bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of an Academic Centre
Authors: Goldberg Alan M.
Published: April 2019

On FRAME's 40th anniversary, I had the opportunity to examine FRAME and CAAT's missions as closely linked to those of their universities. The roles of education, research and service are key, both to the universities and to our two centres. By examining the current programmes, and identifying the needs of the future, the research activities, policy studies and training, it becomes clear that the Three Rs of alternatives contribute significantly to our respective universities’ missions.
Establishing a Three Rs Programme at the Canadian Council on Animal Care
Authors: Griffin Gilly
Published: April 2019

The anniversary of the Fund for the Replacement of Animals in Medical Experiments (FRAME) provides an opportunity to celebrate the Charity's achievements over the past 40 years, and in particular, its contribution to the international acceptance of the Three Rs as the basis of an ethic for animal experimentation, and its role as a leader in the implementation of the Three Rs in the life sciences. The Canadian Council on Animal Care (CCAC) has based its work on the Three Rs, especially since the establishment of its fundamental CCAC Policy Statement on the Ethics of Animal Investigation (1989). Following recommendations from the evaluation of its programmes by external committees established by the national granting agencies, the CCAC recently launched a Three Rs Program. This programme will build on the work of FRAME and other similar Three Rs organisations, to further the promotion and implementation of the Three Rs in Canadian science.
Finding the Courage: The Unconventional Lives of WMS and Claire Russell
Authors: Paskal Cleo
Published: April 2019

WMS and Claire Russell led lives of extraordinary intellectual adventure, spanning from the traditional formal education of Oxford University to the creativity of science fiction and the experimental world of mid-20th Century psychoanalysis. The verbal and pictorial illustrations presented in this article offer an insight into the vast collection of documents related to their lives and work currently being catalogued in the Manuscripts and Special Collections department of the University of Nottingham.
The Institute for Laboratory Animal Research — An International Resource for Promoting the Three Rs
Authors: Zurlo Joanne
Published: April 2019

In 1995, an international group of scientists met in Sheringham, Norfolk, UK, for a workshop entitled The Three Rs: The Way Forward. There, with the participation of William Russell and Rex Burch, the group discussed each of the Three Rs in detail, and made specific recommendations on how to promote them within the scientific community. The Institute for Laboratory Animal Research (ILAR) was established as part of the US National Academy of Sciences, to develop and disseminate information and guidelines for the care and use of laboratory animals. Over the years, the focus of ILAR has been to improve the health, welfare, and psychological well-being of the research animal, using the Three Rs as a foundation. ILAR's programmes include an international component, through which it reaches out to other countries via translations of its report; the ILAR Journal, a quarterly publication that focuses on animal models, animal welfare and protocol review; communications and outreach through the ILAR website and presence at scientific meetings; and special reports in which expert committees make recommendations to improve science and animal welfare. Through the efforts of ILAR and similar organisations, it is now recognised that high standards of humane care lead to better science. However, continued effort is needed to promote the Three Rs in developing countries.
The use of Human Tissues and Cells in Biomedical Research: The Unusual Suspects
Authors: Bennett Andrew
Published: April 2019

There are compelling reasons to search for alternatives to the use of animals in medical and pharmaceutical research. Aside from the obvious animal welfare issues, both the well-established differences between animal models and humans, and the inherent inter-individual variability in human biological responses, indicate that human-based alternatives are urgently required. However, any such alternative must out-perform the animal-based alternative, otherwise there will be little or no uptake and adoption by end-users. Data obtained from inbred animal models is often highly reproducible, and is therefore attractive to researchers in the fields of biomedical and pharmaceutical research. The inter-individual variability observed during human volunteer and human tissue-based studies is often considered to be problematic, and has been highlighted further with the advent of the ‘omics’ technologies, which generate large biological datasets. However, the variability in both baseline data and response to pharmacological or toxicological challenge observed in human tissues potentially contains a veritable gold mine of information, which may be critical for the advancement of drug discovery.
Filter-well Technology for Advanced Three-dimensional Cell Culture: Perspectives for Respiratory Research
Authors: BéruBé Kelly , Pitt Aldo , Hayden Patrick , Prytherch Zoë , Job Claire
Published: April 2019

Cell culture has long been a valuable tool for studying cell behaviour. Classical plastic substrates are two-dimensional, and usually promote cellular proliferation and inhibit differentiation. Understanding cell behaviour within complex multicellular tissues requires the systematic study of cells within the context of specific model microenvironments. A model system must mimic, to a certain degree, the in vivo situation, but, at the same time, can significantly reduce its complexity. There is increasing agreement that moving up to the third dimension provides a more physiologically-relevant and predictive model system. Moreover, many cellular processes (morphogenesis, organogenesis and pathogenesis) have been confirmed to occur exclusively when cells are ordered in a three-dimensional (3D) manner. In order to achieve the desired in vivo phenotype, researchers can use microporous membranes for improved in vitro cell culture experiments. In the present review, we discuss the applications of filter-well technology for the advanced 3-D cell culture of human pulmonary cells.
The Generation of Three-dimensional Tissue Structures with Mesenchymal Stem Cells
Authors: Genever Paul G.
Published: April 2019

