magnetic resonance imaging of ex vivo or post mortem tissue, and are used to compute the spread of current in the tissue. Cardiac virtual tissues are well established and reproduce normal and pathological patterns of cardiac excitation within the atria or ventricles of the human heart. They have been applied to increase the understanding of normal cardiac electrophysiology, to evaluate the candidate mechanisms for re-entrant arrhythmias that lead to sudden cardiac death, and to predict the tissue level effects of mutant or pharmacologically-modified ion channels. The human full-term virtual uterus is still in development. This virtual tissue reproduces the in vitro behaviour of uterine tissue biopsies, and provides possible mechanisms for premature labour.
Accelerating Clinical Insights: How to Use Accelerator Mass Spectrometry to Make Better Early Development Decisions
Authors: Seymour Mark
Published: April 2019

This paper is an overview of the applications of the technique of Accelerator Mass Spectrometry (AMS) in the biomedical drug development field. The work described here has been carried out at Xceleron (York, UK and Germantown, MD, USA), and it aims to apply AMS to provide better information about the human pharmacokinetic/metabolic behaviour of drugs or drug candidates as early as possible. It is hoped that the use of this technique will contribute to the delivery of better, more effective drugs onto the market sooner, which will be good news for all concerned.
Validation of the Educational Potential of a Simulator to Develop Abilities and Skills for the Creation and Maintenance of an Intravenous Cannula
Authors: Perez-Rivero Juan J. , Rendón-Franco Emilio
Published: April 2019

Historically, the method used in veterinary medicine to teach technical procedures which are to be performed on live animals, has taken the form of an apprenticeship. However, in the last decade, there have been several new developments in technologies oriented toward the development of such abilities and skills by students — for example, manuals, videos, pictures, and virtual reality simulators. Unfortunately, these simulators are inaccessible to many, due to their high cost. For this reason, it is necessary to create simulators that are easy to manufacture at low cost, and that are also portable. These simulators also need to be validated with regard to their ability to fulfil the required educational objectives. The validation of a venous simulator is described in this study. Fifty-two veterinary students, with no previous experience in the creation and maintenance of an indwelling venous cannula, were selected at random. They were divided into two groups: one experimental group (n = 35), who had training practice on the simulator, and the remainder (n = 17), who acted as the control group (i.e. they had no training practice on the simulator). The outcome measure was the number of attempts required to successfully cannulate the cephalic vein of an anaesthetised rabbit. The students in the experimental group showed more skill in cannulating the vein, with 45% effectiveness, as compared to 20% effectiveness in the control group. The difference between the groups was statistically significant ( p < 0.05).
Vaginal Irritation Models: The Current Status of Available Alternative and In Vitro Tests
Authors: Costin Gertrude-Emilia , Raabe Hans A. , Priston Robert , Evans Eric , Curren Rodger D.
Published: April 2019

Mucosal surfaces, such as the vaginal epithelium, are natural barriers to infection that are constantly exposed to bacteria and viruses, and are therefore potential sites of entry for numerous pathogens. The vaginal epithelium can be damaged mechanically, e.g. by the incorrect use of objects such as tampons, and by chemicals that are irritating or corrosive. Consequently, this can lead to an increase in susceptibility to further damage or infection. Pharmaceutical, cosmetic and personal care products that are specifically formulated for application onto human external mucosae can occasionally induce undesirable local or systemic side-effects. Therefore, the compatibility of applied materials with this mucosal surface represents a key issue to be addressed by manufacturers. The most frequently used method for assessing vaginal mucosal irritation is the in vivo rabbit vaginal irritation test. However, the current emphasis in the field of toxicology is to use alternative in vitro methods that reduce, refine, and replace the use of animals, and which model and predict human, not animal, responses. Such an approach is of particular interest to the personal care and cosmetic industries in their effort to comply with European legislative measures, such as the 7th Amendment to the EU Cosmetics Directive that does not permit the marketing of cosmetic products if they, or their ingredients, have been tested for irritation responses in animals. The focus of this review is to provide an overview of the alternative and in vitro tests that are currently available for vaginal mucosal irritation assessment, and which are already used, or may become useful, to establish the safety of newly-designed products for human use.
Refinement of the Charcoal Meal Study by Reduction of the Fasting Period
Authors: Prior Helen , Ewart Lorna , Bright Jonathan , Valentin Jean-Pierre
Published: April 2019

