bluuebunny/crossref_metadata_2025 huggingface dataset:
The FRAME Reduction Steering Committee: Reflections on a Decade Devoted to Reducing Animal Use in Biomedical Science
Authors: Hudson Michelle , Howard Bryan
Published: April 2019

Established in 1998, the FRAME Reduction Committee (FRC) (now the FRAME Reduction Steering Committee [FRSC]) has continued to pursue its aim of reducing the number of animals used in bio-medical science. Through its expertise in statistics, experimental design, animal welfare and research on alternatives, it has contributed to raising awareness of the need for reduction and the means of achieving and demonstrating it. In recognising the need for training of scientists to appreciate and understand the concept of reduction, the FRSC has organised dedicated workshops and training schools. Some of the Committee's major achievements are described, and, bearing in mind the current year-on-year increases in the number of scientific procedures on animals, its future activities are outlined.
The Use and Refinement of Rodent Models in Anti-cancer Drug Discovery: A Review
Authors: Curwen Jon O. , Wedge Stephen R.
Published: April 2019

This review describes the changing use of tumour models in rodents (predominantly mice) as employed over the last four decades in anti-cancer drug discovery, and the refinements in the experimental methods used. Such models are required to examine the complexities of cancer biology (e.g. tumour angiogenesis, invasion and metastasis, host immunity factors) and the impact of potential therapies (e.g. drug pharmacokinetics, pharmacodynamics and therapeutic index), and they have produced efficacious human therapeutics. Animal welfare considerations have driven refinements to animal models of cancer over time, with the most dramatic refinements being facilitated by the move away from inherently cytotoxic therapeutic approaches toward targeted inhibitors of disease-related processes. Whereas, four decades ago, the impact of disease burden was used as an endpoint in the absence of defined mechanistic parameters, acute pharmacodynamic measures are now increasingly used to minimise the adverse effects of disease and experimental procedures in a given animal. The changes in the UK guidelines on the use of rodents in preclinical cancer testing are also used as an illustration of the progressive refinement in tumour models and drug testing.
Reflections on Improved Health Status of Rodents Bred for Research: Contributions to the Reduction and Refinement of Animal Use
Authors: Brown Marilyn J. , White William J.
Published: April 2019

Reductions and refinements in the use of animals have steadily occurred over the last century. The need for improved health status has been a catalyst for much of this effort. This has also driven improvements in the housing and husbandry techniques required to maintain the health status of animals produced or used for biomedical research. This has decreased the number of animals used in biomedical research studies, contributed to refinements in animal care and use, and has resulted in better science as well as better animal welfare.
Computer Simulation Models are Implementable as Replacements for Animal Experiments
Authors: Badyal Dinesh K. , Modgill Vikas , Kaur Jasleen
Published: April 2019

It has become increasingly difficult to perform animal experiments, because of issues related to the procurement of animals, and strict regulations and ethical issues related to their use. As a result, it is felt that the teaching of pharmacology should be more clinically oriented and that unnecessary animal experimentation should be avoided. Although a number of computer simulation models (CSMs) are available, they are not being widely used. Interactive demonstrations were conducted to encourage the departmental faculty to use CSMs. Four different animal experiments were selected, that dealt with actions of autonomic drugs. The students observed demonstrations of animal experiments involving conventional methods and the use of CSMs. This was followed by hands-on experience of the same experiment, but using CSMs in small groups, instead of hands-on experience with the animal procedures. Test scores and feedback showed that there was better understanding of the mechanisms of action of the drugs, gained in a shorter time. The majority of the students found the teaching programme used to be good to excellent. CSMs can be used repeatedly and independently by students, and this avoids unnecessary experimentation and also causing pain and trauma to animals. The CSM programme can be implemented in existing teaching schedules for pharmacology undergraduate teaching with basic infrastructure support, and is readily adaptable for use by other institutes.
The Origins and Early Days of the Three Rs Concept
Authors: Balls Michael
Published: April 2019

Some questions are answered concerning the origins of the Three Rs ( Replacement, Reduction, Refinement) concept in relation to animal experimentation, expounded 50 years ago by Russell and Burch in The Principles of Humane Experimental Technique, by reference to some key publications and to correspondence in the W.M.S. and Claire Russell Archive, which is currently being established at the University of Nottingham. Some insight is also given into the relationship between Russell and Burch, the first use of “alternatives” in the Three Rs context, and the background to the publication of the book.
Ethics: Views from IACUC Members
Authors: Houde Lise , Dumas Claude , Leroux Thérèse
Published: April 2019