Mesenchymal stem cells (MSCs) are multipotent stem cells, found in the bone-marrow and other adult tissues, which give rise to various cell lineages. Although MSCs are biologically important, and may have widespread therapeutic potential, they are not well-characterised, particularly in terms of their cell surface receptors and in vivo phenotype. We aimed to develop a three-dimensional (3-D) MSC in vitro model, in order to understand the factors involved in the regulation of lineage specification routes. A suitable model, which replicates the MSC microenvironment as accurately as possible, will allow more-detailed investigations into the phenotype of the cells. Our MSC spheroids appear to have an enhanced mesenchymal differentiation compared to two-dimensional MSC monolayers. With this in vitro system, it is possible to perform real-time analysis of cellular differentiation status. MSC spheroids may also be amenable for use in high-throughput assays. A more-recent research project aims to generate knockout micro-tissues, based on human 3-D MSCs, as an alternative to animal studies.
Development of the EpiOcular™ Eye Irritation Test for Hazard Identification and Labelling of Eye Irritating Chemicals in Response to the Requirements of the EU Cosmetics Directive and REACH Legislation
Authors: Kaluzhny Yulia , Kandárová Helena , Hayden Patrick , Kubilus Joseph , d'Argembeau-Thornton Laurence , Klausner Mitchell
Published: April 2019

The recently implemented 7th Amendment to the EU Cosmetics Directive and the EU REACH legislation have heightened the need for in vitro ocular test methods. To address this need, the EpiOcular™ eye irritation test (EpiOcular-EIT), which utilises the normal (non-transformed) human cell-based EpiOcular tissue model, has been developed. The EpiOcular-EIT prediction model is based on an initial training set of 39 liquid and 21 solid test substances and uses a single exposure period and a single cut-off in tissue viability, as determined by the MTT assay. A chemical is classified as an irritant (GHS Category 1 or 2), if the tissue viability is ≤ 60%, and as a non-irritant (GHS unclassified), if the viability is > 60%. EpiOcular-EIT results for the training set, along with results for an additional 52 substances, which included a range of alcohols, hydrocarbons, amines, esters, and ketones, discriminated between ocular irritants and non-irritants with 98.1% sensitivity, 72.9% specificity, and 84.8% accuracy. To ensure the long-term commercial viability of the assay, EpiOcular tissues produced by using three alternative cell culture inserts were evaluated in the EpiOcular-EIT with 94 chemicals. The assay results obtained with the initial insert and the three alternative inserts were very similar, as judged by correlation coefficients ( r2) that ranged from 0.82 to 0.96. The EpiOcular-EIT was pre-validated in 2007/2008, and is currently involved in a formal, multi-laboratory validation study sponsored by the European Cosmetics Association (COLIPA) under the auspices of the European Centre for the Validation of Alternative Methods (ECVAM). The EpiOcular-EIT, together with EpiOcular's long history of reproducibility and proven utility for ultra-mildness testing, make EpiOcular a useful model for addressing current legislation related to animal use in the testing of potential ocular irritants.
Estimation of the Acute Inhalation Hazards of Chemicals Based on Route-to-route and Local Endpoint Extrapolation: Experience from Bulk Maritime Transport
Authors: Höfer Thomas , James Derek , Syversen Tore , Bowmer Tim
Published: April 2019

Data on acute lethal inhalation toxicity from animal studies are commonly required for assessing the hazards to human health of volatile, gaseous and dusty chemicals or their mixtures. The International Maritime Organisation (IMO) made the provision of acute inhalation toxicity data a mandatory requirement for the carriage of bulk liquid chemicals transported by sea in tank ships, thereby creating the need for inhalation data on many hundreds of chemicals in bulk maritime transport. Taking note of previously published proposals for estimating acute inhalation toxicity hazards for chemicals, and the paucity of measured experimental data, an extrapolation method has been developed by the Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP) to partly fulfil this need. This method should be seen as a pragmatic approach to the challenge of missing measured experimental test data, with the added benefit of reducing tests in experimental animals. The method is based on a route-to-route (i.e. between-route) extrapolation of information on acute oral and/or dermal toxicity, in combination with data on the potential for irritation and/or corrosion to skin and eyes. The validation of this method was based on the individual evaluation of inhalation toxicity studies for 330 chemicals, including mixtures and many important chemical groups, for which the IMO holds public and industry-confidential data. The authors contend that this extrapolation method offers a reliable basis for hazard evaluation in the context of bulk maritime transport, and the ‘GESAMP inhalation toxicity extrapolation method’ has become part of the IMO regulatory system for the carriage of bulk liquids (i.e. noxious liquid substances) on board tank ships.
Comments on UK Options for Transposition of European Directive 2010/63/EU
Authors: Balls Michael , Hudson Michelle
Published: April 2019