The aim of this investigation was to determine whether a shorter fasting period than the one historically employed for the charcoal meal test, could be used when measuring gastric emptying and intestinal transit within the same animal, and to ascertain whether the scientific outcome would be affected by this benefit to animal welfare. Rats and mice were fasted for 0, 3, 6 or 18 hours before the oral administration of vehicle or atropine. One hour later, the animals were orally administered a charcoal meal, then 20 minutes later, they were killed and the stomach and small intestine were removed. Intestinal transit time (the position of the charcoal front as a percentage of the total length of the small intestine) and relative gastric emptying (weight of stomach contents) were measured. Rats and mice fasted for six hours showed results for gastric emptying and intestinal transit which were similar to those obtained in animals fasted for 18 hours. Reducing the fasting period reduced the body weight loss in both species, and mice on shorter fasts could be group-housed, as hunger-induced fighting was lessened. Therefore, a fasting period of six hours was subsequently adopted for charcoal meal studies at our institution.
SNP Genotyping for the Genetic Monitoring of Laboratory Mice by Using a Microarray-based Method with Dual-colour Fluorescence Hybridisation
Authors: Cui Shu-Fang , Zhou Qian , Qu Xiu-Hua
Published: April 2019

Ensuring the genetic homogeneity of the mice used in laboratory experiments contributes to the Reduction aspect of the Three Rs, by maximising the quality of the data obtained from any animals that are used for these purposes, and ultimately reducing the numbers of animals used. Single nucleotide polymorphism (SNP) genotyping is especially suitable for use in the analysis of the genetic purity of model organisms such as the mouse, because bi-allelic markers remain fully informative when used to characterise crosses between inbred strains. Here, we attempted to apply a microarray-based method for a SNP marker to monitor the genetic quality of inbred mouse strains, so as to validate the reliability, stability and applicability of this SNP genotyping panel. The amplified PCR products containing four different SNP loci from four inbred mouse strains were spotted and immobilised onto amino-modified glass slides to generate a microarray. This was then interrogated through hybridisation with dual-colour probes, to determine the SNP genotypes of each sample. The results indicated that this microarray-based method could effectively determine the genotypes of the four selected SNPs with a high degree of accuracy. We have developed a new SNP genotyping technique for effective use in the genetic monitoring of inbred mouse strains.
Do Faculty in Southern Indian Medical Colleges Support Animal use in Postgraduate Education More than in Undergraduate Education?
Authors: Shehnaz Syed Ilyas , Sreedharan Jayadevan , Arifulla Mohamed , Gomathi Kadayam Guruswami
Published: April 2019

In India, animal experiments play an integral role in both undergraduate medical education (UGME) and postgraduate medical education (PGME) in the discipline of Pharmacology. Therefore, we aimed to compare the perceptions of pharmacology faculty members in southern India with regard to the use of animal experiments and alternatives in UGME and in PGME. We also determined the association between these perceptions and the socio-demographic characteristics of the participants. Pharmacology faculty members in 15 medical colleges located in southern India answered a 27-statement, 5-domain questionnaire with a total score of 108. The means of the total, domain and statement scores were analysed by the Wilcoxon signed-rank test. The mean total score obtained for faculty members ( n = 52) was significantly higher ( p < 0.001) for PGME (61.2/108) than that for UGME (51.9/108). Significant differences were observed in the mean total and in the domain scores for PGME when compared to UGME in all of the socio-demographic groups, except for male faculty members and those without an MD or doctoral degree. The mean individual statement scores also indicated that there is more support for animal use in PGME. Overall, it was apparent that pharmacology faculty members in southern Indian medical colleges support animal use in PGME more than in UGME. Increased awareness is required among faculty members concerning alternatives to animal experiments in medical education, especially in PGME.
A Pilot Study on the Refinement of Acute Inhalation Toxicity Studies: The Isolated Perfused Rat Lung as a Screening Tool for Surface-active Substances
Authors: Fischer Monika , Koch Wolfgang , Windt Horst , Dasenbrock Clemens
Published: April 2019