Institutional Animal Care and Use Committee (IACUC) members were interviewed on various ethical matters, including ethics, animal ethics, science and ethics, and the use of animals in research, in order to explore their implicit ethical framework. The results revealed that IACUC members entertain rich and diverse beliefs about ethics, that are part of an implicit ethical framework which relates to different domains of knowledge, such as biology (differences between human and animals), psychology (e.g. affective relationships with pets), and so on. The results also revealed that IACUC members hold quite a restrictive view on both animal ethics and animal use in research, and that they apply implicit ethical notions, such as respect and justice, to some elements (e.g. ethical rules) of the explicit ethical framework they are provided with when performing ethical evaluations of animal use. The study suggests that IACUC members should be provided with more up-to-date information on topics such as animal ethics and animal use in research.
Assessing the Search for and Implementation of the Three Rs: A Survey among Scientists
Authors: Leenaars Marlies , Savenije Bart , Nagtegaal Anne , van der Vaart Lilian , Ritskes-Hoitinga Merel
Published: April 2019

A survey among scientists into the current practice of searching for Replacement, Reduction and Refinement (Three Rs) alternatives, highlights the gap between the statutory required need to apply the Three Rs concept whenever possible and the lack of criteria for searching for Three Rs alternatives. A questionnaire was distributed to 342 scientists (Federation of European Laboratory Animal Science Associations [FELASA] Category C and B individuals), of which 67 responded. These scientists are customers of the Central Animal Laboratory of Radboud University Nijmegen Medical Centre. The results indicate that there is room for improvement in searching effectively for the Three Rs: skills in searching biomedical databases for Three Rs alternatives are limited, knowledge of specialised Three Rs databases is very limited, and satisfaction on the availability and accessibility of Three Rs information is low. None of the respondents allocate budget for a specific Three Rs alternatives search, although 50% do spend, on average, two hours engaged in this search for each application to their animal ethics committees. The majority of the respondents expressed the wish that the search for alternatives could be easier and less time consuming, and prefer to achieve this through the service offered by specialists at the Central Animal Laboratory. On the basis of the results from the questionnaire, the 3R Research Centre was established, with the aim of providing services and support for bio-medical scientists, to improve the search for, and subsequent implementation of, the Three Rs.
Animal Use in the Chemical and Product Manufacturing Sectors — Can the Downtrend Continue?
Authors: Curren Rodger
Published: April 2019

During the 1990s and early 2000s, a number of manufacturing companies in the cosmetic, personal care and household product industries were able to substantially reduce their use of animals for testing (or to not use animals in the first place). These reductions were almost always the result of significant financial contributions to either direct, in-house alternatives research, or to support personnel whose duties were to understand and apply the current state-of-the-art for in vitro testing. They occurred almost exclusively in non-regulatory areas, and primarily involved acute topical toxicities. Over the last few years, the reduction in animal use has been much less dramatic, because some companies are still reluctant to change from the traditional animal studies, because systemic, repeat-dose toxicity is more difficult to model in vitro, and because many products still require animal testing for regulatory approval. Encouragingly, we are now observing an increased acceptance of non-animal methods by regulatory agencies. This is due to mounting scientific evidence from larger databases, agreement by companies to share data and testing strategies with regulatory agencies, and a focus on smaller domains of applicability. These changes, along with new emphasis and financial support for addressing systemic toxicities, promise to provide additional possibilities for industry to replace animals with in vitro methods, alone or in combination with in silico methods. However, the largest advance will not occur until more companies commit to using the non-animal test strategies that are currently available.
The Way Forward in Reproductive/Developmental Toxicity Testing
Authors: Spielmann Horst
Published: April 2019