The British Government's proposals for the transposition of European Directive 2010/63/EU are discussed under five main headings: direct transposition without major effects on the UK legislation, introduction of stricter requirements in the Directive, retention of stricter controls in the Animals [Scientific Procedures] Act 1986, questions requiring further consideration, and matters of concern. The Home Office had published a consultation on the options in 2011, which resulted in 98 responses from organisations and 13,458 responses from individuals. Our main concerns relate to the use of non-human primates, the annual publication of the UK statistics on laboratory animal use, and the provision of greater transparency on how animals are used, and why. Finally, we conclude that the new Directive and its transposition into the national laws of the Member states provide a renewed opportunity for genuine commitment to the Three Rs, leading to progressive and significant Reduction, Refinement and Replacement.
Arguments for considering Uncertainty in QSAR Predictions in Hazard and Risk Assessments
Authors: Sahlin Ullrika , Golsteijn Laura , Iqbal M. Sarfraz , Peijnenburg Willie
Published: April 2019

Chemical regulation allows non-in vivo testing (i.e. in silico-derived and in vitro-derived) information to replace experimental values from in vivo studies in hazard and risk assessments. Although non-in vitro testing information on chemical activities or properties is subject to added uncertainty as compared to in vivo testing information, this uncertainty is commonly not (fully) taken into account. Considering uncertainty in predictions from quantitative structure–activity relationships (QSARs), which are a form of non-in vivo testing information, may improve the way that QSARs support chemical safety assessment under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system. We argue that it is useful to consider uncertainty in QSAR predictions, as it: a) supports rational decision-making; b) facilitates cautious risk management; c) informs uncertainty analysis in probabilistic risk assessment; d) may aid the evaluation of QSAR predictions in weight-of-evidence approaches; and e) provides a probabilistic model to verify the experimental data used in risk assessment. The discussion is illustrated by using case studies of QSAR integrated hazard and risk assessment from the EU-financed CADASTER project.
Official Batch Control of Rabies Veterinary Vaccines: Current Situation and Perspectives in the European Union
Authors: Servat Alexandre , Cliquet Florence
Published: April 2019

The use of seroneutralisation techniques is the first concrete step toward the replacement of the mouse challenge test and its derivatives for the batch testing of rabies vaccines
Definition of the Applicability Domain of the Short Time Exposure (STE) Test for Predicting the Eye Irritation of Chemicals
Authors: Hayashi Kazuhiko , Abo Takayuki , Nukada Yuko , Sakaguchi Hitoshi
Published: April 2019

The Short Time Exposure (STE) test is a simple and easy-to-perform in vitro eye irritation test, that uses the viability of SIRC cells (a rabbit corneal cell line) treated for five minutes as the endpoint. In this study, our goal was to define the applicability domain of the STE test, based on the results obtained with a set of 113 substances. To achieve this goal, chemicals were selected to represent both different chemical classes and different chemical properties, as well as to cover, in a balanced manner, the categories of eye irritation potential according to the Globally Harmonised System (GHS). Accuracy analysis indicated that the rates of false negatives for organic/inorganic salts (75.0%), hydrocarbons (33.3%) and alcohols (23.5%) were high. Many of the false negative results were for solid substances. It is noteworthy that no surfactant resulted in a false negative result in the STE test. Further examination of the physical property data and performance showed a significant improvement in the predictive accuracy, when substances with vapour pressures over 6kPa were excluded from the analyses. Our results indicate that several substances — i.e. certain solids such as salts, alcohols, hydrocarbons, and volatile substances with a vapour pressure over 6kPa — do not fall within the applicability domain of the STE test. Overall, we are encouraged by the performance and improved accuracy of the STE test.
Resistance to Cancer in Amphibians: A Role for Apoptosis?
Authors: Ruben Laurens N. , Johnson Rachel O. , Clothier Richard H. , Balls Michael
Published: April 2019

The rarity of spontaneous cancer in amphibians, and the difficulty of inducing cancer in these lower vertebrates, suggest that they possess an effective system for resistance to the development of cancer. The first part of this narrative presents evidence for cancer resistance in amphibians, and then a variety of studies designed to help understand the physiological basis for this resistance are reviewed. Here, our emphasis is on evidence with regard to the role that apoptosis might play.
Is Phenylbutazone a Genotoxic Carcinogen? A Weight-of-Evidence Assessment
Authors: Combes Robert D.
Published: April 2019