New surface-active agents in waterproofing sprays are frequently tested for acute inhalation toxicity in vivo according to OECD Test Guideline 403. In order to refine and reduce the number of acute inhalation tests performed, we propose a screening test that uses isolated lungs. The test consists of the exposure of isolated, ventilated and perfused rat lungs, to aerosolised formulations of waterproofing agents (mass median aerodynamic diameter = 1μm), and online monitoring of respiratory parameters and gross pathology analysis. A pilot evaluation of the isolated perfused rat lung model for use in a screening test was carried out by blind testing 12 surface-active substances. The results obtained compared well with data available from in vivo acute inhalation studies. Substances that triggered harmful effects, such as impaired lung compliance and atelectasis of the isolated perfused lung, were also found to cause changes in respiratory parameters, some of which would be severe enough to lead to death in in vivo tests with rats. The changes in respiratory parameters suggest that the mode-of-action is associated with impairment of the surfactant layer. Therefore, pre-testing in the isolated perfused rat lung allows the identification of surface-active substances with the potential for causing acute inhalation toxicity.
The Botulinum Toxin LD50 Potency Assay — Another Chapter, Another Mystery
Authors: Pickett Andy
Published: April 2019

Observers of the potency assay used for botulinum toxin were greeted last year with the news that one company had an alternative, non-animal alternative in place. But all was not as it seemed from the press release, and over a year later, information is still lacking.
Factors Affecting People's Acceptance of the Use of Zebrafish and Mice in Research
Authors: Ormandy Elisabeth H. , Schuppli Catherine A. , Weary Daniel M.
Published: April 2019

The species of laboratory animal used is known to influence people's willingness to support animal-based research. An online experiment was used to test people's willingness to accept the use of zebrafish or mice, two of the most commonly used species, in research involving either induced mutation (specifically, ethyl -N-nitrosourea [ENU] mutagenesis) or genetic modification, with and without regulatory oversight. Participants who were willing to support research on zebrafish (31.9%) were also willing to support the same research on mice. The participants expressed low levels of support for research involving ENU mutagenesis of zebrafish in both unregulated (30.7%) and regulated (38.5%) research programmes. A reason for the rejection of ENU mutagenesis was the perception that the procedure is painful. Some participants expressed a preference for the use of genetically-modified (GM) animal models over ENU mutagenesis, based on the belief that the former involves less pain and improves both the accuracy and efficiency of the animal models. Better informing the public about scientific practice, and scientists about public attitudes, may help reduce the disconnect between scientific practice and societal values.
2. The Wages of Inhumanity
Authors: Balls Michael
Published: April 2019

Inhumanity in the treatment of laboratory animals leads to ambiguous and otherwise unsatisfactory results
Evaluation of CADASTER QSAR Models for the Aquatic Toxicity of (Benzo)triazoles and Prioritisation by Consensus Prediction
Authors: Cassani Stefano , Kovarich Simona , Papa Ester , Roy Partha Pratim , Rahmberg Magnus , Nilsson Sara , Sahlin Ullrika , Jeliazkova Nina , Kochev Nikolay , Pukalov Ognyan , Tetko Igor V. , Brandmaier Stefan , Durjava Mojca Kos , Kolar Boris , Peijnenburg Willie , Gramatica Paola
Published: April 2019

QSAR regression models of the toxicity of triazoles and benzotriazoles ([B]TAZs) to an alga (Pseudokirchneriella subcapitata), Daphnia magna and a fish (Onchorhynchus mykiss), were developed by five partners in the FP7-EU Project, CADASTER. The models were developed by different methods —Ordinary Least Squares (OLS), Partial Least Squares (PLS), Bayesian regularised regression and Associative Neural Network (ASNN) — by using various molecular descriptors (DRAGON, PaDEL-Descriptor and QSPR-THESAURUS web). In addition, different procedures were used for variable selection, validation and applicability domain inspection. The predictions of the models developed, as well as those obtained in a consensus approach by averaging the data predicted from each model, were compared with the results of experimental tests that were performed by two CADASTER partners. The individual and consensus models were able to correctly predict the toxicity classes of the chemicals tested in the CADASTER project, confirming the utility of the QSAR approach. The models were also used for the prediction of aquatic toxicity of over 300 (B)TAZs, many of which are included in the REACH pre-registration list, and were without experimental data. This highlights the importance of QSAR models for the screening and prioritisation of untested chemicals, in order to reduce and focus experimental testing.
Read-across Estimates of Aquatic Toxicity for Selected Fragrances
Authors: Rorije Emiel , Aldenberg Tom , Peijnenburg Willie
Published: April 2019