The use of experimental animals in reproductive toxicity testing is critically reviewed on the occasion of the 50th anniversary of the publication of the Three Rs concept by Russell and Burch, since there is major concern that reproductive toxicity testing will significantly increase due to the requirements of the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system. A comparison of the test guidelines for drugs, agrochemicals and industrial chemicals shows that, for historical reasons, significantly different testing strategies are applied. The current status of development and validation of in vitro tests in reproductive toxicology is also critically evaluated. The mouse embryonic stem cell test (mEST) is the most advanced and promising of the in vitro tests. Although it has not yet been accepted for regulatory purposes, its use in preclinical drug development is well established. Moreover, promising molecular endpoints have been established in the mEST, including proteomic and toxicogenomic endpoints. Preliminary results have been obtained with a human EST (hEST). In addition, an overview is given on new in vitro reproductive toxicity tests that are currently being developed in the EU FP6 project, ReProTect, since the ReProTect test battery, which covers the essential steps of female and male fertility, implantation and embryotoxicity, holds promise for use as a screening assay for reproductive toxicity testing according to the EU REACH legislation. However, since validated in vitro methods will not be available in the short term, opportunities for the refinement of the standard in vivo tests are discussed, in order to reduce the numbers of animal used in reproductive toxicity testing. Finally, recommendations for toxicity testing in the 21st century call for the harmonisation of test methods across all areas of regulatory testing as a first step. Since the REACH system testing framework for industrial chemicals is driven by the reproductive safety testing requirements of agro-chemicals, a shift is proposed to exposure-driven testing of industrial chemicals. In particular, the implementation of a new ‘extended one-generation reproductive toxicity study’ (EOGRTS), which includes triggers for additional testing for fertility, developmental neurotoxicity and immunotoxicity, would significantly reduce test animal numbers. It is concluded that in vitro methods hold great promise for reproductive toxicity testing in the 21st century, e.g. the ReProTect in vitro battery and the embryonic stem cell (ESC) technology focusing on molecular endpoints in both the mEST and the hEST.
The Best Model for Humans is Human — How to Accelerate Early Drug Development Safely
Authors: Seymour Mark
Published: April 2019

Traditionally, the choice of which candidate compounds to take forward into development has been based on pre-clinical data. However, lack of predictivity of the human clinical situation in the models used has led to poor decision-making, and the later in the development process that such mistakes are realised, the more costly and time-consuming it is to correct them. Furthermore, compounds that may have made perfectly good drugs, have been dropped due to poor pharmacokinetics in animal models. Accelerator mass spectrometry (AMS) is an ultra-sensitive detection technique that can be used to quantify carbon-14. By administering very small amounts of 14C-labelled compounds, AMS can be used to obtain human clinical data very early in the drug development process. Such studies: a) can be helpful in understanding human pharmacokinetics using microdosing; b) can provide early human metabolism information, to validate the choice of animal species used in pre-clinical safety testing and identify unique or disproportionate human metabolites during Phase 1; and c) can provide fundamental human pharmacokinetic data, including absolute bioavailability, by facilitating a scientifically optimal and cost-effective study design. The provision of these clinical insights at the earliest possible opportunity can lead to improved decision-making, and therefore can reduce the time and cost involved in the drug development process.
Toxicogenomics in Drug Discovery and Development — Making an Impact
Authors: Wills Quin , Mitchell Cathrine
Published: April 2019

As a branch of pharmacogenomics aimed at predicting drug safety concerns, toxicogenomics drew much excitement with the emergence of technologies such as gene expression microarrays. A few years down the line, the evidence is scant that current approaches to toxicogenomics are really making an impact in areas such as preclinical toxicology. It has been argued that there needs to be a re-focus of application toward high-throughput approaches which combine the best of tissue and genomic modelling. This commentary gives a brief introduction to in vitro toxicogenomics, drawn from the perspectives of the specialist toxicogenomics company, SimuGen.
“I'm Not Going to Make a Song and Dance about It…”
Authors: Richmond Jon
Published: April 2019

Having been involved with the biomedical sciences and alternatives for 30 of the 50 years since Russell and Burch published The Principles of Humane Experimental Technique, I feel well placed to speak of the contributions made by FRAME over the last 40 years. FRAME has promoted the development and use of alternatives by presenting the “better science” arguments to scientists, promoting informed discussion, changing opinions and changing behaviours. FRAME has also contributed constructively to the political debate, and was instrumental in promoting legislation which incorporated the principles of the Three Rs. Times are changing, and the Three Rs are increasing being politicised: and FRAME is particularly well placed to ensure that good science and good welfare, rather than ideology, remain the basis for interest in and progress with the Three Rs.
Application of the Improved BALB/c 3T3 Cell Transformation Assay to the Examination of the Initiating and Promoting Activities of Chemicals: The Second Inter-laboratory Collaborative Study by the Non-genotoxic Carcinogen Study Group of Japan
Authors: Tsuchiya Toshiyuki , Umeda Makoto , Tanaka Noriho , Sakai Ayako , Nishiyama Hiroshi , Yoshimura Isao , Ajimi Syozo , Asada Shin , Asakura Masumi , Baba Hiroshi , Dewa Yasuaki , Ebe Youji , Fushiwaki Yuichi , Hagiwara Yuji , Hamada Shuichi , Hamamura Tetsuo , Iwase Yumiko , Kajiwara Yoshitsugu , Kasahara Yasushi , Kato Yukihiko , Kawabata Masayoshi , Kitada Emiko , Kaneko Kazuko , Kizaki Yuko , Kubo Kinya , Miura Daisaku , Mashiko Kaori , Mizuhashi Fukutaro , Muramatsu Dai , Nakajima Madoka , Nakamura Tetsu , Oishi Hidetoshi , Sasaki Toshiaki , Shimada Sawako , Takahashi Chitose , Takeda Yuko , Wakuri Sinobu , Yajima Nobuhiro , Yajima Satoshi , Yatsushiro Tomoko
Published: April 2019