Published in silico, in vitro, in vivo laboratory animal and human data, together with information on biotransformation and data from structure-activity analyses with two decision-tree systems (ACToR and Toxtree), have been used in a weight-of-evidence (WoE) assessment to determine whether phenylbutazone (PBZ) is a genotoxic or a non-genotoxic carcinogen. This was undertaken to facilitate the risk assessment of human exposure to this veterinary drug via the consumption of horsemeat from treated animals. Despite problems with data interpretation at all tiers of the database, it was concluded that PBZ behaves like a genotoxic carcinogen with a threshold dose. This conclusion is based mainly on the results of a definitive rodent bioassay, and on the following observations: a) that PBZ has weak in vitro activity only at high concentrations in some genotoxicity assays, accompanied by high levels of cytotoxicity; b) that it (and a major metabolite) is able to cause sister chromatid exchanges in vivo in rodents; and c) that it can induce cytogenetic effects in vivo in humans. It also takes into account the known and predicted activities of the parent drug, some of its metabolites and two structural analogues, and, importantly, several of the drug's other biochemical effects that are unrelated to toxicity. However, this conclusion is not fully supported by all the evidence, and much of the information is based on old papers. Therefore, more studies are required to establish whether the concentration thresholds seen in vitro would translate to dose thresholds for carcinogenicity, such that a safe dose-level could be defined for the purposes of assessing risk. It was disappointing that a WoE approach to evaluating all of the available hazard data, as is increasingly being advocated to improve the hazard identification paradigm, was unable to provide definitive answers in this case, particularly in view of the large numbers of animals that had been used to provide much of the information.
An Opportunity to Refocus on the ‘Humane’ in Experimental Endpoints: Moving beyond Directive 2010/63/EU
Authors: Ashall Vanessa , Millar Kate
Published: April 2019

Humane endpoints are a core refinement concept in animal experimentation. This paper identifies an urgent requirement for individuals and institutions to refocus on humane endpoints as part of the transposition of Directive 2010/63/EU into the national laws of the Member States, and to go beyond their legal construction when setting new guidance or applying humane endpoints in practice. It will be argued that requirements for humane endpoints within the Directive appear not to promote recent advances in best practice, but seem reliant on a narrow and potentially outdated definition of the term. We describe progress that has been made in encouraging change in the construction and application of humane endpoints, and suggest that Directive 2010/63/EU does not sufficiently acknowledge the conceptual complexity of this refinement strategy. For example, a useful development representing recent consensual views of best practice has been proposed by an EU consortium (in 2012). A complex approach to humane endpoints may place additional demands on institutions and raise challenges that would, unfortunately, not need to be overcome in order to remain within the Directive's current requirements regarding humane endpoints. We argue that there is now a need for a practical tool to help structure appropriate ethical reflection during research planning and experimentation, in order to facilitate best practice in the application of this important refinement concept.
The Labelling of All Medicinal Products as Dependent on Animal Research would be a Minefield to be Avoided at All Costs
Authors: Balls Michael
Published: April 2019

The Medicinal Labelling Bill should be rejected by Parliament, as ill-conceived and an unworthy and transparent attempt to promote laboratory animal experimentation
ESNATS Conference — The use of Human Embryonic Stem Cells for Novel Toxicity Testing Approaches
Authors: Rovida Costanza , Vivier Manon , Garthoff Bernward , Hescheler Jürgen
Published: April 2019

The main achievements and results of the ESNATS project (Embryonic Stem Cell-based Novel Alternative Testing Strategies) were presented at the final project conference that was held on 15 September 2013, the day before the traditional EUSAAT (European Society for Alternatives to Animal Testing) Congress in Linz, Austria. The ESNATS project was an FP7 European Integrated Project, running from 2008 to 2013, the aim of which was to develop a novel toxicity testing platform based on embryonic stem cells (ESCs), and in particular, human ESC (hESCs), to accelerate drug development, reduce related R&D costs, and propose a powerful alternative to animal tests in the spirit of the Three Rs principles. Altogether, ESNATS offered the first proof of concept that hESCs can be used to create robust, reproducible and ready-to use test assays for predicting human toxicity. In the end, essentially five test systems were developed to an adequate level for entering possible pre-validation procedures. These methods are based on hESCs, and can be combined to study the possible effects, on the human embryo, of exposure to a chemical during the early stages of development. In addition to the presentations by the main project partners, external speakers were invited to give lectures on relevant topics, both in the field of neurotoxicity and, more generally, on the applicability of hESCs in the development of advanced in vitro tests.
Assessing the Effects of Environmental Enrichment on Behavioural Deficits in C57BL Mice
Authors: Coombs Ellen J.
Published: April 2019

A study on the effects of environmental enrichment on behaviour and cognitive and motor functioning in a standard mouse model and a strain known to have behavioural deficits, suggests that environmental enrichment can positively influence natural functioning and natural behaviour
Human Bioengineered Artery Models for In Vitro Atherosclerosis Research: Fact or Fiction?
Authors: Weber Benedikt , Hoerstrup Simon P.
Published: April 2019

Human bioengineered artery equivalents may represent a first step toward the future replacement of the animal models used for studying the initial phases of atherosclerosis
11. The Analysis of Direct Inhumanity
Authors: Balls Michael
Published: April 2019

The incidence, character and severity of direct inhumanity should be seen as separate, but indivisible, parts of the same whole concept
Monkey-based Research on Human Disease: The Implications of Genetic Differences
Authors: Bailey Jarrod
Published: April 2019