Read-across as a non-animal testing alternative for the generation of risk assessment data can be useful in those cases where quantitative structure–activity relationship (QSAR) models are not available, or are less well developed. This paper provides read-across case studies for the estimation of the aquatic toxicity of five different fragrance substances, and proposes a pragmatic approach for expressing uncertainty in read-across estimates. The aquatic toxicity estimates and their uncertainties are subsequently used to estimate fresh water compartment Predicted No-Effect Concentrations (PNECs), with their two-sided 90% Confidence Intervals (CIs). These PNECs can be used directly in risk assessment. The results of the musk fragrance read-across cases (musk xylene, musk ketone and galaxolide) are compared to experimentally derived PNEC values. The read-across estimates made by using similarity in a hypothesised mechanism of action for (acute) toxicity of musk xylene gave a PNEC of 2μg/L (90% CI 0.0004–13.5μg/L) with the Species Sensitivity Distribution (SSD) approach. This estimated value is 1.8 times above the experimentally-based fresh water PNEC of 1.1μg/L. For musk ketone and galaxolide, the PNEC values based on the SSD approach and employing a toxicity mechanism-based read-across were 2.0 times greater, and 4.9 times below the experimentally derived PNEC values, respectively.
Animal Ethics Training for Postgraduates in Medical Schools in India: Catch Them Young!
Authors: Shehnaz Syed Ilyas , Agarwal Anoop Kumar
Published: April 2019

Adequate training in ethics for young scientists will serve as an important means of improving animal-based research in India
Is the Ethical Appraisal of Protocols Enough to Ensure Best Practice in Animal Research?
Authors: Franco Nuno H. , Olsson I. Anna S.
Published: April 2019

The common-held belief is that the ethical appraisal of protocols ensures good practice in animal-based research, but a large number of ethically approved studies allow animals to endure avoidable and severe suffering
Lost in Transit — A Forgotten Rodent Welfare Issue?
Authors: Quigley Caroline , Quigley Caroline
Published: April 2019

The transport of large numbers of rodent arouses animal welfare concerns, which need to be investigated; embryo and sperm transport offers an ethical alternative, but some technical issues need to be resolved
Progress in Promoting the Three Rs in Korea: Efficiency and Consistency
Authors: Choe Byung In , Lee Gwi Hyang , Braunschweiger Paul G. , Hart Lynette A.
Published: April 2019

The Korea National Information Center for the 3Rs was established in August 2011, to enhance humane science and animal welfare in Korea. It is a national Three Rs platform, with the goal of exchanging knowledge and sharing examples of best practice, in order to help replace laboratory animal use in education and training. This paper briefly summarises the progress made and the challenges that have become apparent during the initial operation of the collaborative project. There is a need to recognise and manage the challenges resulting from policy changes, especially misinterpretation of the objectives by the decision makers at the participating academic institutions and the government. The collective power of this type of collaborative project can be used to leverage an ongoing commitment to essentials such as consistent funding for operational and support staff, as well as allocation of the required space.
Transparency and Public Accountability on the Use of Non-Human Primates as Laboratory Animals Needs Actions, as Well as Words
Authors: Balls Michael
Published: April 2019

Living up to the promise of greater openness in the application of Directive 2010/63/EU will be very demanding, given the conclusions of an analysis of publicly-available information on NHP use in EU FP7-funded projects
Laboratory Animal Welfare and the Three Rs in Korea
Authors: Han Jin Soo , Lee Gwi Hyang
Published: April 2019

Ten years ago, Korea could be viewed as a “paradise for animal experimenters”; but this situation has changed in favour of the animals, as a result of tireless efforts
An Analysis of the Use of Dogs in Predicting Human Toxicology and Drug Safety
Authors: Bailey Jarrod , Thew Michelle , Balls Michael
Published: April 2019