The Non-genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan organised the second step of the inter-laboratory collaborative study on one-stage and two-stage cell transformation assays employing BALB/c 3T3 cells, with the objective of confirming whether the respective laboratories could independently produce results relevant to initiation or promotion. The method was modified to use a medium consisting of DMEM/F12 supplemented with 2% fetal bovine serum and a mixture of insulin, transferrin, ethanolamine and sodium selenite, at the stationary phase of cell growth. Seventeen laboratories collaborated in this study, and each chemical was tested by three to five laboratories. Comparison between the one-stage and two-stage assays revealed that the latter method would be beneficial in the screening of chemicals. In the test for initiating activity with the two-stage assay (post-treated with 0.1μg/ml 12- O-tetradecanoylphorbol-13-acetate), the relevant test laboratories all obtained positive results for benzo[ a]pyrene and methylmethane sulphonate, and negative results for phenanthrene. Of those laboratories assigned phenacetin for the initiation phase, two returned positive results and two returned negative results, where the latter laboratories tested up to one dose lower than the maximum dose used by the former laboratories. In the exploration of promoting activity with the two-stage assay (pretreated with 0.2μg/ml 3-methylcholanthrene), the relevant test laboratories obtained positive results for mezerein, sodium orthovanadate and TGF-β1, and negative results for anthralin, phenacetin and phorbol. Two results returned for phorbol 12,13-didecanoate were positive, but one result was negative — again, the maximum dose to achieve the latter result was lower than that which produced the former results. These results suggest that this modified assay method is relevant, reproducible and transferable, provided that dosing issues, such as the determination of the maximum dose, are adequately considered. The application of this two-stage assay for screening the initiating and promoting potential of chemicals is recommended for consideration by other research groups and regulatory authorities.
Comparing In Vivo, In Vitro and In Silico Methods and Integrated Strategies for Chemical Assessment: Problems and Prospects
Authors: Benfenati Emilio , Gini Giuseppina , Hoffmann Sebastian , Luttik Robert
Published: April 2019

The RAINBOW workshop addressed the background for the integration of in vivo, in vitro and computer-based ( in silico) methods, to facilitate the study of the toxic properties of chemicals. On the basis of these discussions, we prepared the present paper, outlining the strengths, weaknesses, opportunities and threats of each approach, both alone and integrated into a single testing strategy. The current scheme for evaluation of chemicals needs to be reshaped, in the face of the much larger numbers of chemicals which need to be examined and the availability of a diversified set of tools.
Further Verification of an In Vitro Tier System for the Identification of Cosmetic Ingredients that are Not Ocular Irritants
Authors: Hagino Shigenobu , Okazaki Yuuko , Kitagaki Masato , Itagaki Hiroshi
Published: April 2019

A tier evaluation system for the identification of cosmetic ingredients that are not ocular irritants was applied to 59 cosmetic ingredients, for which in vivo data were available. The tier system employs monolayer cultures of SIRC cells, an established cell line originally derived from rabbit cornea, and a three-dimensional living dermal model (LDM; MATREX™), which consists of human dermal fibroblasts in a contracted collagen lattice. The effects of ingredients on monolayer cultures of SIRC cells were determined by Crystal Violet staining (in the SIRC-CVS assay), and the effects on the LDM were measured by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (in the LDM-MTT assay). The classifications of eye irritancy predicted by the in vitro system were compared with previously reported data obtained with the in vivo Draize rabbit eye test. The in vivo classification was based on appearance of corneal damage, or a maximal average score (MAS) of 15 as the cut-off point. The SIRC-CVS assay was effective in the prediction of compounds that would be non-irritants at a concentration of 10%, while the subsequent LDM-MTT assay could predict non-irritancy at various lower and higher concentrations, including 10%. The tier system gave very few false-negative predictions, though false positives were unavoidable. Performing the LDM-MTT assay with an additional 73 ingredients gave similar results in the prediction of non-irritancy at various concentrations. Our findings indicate that the tier system may be suitable for the safety assessment of eye irritancy of ingredients intended to be used in cosmetics and medicated cosmetics in Japan.
A Gold Standard Publication Checklist to Improve the Quality of Animal Studies, to Fully Integrate the Three Rs, and to Make Systematic Reviews More Feasible
Authors: Hooijmans Carlijn R. , Leenaars Marlies , Ritskes-Hoitinga Merel
Published: April 2019