Assertions that the use of monkeys to investigate human diseases is valid scientifically are frequently based on a reported 90–93% genetic similarity between the species. Critical analyses of the relevance of monkey studies to human biology, however, indicate that this genetic similarity does not result in sufficient physiological similarity for monkeys to constitute good models for research, and that monkey data do not translate well to progress in clinical practice for humans. Salient examples include the failure of new drugs in clinical trials, the highly different infectivity and pathology of SIV/HIV, and poor extrapolation of research on Alzheimer's disease, Parkinson's disease and stroke. The major molecular differences underlying these inter-species phenotypic disparities have been revealed by comparative genomics and molecular biology — there are key differences in all aspects of gene expression and protein function, from chromosome and chromatin structure to post-translational modification. The collective effects of these differences are striking, extensive and widespread, and they show that the superficial similarity between human and monkey genetic sequences is of little benefit for biomedical research. The extrapolation of biomedical data from monkeys to humans is therefore highly unreliable, and the use of monkeys must be considered of questionable value, particularly given the breadth and potential of alternative methods of enquiry that are currently available to scientists.
Human Embryonal Carcinoma Cells in Serum-free Conditions as an In Vitro Model System of Neural Differentiation
Authors: Jasnic-Savovic Jovana , Klajn Andrijana , Milivojevic Milena , Mojsin Marija , Nikcevic Gordana
Published: April 2019

Serum is generally regarded as an essential component of many eukaryotic cell culture media, despite the fact that serum composition varies greatly and may be the source of a wide range of artefacts. The objective of this study was to assess serum-free growth conditions for the human embryonal carcinoma cell line, NT2/D1. These cells greatly resemble embryonic stem cells. In the presence of retinoic acid (RA), NT2/D1 cells irreversibly differentiate along the neuronal lineage. We have previously shown that the early phases of neural induction of these cells by RA involve the up-regulation of SOX3 gene expression. Our goal was to compare RA-induced differentiation of NT2/D1 cells in serum-containing and serum-free media, by using SOX3 protein levels as a marker of differentiation. We found that NT2/D1 cells can be successfully grown under serum-free conditions, and that the presence or absence of serum does not affect the level of SOX3 protein after a 48-hour RA induction. However, six days of RA treatment resulted in a marked increase in SOX3 protein levels in serum-free media compared to serum-containing media, indicating that serum might have an inhibitory effect on the expression of this neural differentiation marker. This finding is important for both basic and translational studies that hope to exploit cell culture conditions that are free of animal-derived products.
Better Science with Human Cell-based Organ and Tissue Models
Authors: Heinonen Tuula
Published: April 2019

At present, animal-based models are the major test systems for assessing the tolerability and safety of chemical substances for regulatory purposes, and also for pivotal efficacy testing in pharmaceutical development. In spite of the high genetic similarity between many laboratory animals and humans, animal models are very poor predictors of human health effects and pathophysiological processes. Thus, models and testing strategies that are more relevant to human biology, are needed for these purposes. The best predictability is achieved with human organotypic models that mimic the microenvironment of human tissues. During their development, such models have to be characterised at the structural, genetic and functional levels, and compared to the respective human tissues. Their predictivity should be confirmed by using known reference chemicals with corresponding human data. The use of these methods in safety assessment and biomedical research, combined with the knowledge gained of the underlying biological processes on gene and protein expression, as well as on cellular signalling, will ultimately lead to better human science and animal welfare.
Participation of Brazil in the World Congresses on Alternatives and Animal Use in the Life Sciences: An Increase in Commitment to the Three Rs
Authors: Presgrave Octavio , Caldeira Cristiane , Moura Wlamir , Cruz Mayara , Méier Gisele , Santos Elisabete dos , Boas Maria H.V.
Published: April 2019

Many Brazilian researchers have long been interested in the development and use of alternative methods. Most of their research groups work in isolation, due to the lack of funding for collaborative studies. Despite these problems, since the Third World Congress on Alternatives and Animal Use in the Life Sciences, Brazilian researchers have strongly participated, not only by presenting posters and oral presentations, but also by being involved in the World Congress Committees. The Brazilian Center for the Validation of Alternative Methods (BraCVAM) must play an important role in the development and validation of alternative methods, through the active participation of the National Network of Alternative Methods (ReNaMA). In Brazil, Law 11,794/2008 regulates the use of animals in experimentation and education, and Law 9,605/1998 clearly states that use of the original animal test is not permitted, if an alternative method is available. Therefore, given the current legal framework, it is very important that all the Ministries involved with animal use, and the organisations responsible for funding researchers, strive to increase the financial support of those groups that are involved in the development and use of alternative methods in Brazil.
Comparative Costs of the Mouse Inoculation Test (MIT) and Virus Isolation in Cell Culture (VICC) for Use in Rabies Diagnosis in Brazil
Authors: Bones Vanessa C. , Gameiro Augusto H. , Castilho Juliana G. , Molento Carla F.M.
Published: April 2019