Dogs remain the main non-rodent species in preclinical drug development. Despite the current dearth of new drug approvals and meagre pipelines, this continues, with little supportive evidence of its value or necessity. To estimate the evidential weight provided by canine data to the probability that a new drug may be toxic to humans, we have calculated Likelihood Ratios (LRs) for an extensive dataset of 2,366 drugs with both animal and human data, including tissue-level effects and Medical Dictionary for Regulatory Activities (MedDRA) Level 1–4 biomedical observations. The resulting LRs show that the absence of toxicity in dogs provides virtually no evidence that adverse drug reactions (ADRs) will also be absent in humans. While the LRs suggest that the presence of toxic effects in dogs can provide considerable evidential weight for a risk of potential ADRs in humans, this is highly inconsistent, varying by over two orders of magnitude for different classes of compounds and their effects. Our results therefore have important implications for the value of the dog in predicting human toxicity, and suggest that alternative methods are urgently required.
A Critical Review of Anaesthetised Animal Models and Alternatives for Military Research, Testing and Training, with a Focus on Blast Damage, Haemorrhage and Resuscitation
Authors: Combes Robert D.
Published: April 2019

Military research, testing, and surgical and resuscitation training, are aimed at mitigating the consequences of warfare and terrorism to armed forces and civilians. Traumatisation and tissue damage due to explosions, and acute loss of blood due to haemorrhage, remain crucial, potentially preventable, causes of battlefield casualties and mortalities. There is also the additional threat from inhalation of chemical and aerosolised biological weapons. The use of anaesthetised animal models, and their respective replacement alternatives, for military purposes — particularly for blast injury, haemorrhaging and resuscitation training — is critically reviewed. Scientific problems with the animal models include the use of crude, uncontrolled and non-standardised methods for traumatisation, an inability to model all key trauma mechanisms, and complex modulating effects of general anaesthesia on target organ physiology. Such effects depend on the anaesthetic and influence the cardiovascular system, respiration, breathing, cerebral haemodynamics, neuroprotection, and the integrity of the blood–brain barrier. Some anaesthetics also bind to the NMDA brain receptor with possible differential consequences in control and anaesthetised animals. There is also some evidence for gender-specific effects. Despite the fact that these issues are widely known, there is little published information on their potential, at best, to complicate data interpretation and, at worst, to invalidate animal models. There is also a paucity of detail on the anaesthesiology used in studies, and this can hinder correct data evaluation. Welfare issues relate mainly to the possibility of acute pain as a side-effect of traumatisation in recovered animals. Moreover, there is the increased potential for animals to suffer when anaesthesia is temporary, and the procedures invasive. These dilemmas can be addressed, however, as a diverse range of replacement approaches exist, including computer and mathematical dynamic modelling of the human body, cadavers, interactive human patient simulators for training, in vitro techniques involving organotypic cultures of target organs, and epidemiological and clinical studies. While the first four of these have long proven useful for developing protective measures and predicting the consequences of trauma, and although many phenomena and their sequelae arising from different forms of trauma in vivo can be induced and reproduced in vitro, non-animal approaches require further development, and their validation and use need to be coordinated and harmonised. Recommendations to these ends are proposed, and the scientific and welfare problems associated with animal models are addressed, with the future focus being on the use of batteries of complementary replacement methods deployed in integrated strategies, and on greater transparency and scientific cooperation.
Implementing the In Vitro Pyrogen Test: One More Step Toward Replacing Animal Experimentation
Authors: Hennig Ulrike
Published: April 2019

The MAT-based in vitro pyrogen test should be GLP-accredited for use in medical device testing
The Need to Improve Experimental Design
Authors: Fry Derek J.
Published: April 2019

Good experimental design is a valuable strategy to address the reduction and refinement principles of the Three Rs
A Toolbox for Animal Welfare Education
Authors: Coombs Ellen
Published: April 2019

A new online resource, aimed at qualified vets and veterinary science students, is being developed by WSPA
A Survey of Knowledge of the Three Rs Concept among Lecturers and Postgraduate Students in Brazil
Authors: Tréz Thales de A. e
Published: April 2019