Systematic reviews are generally regarded by professionals in the field of evidence-based medicine as the highest level of medical evidence, and they are already standard practice for clinical studies. However, they are not yet widely used nor undertaken in the field of animal experimentation, even though there is a lot to be gained from the process. Therefore, a gold standard publication checklist (GSPC) for animal studies is presented in this paper. The items on the checklist have been selected on the basis of a literature analysis and the resulting scientific evidence that these factors are decisive in determining the outcome of animal studies. In order to make future systematic reviews and meta-analyses of animal studies possible, to allow others to replicate and build on work previously published, diminish the number of animals needed in animal experimentation ( reduction), improve animal welfare ( refinement) and, above all, improve the quality of scientific papers on animal experimentation, this publication checklist needs to be used and followed. We have discussed and optimised this GSPC through feedback from interviews with experts in the field of animal experimentation. From these interviews, it became clear that scientists will adopt this GSPC when journals demand it. The GSPC was compared with the current instructions for authors from nine different journals, selected on the basis that they featured a high number of publications on animal studies. In general, the journals’ demands for the description of the animal studies are so limited that it is not possible to repeat the studies, let alone carry out a systematic review. By using the GSPC for animal studies, the quality of scientific papers will be improved. The use of the GSPC and the concomitant improvement in the quality of scientific papers will also contribute to decreased variation and increased standardisation and, as a consequence, a reduction in the numbers of animals used and a more reliable outcome of animal studies. It is of major importance that journal editors become convinced of and adopt these recommendations, because only then will scientists follow these guidelines to the full extent.
Artificial Human Tissues from Cord and Cord Blood Stem Cells for Multi-organ Regenerative Medicine: Viable Alternatives to Animal In Vitro Toxicology
Authors: Jurga Marcin , Forraz Nico , McGuckin Colin P.
Published: April 2019

New medicinal products and procedures must meet very strict safety criteria before being applied for use in humans. The laboratory procedures involved require the use of large numbers of animals each year. Furthermore, such investigations do not always give an accurate translation to the human setting. Here, we propose a viable alternative to animal testing, which uses novel technology featuring human cord and cord blood stem cells. With over 130 million children born each year, cord and cord blood remains the most widely available alternative to the use of animals or cadaveric human tissues for in vitro toxicology.
Implementing Scientifically-robust and Humane Shellfish Toxicity Testing: We're Still Waiting
Authors: Buckland Gemma
Published: April 2019

The response to a Parliamentary Question put to the then-Home Office Minister on 8 March 2006, was that “All protocols for the detection of toxins in shellfish intended for human consumption were assigned a substantial severity limit”, and that “A total of 6,468 animals were used in the relevant procedures [for the testing of shellfish toxins in the UK] during 2004”. The official European Union (EU) method for shellfish toxin testing is the Mouse Bioassay (MBA). The MBA is the primary method, although the Rat Bioassay (RBA) is permitted for some toxins. Six years later, following the completion of ten reports from the European Food Safety Authority (EFSA) stating that current reliance on the MBA is scientifically inappropriate, the regulatory climate for testing is almost unchanged, despite the availability of alternatives. The reliance on such a scientifically questionable method, and the welfare concerns for the animals used, highlight the extent of the clash between policy and science. The ongoing struggle to persuade the European Commission to formally adopt non-animal testing methods for all of the relevant toxins has been fruitless, and evidence remains that thousands of mice are used every year in lethal tests that could be replaced. There is an absolute requirement for advanced scientific methods to replace questionable methods which rely on outdated, inaccurate animal tests; in this case, marine biotoxin testing has surely been waiting in line for far too long.
Abattoir-sourced Isolated Ileum from Gallus gallus domesticus as an Experimental Tool
Authors: Kaithwas Gaurav , Sharma Satya D. , Majumdar Dipak K.
Published: April 2019