The decision to use laboratory animals rather than in vitro methods is frequently based on the financial costs involved, so the objective of our study was to compare the costs of performing the Mouse Inoculation Test (MIT) and Virus Isolation in Cell Culture (VICC) for use in rabies diagnosis in Brazil. Based on observations of laboratory routines at the Pasteur Institute, São Paulo, we listed the fixed cost (FC) and variable cost (VC) items necessary to perform both tests. Considering that 200 MITs are equivalent to 350 VICC assays, in terms of facilities and staff-hours needed per month, we calculated, for both tests, the average total cost per sample, the costs of the implementation of the laboratory structure, and the costs of routine use. With regard to absolute values, the total cost was mainly influenced by FC items, as they represented 60% of the cost for the MIT and 86% of the cost for VICC. A sample analysed by the MIT costs around 205% more than one analysed by using VICC. The MIT costs 74% and 406% more than VICC, when implementation costs and routine use per month, respectively, are taken into account. Our results can assist in the resolution of costing disputes that could hinder the replacement of animals for rabies diagnosis in Brazil. The method demonstrated here might also be useful for cost comparisons in other situations where animal use still continues when validated alternatives exist.
Conditions and Possible Mechanisms of VCD-induced Ovarian Failure
Authors: Liu Wei , Wang Ling-Yan , Xing Xiao-Xue , Fan Guan-Wei
Published: April 2019

Perimenopause is an important period in women's lives, in which they experience a series of physiological changes. Current animal models of perimenopause fail to adequately replicate this particular stage in female life, while current in vitro models are too simplistic and cannot account for systemic effects. Neither the naturally-ageing animal model, nor the ovariectomised animal model, mimic the natural transitional process that is the menopause. In vivo and in vitro studies have confirmed that the occupational chemical, 4-vinylcyclohexene diepoxide (VCD), can cause selective destruction of the ovarian primordial and primary follicles of rats and mice by accelerating the apoptotic process, which successfully mimics the perimenopausal state in women. However, it is the in vivo VCD-induced rodent perimenopausal models that are currently the most widely used in research, rather than any of the available in vitro models. Studies on the mechanisms involved have found that VCD induces ovotoxicity via interference with the c-kit/kit ligand and apoptotic signalling pathways, among others. Overall, the VCD-induced perimenopausal animal models have provided some insight into female perimenopause, but they are far from ideal models of the human situation.
20. On Replacing the Concept of Replacement
Authors: Balls Michael
Published: April 2019

In The Principles of Humane Experimental Technique, Russell and Burch gave us the Three Rs concept – but more important than this was their gift of The Humanity Criterion
Brazilian Center for the Validation of Alternative Methods (BraCVAM) and the Process of Validation in Brazil
Authors: Presgrave Octavio , Moura Wlamir , Caldeira Cristiane , Pereira Elisabete , Bôas Maria H. Villas , Eskes Chantra
Published: April 2019

The need for the creation of a Brazilian centre for the validation of alternative methods was recognised in 2008, and members of academia, industry and existing international validation centres immediately engaged with the idea. In 2012, co-operation between the Oswaldo Cruz Foundation (FIOCRUZ) and the Brazilian Health Surveillance Agency (ANVISA) instigated the establishment of the Brazilian Center for the Validation of Alternative Methods (BraCVAM), which was officially launched in 2013. The Brazilian validation process follows OECD Guidance Document No. 34, where BraCVAM functions as the focal point to identify and/or receive requests from parties interested in submitting tests for validation. BraCVAM then informs the Brazilian National Network on Alternative Methods (RENaMA) of promising assays, which helps with prioritisation and contributes to the validation studies of selected assays. A Validation Management Group supervises the validation study, and the results obtained are peer-reviewed by an ad hoc Scientific Review Committee, organised under the auspices of BraCVAM. Based on the peer-review outcome, BraCVAM will prepare recommendations on the validated test method, which will be sent to the National Council for the Control of Animal Experimentation (CONCEA). CONCEA is in charge of the regulatory adoption of all validated test methods in Brazil, following an open public consultation.
Autologous Co-culture of Primary Human Alveolar Macrophages and Epithelial Cells for Investigating Aerosol Medicines. Part II: Evaluation of IL-10-loaded Microparticles for the Treatment of Lung Inflammation
Authors: Hittinger Marius , Mell Nico Alexander , Huwer Hanno , Loretz Brigitta , Schneider-Daum Nicole , Lehr Claus-Michael
Published: April 2019