The use of animals in science is a widespread practice, despite growing concern about its moral justification and scientific relevance. In this scenario, the Three Rs concept might be considered to be a motivation for the establishment of a new scientific approach to the use of experimental animals and to research itself. The main objective of this survey-based study was to identify the level of knowledge about this concept among lecturers (i.e. tenure-track professors) and postgraduate students in the physiological and pharmaceutical sciences in Brazilian universities. A questionnaire was completed by 185 lecturers from 16 universities, and 140 postgraduate students from five of these universities. The results indicate that the concept of the Three Rs is widespread among lecturers and students in the areas of physiology and pharmacology, throughout Brazilian universities, but that its interpretation generally attributes more importance to refinement than to reduction and replacement.
Every Silver Lining has a Cloud: The Scientific and Animal Welfare Issues Surrounding a New Approach to the Production of Transgenic Animals
Authors: Combes Robert D. , Balls Michael
Published: April 2019

The scientific basis and advantages of using recently developed CRISPR/Cas-9 technology for transgenesis have been assessed with respect to other production methods, laboratory animal welfare, and the scientific relevance of transgenic models of human diseases in general. As the new technology is straightforward, causes targeted DNA double strand breaks and can result in homozygous changes in a single step, it is more accurate and more efficient than other production methods and speeds up transgenesis. CRISPR/Cas-9 also obviates the use of embryonic stem cells, and is being used to generate transgenic non-human primates (NHPs). While the use of this method reduces the level of animal wastage resulting from the production of each new strain, any long-term contribution to reduction will be offset by the overall increase in the numbers of transgenic animals likely to result from its widespread usage. Likewise, the contribution to refinement of using a more-precise technique, thereby minimising the occurrence of unwanted genetic effects, will be countered by a probable substantial increase in the production of transgenic strains of increasingly sentient species. For ethical and welfare reasons, we believe that the generation of transgenic NHPs should be allowed only in extremely exceptional circumstances. In addition, we present information, which, on both welfare and scientific grounds, leads us to question the current policy of generating ever-more new transgenic models in light of the general failure of many of them, after over two decades of ubiquitous use, to result in significant advances in the understanding and treatment of many key human diseases. Because this unsatisfactory situation is likely to be due to inherent, as well as possibly avoidable, limitations in the transgenic approach to studying disease, which are briefly reviewed, it is concluded that a thorough reappraisal of the rationale for using genetically-altered animals in fundamental research and by the pharmaceutical industry, and for its support by funding bodies, should be undertaken. In the meantime, the use of CRISPR/Cas-9 to generate new transgenic cells in culture is to be guardedly encouraged.
Locating the Middle Ground
Authors: Simpson Matthew
Published: April 2019

A ‘middle ground’ exists, where the advocates and the opponents of vivisection can usefully negotiate, but where exactly is it?
10. The Use of Lower Organisms
Authors: Balls Michael
Published: April 2019

Lower organisms can be particularly useful as high discrimination models in fundamental research, but cannot offer the high fidelity required when specific questions are asked about humans in health and disease, and about their responses to drugs and other chemicals
FRAME's Online Alternatives Timeline Goes Live
Authors: Coll Kevin
Published: April 2019

A new online resource aims to collect a range of useful Three Rs-relevant information in one place and put it in its true historical context
Statistical Methods and Software for Validation Studies on New In Vitro Toxicity Assays
Authors: Schaarschmidt Frank , Hothorn Ludwig A.
Published: April 2019

When a new in vitro assay method is introduced, it should be validated against the best available knowledge or a reference standard assay. For assays resulting in a simple binary outcome, the data can be displayed as a 2 × 2 table. Based on the estimated sensitivity and specificity, and the assumed prevalence of true positives in the population of interest, the positive and negative predictive values of the new assay can be calculated. We briefly discuss the experimental design of validation experiments and previously published methods for computing confidence intervals for predictive values. The application of the methods is illustrated for two toxicological examples, by using tools available in the free software, namely, R: confidence intervals for predictive values are computed for a validation study of an in vitro test battery, and sample size calculation is illustrated for an acute toxicity assay. The R code necessary to reproduce the results is given.
‘One R’ is the new ‘Three Rs’
Authors: Redmond Craig
Published: April 2019

The Lush Prize, which rewards initiatives to end animal testing, believes that more attention needs to be given to the ‘One R’ of absolute replacement, and that research methods that exploit animals in any way (including tissues and cells) should not be considered as ‘alternatives’