A study was undertaken to determine the longevity of active muscarinic receptors on abattoir-sourced isolated ileum preparations from Gallus gallus domesticus, with a view to using the tissue as an experimental tool for functional response assays in laboratory experiments. A concentration–response curve for acetylcholine (1–256μM) was plotted, in the presence and absence of 1, 3 and 6nM atropine. In a second experiment, unknown concentrations of acetylcholine samples were determined by using an interpolation method. In this experiment, four sample concentrations were used and the calculated values were found to be almost equal to the actual values. Finally, an experiment was carried out to elucidate the effects of post-sacrifice time on the contractile response of the tissue. The results showed that the tissue maintained considerable contractile response at the 6-hour post-sacrifice time-point. Competitive antagonistic activity was observed between acetylcholine and atropine on the chicken ileum, and the pA2 value was calculated to be 9.21 by using an Arunlakshana–Schild plot. The results suggest that isolated ileum preparations of Gallus gallus domesticus, obtained from a meat abattoir, can be used as a basic experimental tool for bioassays in routine laboratory experiments. However, its potential as a research tool still needs to be confirmed.
Biomimetic Design of Artificial Micro-vasculatures for Tissue Engineering
Authors: Barber Robert W. , Emerson David R.
Published: April 2019

Over the last decade, highly innovative micro-fabrication techniques have been developed that are set to revolutionise the biomedical industry. Fabrication processes, such as photolithography, wet and dry etching, moulding, embossing and lamination, have been developed for a range of biocompatible and biodegradable polymeric materials. One area where these fabrication techniques could play a significant role is in the development of artificial micro-vasculatures for the creation of tissue samples for drug screening and clinical applications. Despite the enormous technological advances in the field of tissue engineering, one of the major challenges is the creation of miniaturised fluid distribution networks to transport nutrients and waste products, in order to sustain the viability of the culture. In recent years, there has been considerable interest in the development of microfluidic manifolds that mimic the hierarchical vascular and parenchymal networks found in nature. This article provides an overview of microfluidic tissue constructs, and also reviews the hydrodynamic scaling laws that underpin the fluid mechanics of vascular systems. It shows how Murray's law, which governs the optimum ratio between the diameters of the parent and daughter branches in biological networks, can be used to design the microfluidic channels in artificial vasculatures. It is shown that it is possible to introduce precise control over the shear stress or residence time in a hierarchical network, in order to aid cell adhesion and enhance the diffusion of nutrients and waste products. Finally, the paper describes the hydrodynamic extensions that are necessary in order to apply Murray's law to the rectangular channels that are often employed in artificial micro-vasculatures.
The Use of Functional Human Tissues in Drug Development
Authors: Bunton David
Published: April 2019

Drug development currently depends on animal models to provide an accurate prediction of human physiology and pathophysiology. However, as is clear from clinical trial failures during phases II and III, such in vivo models do not always predict the effects that a drug can elicit in humans. Tests with human tissues, which are obviously considered to be the closest model of human in vivo function, could fill the gap between animal-based tests and trials in patients. Despite clear advantages, logistical and ethical barriers prevent fresh human tissues from being widely used during drug development. Biopta is aiming to make human tissue testing a regular element of drug development, and works to lower the barriers surrounding the availability of tissue and practicalities of experimental work.
The Development of Cell Line Models of Childhood Brain Tumours
Authors: Grundy Richard
Published: April 2019

Childhood brain cancers have a significant impact on society. Currently, it is possible to make sophisticated diagnoses, but the treatments do not reflect patient differences and are out-dated. In order to develop better therapies and improve the outcome, we must first understand the underlying biology of brain cancer and how cells influence the disease process. For that purpose, several lines of brain cancer stem cells have been isolated, which have retained the characteristics of their original tissues. These in vitro human cell models are a much-needed addition to research on childhood brain cancers.
Development of a Cell-based Diabetic Wound Assay
Authors: Stephens Phil
Published: April 2019

Chronic wounds require prolonged healthcare and adversely affect the quality of life of patients. They are particularly prominent in patients with diabetes, and their relative numbers are set to increase with the rise of diabetes within our population. Research is still needed to understand the factors leading to such wounds, to understand why they persist for such long periods of time, and also to develop new and efficacious treatment strategies. One problem facing this research is a lack of adequate animal models, as the current models do not truly reflect the human condition and often lead to much animal suffering. Hence, over the past four years, our group has been trying to develop a human-based in vitro diabetic wound model, which could be used as a high-throughput screening system to pre-screen potential chronic diabetic wound healing agents and to reduce unnecessary animal pain and suffering. To this end, we have isolated healthy and diseased skin fibroblasts from patient tissue biopsies. Crucially, to create a cell reporter system that can be widely used in the future, the cells were immortalised in order to escape senescence. By using microarray analysis, gene expression pattern differences have been identified between healthy and diseased cells, and disease-specific ‘reporter’ genes have been selected for further studies. The promoters of these reporter genes have been coupled to fluorescent reporter constructs and inserted back into the diseased fibroblasts, so that we now have proof-of-concept for a real-time diabetic reporter system for future exploitation.
Integrated Testing Strategies for Toxicity Employing New and Existing Technologies
Authors: Combes Robert D. , Balls Michael
Published: April 2019