Acute respiratory distress syndrome is linked to inflammatory processes in the human lung. The aim of this study was to mimic in vitro the treatment of lung inflammation by using a cell-based human autologous co-culture model. As a potential trial medication, we developed a pulmonary dry powder formulation loaded with interleukin-10 (IL-10), a potent anti-inflammatory cytokine. The inflammatory immune response was stimulated by lipopolysaccharide. The co-culture was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures (PADDOCC), to deposit the IL-10-loaded microparticles on the inflamed co-culture model at the air–liquid interface. This treatment significantly reduced the secretion of interleukin-6 and tumour necrosis factor, as compared to the deposition of placebo (unloaded) particles. Our results show that the alveolar co-culture model, in combination with a deposition device such as the PADDOCC, may serve as a powerful tool for testing the safety and efficacy of dry powder formulations for pulmonary drug delivery.
A Multi-faceted Approach to Achieving the Global Acceptance of Animal-free Research Methods
Authors: Melbourne Jodie , Bishop Patricia , Brown Jeffrey , Stoddart Gilly
Published: April 2019

In 2015, the PETA International Science Consortium Ltd. was awarded the Lush Training Prize for its broad approach to education and training on the effective use of human-relevant, non-animal research techniques. The prize was awarded for work that included hosting workshops and webinars, initiating in-person training sessions and developing educational resources. The Consortium works closely with industry and regulatory agencies to identify and overcome barriers to the validation and use of alternatives to animal testing, by using an approach that identifies, promotes and verifies the implementation of these methods. The Consortium's recent activities toward replacing tests on animals for nanomaterials, pesticides and medical devices, are described, as examples of projects with broad applicability aimed at large-scale regulatory change.
Nrf2 Activation as a Key Event Triggered by Skin Sensitisers: The Development of the Stable KeratinoSens Reporter Gene Assay
Authors: Natsch Andreas , Emter Roger
Published: April 2019

The 21st century paradigm for toxicology and the adverse outcome pathway concept envisage a future toxicology largely based on mechanistic in vitro assays and relying mainly on cellular models. In the skin sensitisation field, this concept was not intuitive at the beginning. Given the high structural diversity of skin sensitising molecules, classical receptor binding as the molecular initiating event in a cell-based assay could be excluded from the start, leaving the question of how cells could sense potential skin sensitising chemicals and be able to differentiate them from non-sensitisers. When we entered this field in 2006, we realised that, in another emerging field of toxicology, detailed work on the antioxidant/electrophile sensing pathway Keap1/Nrf2/ARE was being performed. We postulated that, based on their intrinsic electrophilicity, a large structural variety of skin sensitisers would activate this pathway. This was demonstrated in a preliminary pilot study with an existing, breast cancer-derived reporter cell line. Broader confirmation of this initial hypothesis then came from a multitude of genome-wide studies, in which sensitiser-induced changes to the transcriptome were investigated. The results showed that this regulatory pathway is indeed the most common regulatory pathway activated by sensitisers at the gene expression level, and the underlying event in keratinocytes has become formalised as a Key Event in the Organisation for Economic Co-operation and Development (OECD) Adverse Outcome Pathway for sensitisation. These studies led to the development of the KeratinoSens® assay, which became the first cell-based in vitro test for skin sensitisation to be endorsed by a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) statement and an OECD Test Guideline. More recently, a number of studies have further developed this approach into 3-D skin models. Here, we review the underlying mechanism and the development of the KeratinoSens assay. We also present data on the stability of the assay over time, which is a key requirement for a cell-based biological assay to be endorsed in a regulatory context.
No Time like the Present — Two Hundred Years of Parkinson's Disease
Authors: Combes Robert
Published: April 2019

Two hundred years after his seminal publication on the ‘shaking palsy’, what would James Parkinson now make of progress achieved in understanding and treating the disease that bears his name?
Some Personal Reminiscences of the Long Road to Acceptance of the Three Rs
Authors: Balls Michael
Published: April 2019

Recent events have revived memories of events and people of great significance in my life and in the world of the Three Rs
In Vitro Models as a Platform to Investigate Traumatic Brain Injury
Authors: Kumaria Ashwin
Published: April 2019

Traumatic Brain Injury (TBI) remains a significant cause of mortality and morbidity, affecting individuals of all age groups. Much remains to be learned about its complex pathophysiology, with a view to designing effective neuroprotective strategies to protect sublethally injured brain tissue that would otherwise die in secondary injury processes. Experimental in vivo models offer the potential to study TBI in the laboratory, however, treatments that were neuroprotective in animals have, thus far, largely failed to translate in human clinical studies. In vitro models of neurotrauma can be used to study specific pathophysiological cascades — individually and without confounding factors — and to test potential neuroprotective strategies. These in vitro models include transection, compression, barotrauma, acceleration, hydrodynamic, chemical injury and cell-stretch methodologies. Various cell culture systems can also be utilised, including brain-on-a-chip, immortalised cell lines, primary cultures, acute preparations and organotypic cultures. Potential positive outcomes of the increased use of in vitro platforms to study TBI would be the refinement of in vivo experiments, as well as enhanced translation of the results into clinically meaningful neuroprotective strategies for the future. In addition, the replacement of in vivo experiments by suitable in vitro studies would lead to a welcome reduction in the numbers of animal procedures in this ethically-challenging field.
On the Journey toward Humane Education in Brazil: First Request for a Total Ban of Harmful Animal Use in Professional and Higher Education
Authors: Bachinski Róber , Alves Gutemberg , de Almeida Souza Mariângela Freitas , Bones Vanessa Carli , Garcia Rita de Cassia Maria , Gebara Rosangela , Ruiz Valeska Regina Reque , da Silva Alonso Luciano , Tréz Thales , Oliveira Simone Tostes , Rocha Alexandro Aluisio , Paixño Rita Leal , Klein Roseli Pizzigatti , Gasparetto Débora , Jukes Nick , Matera Júlia Maria
Published: April 2019

The Brazilian Network for Humane Education (RedEH) is an independent and self-managed group comprised of academics from ten different Brazilian states and a number of international collaborators. In 2016, in a concerted effort to change the educational field in Brazil and propagate humane education, RedEH sent a request to the Brazilian National Council for the Control of Animal Experimentation (CONCEA), asking that harmful animal use in education in professional and undergraduate courses be banned. This was the first formal request for a total replacement of harmful animal use in education in Brazil, and represented a major historic landmark in the advancement of Brazilian science education. This paper presents the full text of the request, as well as outlining its national and international repercussions. The request was supported by InterNICHE and representatives of 18 other international organisations. A major national impact of the request was its recognition by the Federal Council of Veterinary Medicine. With this action, academics and researchers took a potentially revolutionary step in the Brazilian education arena, with regard to advancing and supporting a higher quality, ethical and democratic educational system.
High-throughput Prediction of Nephrotoxicity in Humans
Authors: Loo Lit-Hsin , Zink Daniele
Published: April 2019

The Lush Science Prize 2016 was awarded to Daniele Zink and Lit-Hsin Loo for the interdisciplinary and collaborative work between their research groups in developing alternative methods for the prediction of nephrotoxicity in humans. The collaboration has led to the establishment of a series of pioneering alternative methods for nephrotoxicity prediction, which includes: predictive gene expression markers based on pro-inflammatory responses; predictive in vitro cellular models based on pluripotent stem cell-derived proximal tubular-like cells; and predictive cellular phenotypic markers based on chromatin and cytoskeletal changes. A high-throughput method was established for chemical testing, which is currently being used to predict the potential human nephrotoxicity of ToxCast compounds in collaboration with the US Environmental Protection Agency. Similar high-throughput imaging-based methodologies are currently being developed and adapted by the Zink and Loo groups, to include other human organs and cell types. The ultimate goal is to develop a portfolio of methods accepted for the accurate prediction of human organ-specific toxicity and the consequent replacement of animal experiments.
Harmonisation of Animal Testing Alternatives in China
Authors: Cheng Shujun , Qu Xiaoting , Qin Yao
Published: April 2019

More and more countries are lining up to follow the EU's approach and implement a full ban on the sale of cosmetics that have been tested on animals, which has been the case in the EU since 2013. Besides animal welfare considerations, the need for mutual acceptance of data (MAD) and harmonisation of the global market have made the move toward non-animal testing a desirable general trend for countries worldwide. Over the last 10 years, the concept of alternative methods has been gradually developing in China. This has seen the harmonisation of relevant legislation, the organisation of various theoretical and hands-on training sessions, the exploration of method validation, the adoption of internationally recognised methods, the propagation of alternative testing standards, and an in-depth investigation into the potential use of in vitro methods in the biosciences. There are barriers to this progress, including the demand for a completely new infrastructure, the need to build technology capability, the requirement for a national standardisation system formed through international co-operation, and the lack of technical assistance to facilitate self-innovation. China is now increasing speed in harmonising its approach to the use of non-animal alternatives, accelerating technological development and attempting to incorporate non-animal, in vitro, testing methods into the national regulatory system.
Does the Stress of Laboratory Life and Experimentation on Animals Adversely Affect Research Data? A Critical Review
Authors: Bailey Jarrod
Published: April 2019

Recurrent acute and/or chronic stress can affect all vertebrate species, and can have serious consequences. It is increasingly and widely appreciated that laboratory animals experience significant and repeated stress, which is unavoidable and is caused by many aspects of laboratory life, such as captivity, transport, noise, handling, restraint and other procedures, as well as the experimental procedures applied to them. Such stress is difficult to mitigate, and lack of significant desensitisation/habituation can result in considerable psychological and physiological welfare problems, which are mediated by the activation of various neuroendocrine networks that have numerous and pervasive effects. Psychological damage can be reflected in stereotypical behaviours, including repetitive pacing and circling, and even self-harm. Physical consequences include adverse effects on immune function, inflammatory responses, metabolism, and disease susceptibility and progression. Further, some of these effects are epigenetic, and are therefore potentially transgenerational: the biology of animals whose parents/grandparents were wild-caught and/or have experienced chronic stress in laboratories could be altered, as compared to free-living individuals. It is argued that these effects must have consequences for the reliability of experimental data and their extrapolation to humans, and this may not be recognised sufficiently among those who use animals in experiments.