We have developed individual, integrated testing strategies (ITS) for predicting the toxicity of general chemicals, cosmetics, pharmaceuticals, inhaled chemicals, and nanoparticles. These ITS are based on published schemes developed previously for the risk assessment of chemicals to fulfil the requirements of REACH, which have been updated to take account of the latest developments in advanced in chemico modelling and in vitro technologies. In addition, we propose an ITS for neurotoxicity, based on the same principles, for incorporation in the other ITS. The technologies are deployed in a step-wise manner, as a basis for decision-tree approaches, incorporating weight-of-evidence stages. This means that testing can be stopped at the point where a risk assessment and/or classification can be performed, with labelling in accordance with the requirements of the regulatory authority concerned, rather than following a checklist approach to hazard identification. In addition, the strategies are intelligent, in that they are based on the fundamental premise that there is no hazard in the absence of exposure — which is why pharmacokinetic modelling plays a key role in each ITS. The new technologies include the use of complex, three-dimensional human cell tissue culture systems with in vivo-like structural, physiological and biochemical features, as well as dosing conditions. In this way, problems of inter-species extrapolation and in vitro/ in vivo extrapolation are minimised. This is reflected in the ITS placing more emphasis on the use of volunteers at the whole organism testing stage, rather than on existing animal testing, which is the current situation.
Refinement and Reduction of Acute Oral Toxicity Testing: A Critical Review of the Use of Cytotoxicity Data
Authors: Schrage Arnhild , Hempel Katja , Schulz Markus , Kolle Susanne N. , van Ravenzwaay Bennard , Landsiedel Robert
Published: April 2019

Acute oral toxicity testing is still required for the classification and labelling of chemicals, agrochemicals and related formulations. There have been increasing efforts over the last two decades to reduce the number of animals needed for this testing, according to the Three Rs concept. To evaluate the utility of an in vitro cytotoxicity test in our routine testing for acute oral toxicity, we have implemented in our laboratory the neutral red uptake (NRU) method, with Balb/c 3T3 fibroblasts after a 48-hour exposure, which was recommended in ICCVAM Report 07-4519, 2006. Initially, we tested 16 substances that had existing in vivo and in vitro data available, to prove our technical proficiency with the in vitro test. Then, testing was performed with 187 test substances, including a broad variety of chemicals, agrochemicals and formulations. The starting dose for acute oral systemic toxicity assays in rats (LD50) was estimated by using the prediction model presented in the ICCVAM validation study, and subsequently compared to the results obtained by in vivo testing performed according to, or similar to, OECD Test Guideline 423. Comparison of all of the 203 predicted LD50 values that were deduced from the in vitro IC50 values, with the in vivo results from oral toxicity studies in rats, resulted in a low overall concordance of 35%. The in vitro cytotoxicity assay achieved a good concordance of 74%, only for the weakly toxic substances (EU-GHS Cat. 4). However, it must be noted that 71% of the substances tested (i.e. 145/203) were classified as being weakly toxic in vitro. We further analysed the utility of the in vitro test for predicting the starting dose for an in vivo study, and the potential reduction in animal usage that this would engender. In this regard, the prediction by the cytotoxicity test was useful for 59% of the substances. However, the use of a standard starting dose of 300mg/kg bw by default (without previous cytotoxicity testing) would have been almost as useful (50%). In contrast, the prediction by an experienced toxicologist was correct for 95% of the substances. However, this was only performed for 40% of the substances, mainly those of no to low toxicity. Calculating the theoretical animal numbers needed in several scenarios supported these results. The additional analysis, considering some physicochemical data (solubility, molecular weight, log POW), substance class and mode of action, revealed no specific applicability domains. In summary, the use of the 3T3 NRU cytotoxicity data alone did not sufficiently contribute to refinement and reduction in the acute oral toxicity testing of the substance portfolio tested routinely in our laboratory.
In-house Validation of the EpiOcular™ Eye Irritation Test and its Combination with the Bovine Corneal Opacity and Permeability Test for the Assessment of Ocular Irritation
Authors: Kolle Susanne N. , Kandárová Helena , Wareing Britta , van Ravenzwaay Bennard , Landsiedel Robert
Published: April 2019

In 2009, the Bovine Corneal Opacity and Permeability (BCOP) test was accepted by the regulatory bodies for the identification of corrosive and severe ocular irritants (Global Harmonised System [GHS] Category 1). However, no in vitro test is currently accepted for the differentiation of ocular irritants (GHS Category 2) and non-irritants (GHS No Category). Human reconstructed tissue models have been suggested for incorporation into a tiered testing strategy to ultimately replace the Draize rabbit eye irritation test (OECD TG 405). The purpose of this study was to evaluate whether the EpiOcular™ reconstructed cornealike tissue model and the COLIPA pre-validated EpiOcular Eye Irritation Test (EpiOcular-EIT) could be used as suitable components of this testing strategy. The in-house validation of the EpiOcular-EIT was performed by using 60 test substances, including a broad variety of chemicals and formulations for which in vivo data (from the Draize rabbit eye irritation test) were available. The test substances fell into the following categories: 18 severe irritants/corrosives (Category 1), 21 irritants (Category 2), and 21 non-irritants (No Category). Test substances that decreased tissue viability to = 60% (compared to the negative control tissue) were considered to be eye irritants (Category 1/2). Test substances resulting in tissue viability of > 60% were considered to be non-irritants (No Category). For the assessed dataset and the classification cut-off of 60% viability, the EpiOcular-EIT provided 98% and 84% sensitivity, 64% and 90% specificity, and 85% and 86% overall accuracy for the literature reference and BASF proprietary substances, respectively. Applying a 50% tissue viability cut-off to distinguish between irritants and non-irritants resulted in 93% and 82% sensitivity, 68% and 100% specificity, and 84% and 88% accuracy for the literature reference and BASF proprietary substances, respectively. Further, in the EpiOcular-EIT (60% cut-off), 100% of severely irritating substances under-predicted by the BCOP assay were classified as Category 1/2. The results obtained in this study, based on 60 test substances, indicate that the EpiOcular-EIT and the BCOP assay can be combined in a testing strategy to identify strong/severe eye irritants (Category 1), moderate and mild eye irritants (Category 2), and non-irritants (No Category) in routine testing. In particular, when the bottom-up strategy with the 60% viability cut-off was employed, none of the severely irritating substances (Category 1) were under-predicted to be non-irritant. Sensitivity for Category 1/2 substances was 100% for literature reference substances and 89% for BASF SE proprietary substances.
Cell Transformation Assays: Are we Barking up the Wrong Tree?
Authors: Combes Robert D.
Published: April 2019

There has been a current resurgence of interest in the use of cell transformation for predicting carcinogenicity, which is based mainly on rodent carcinogenicity data. In view of this renewed interest, this paper critically reviews the published literature concerning the ability of the available assays to detect IARC Group 1 agents (known human carcinogens) and Group 2A agents (probable human carcinogens). The predictivity of the available assays for human and rodent non-genotoxic carcinogens (NGCs), in comparison with standard and supplementary in vitro and in vivo genotoxicity tests, is also discussed. The principal finding is that a surprising number of human carcinogens have not been tested for cell transformation across the three main assays (SHE, Balb/c 3T3 and C3H10T1/2), confounding comparative assessment of these methods for detecting human carcinogens. This issue is not being addressed in the ongoing validation studies for the first two of these assays, despite the lack of any serious logistical issues associated with the use of most of these chemicals. In addition, there seem to be no plans for using exogenous bio-transformation systems for the metabolic activation of pro-carcinogens, as recommended in an ECVAM workshop held in 1999. To address these important issues, it is strongly recommended that consideration be given to the inclusion of more human carcinogens and an exogenous source of xenobiotic metabolism, such as an S9 fraction, in ongoing and future validation studies. While cell transformation systems detect a high level of NGCs, it is considered premature to rely only on this endpoint for screening for such chemicals, as recently suggested. This is particularly important, in view of the fact that there is still doubt as to the relevance of morphological transformation to tumorigenesis in vivo, and the wide diversity of potential mechanisms by which NGCs are known to act. Recent progress with regard to increasing the objectivity of scoring the transformed phenotype, and prospects for developing human cell-based transformation assays, are reviewed.
Statistics of Scientific Procedures on Living Animals 2011: Another Increase in Experimentation, but is there a Shift in Emphasis?
Authors: Hudson-Shore Michelle
Published: April 2019

The 2011 Statistics of Scientific Procedures on Living Animals reveal that the level of animal experimentation in Great Britain continues to rise, with almost 3.8 million procedures being conducted. Unlike those in previous years, this increase is not exclusively due to the breeding and utilisation of genetically altered animals, although they are still involved in the greatest proportion of procedures. That a shift toward fundamental research may have become the primary cause of increases in animal experiments is discussed. The general trends in the species used, and the numbers and types of procedures, are reviewed. In addition, some areas of concern and optimism are outlined.
FRAME and the Pharmaceutical Industry
Authors: Balls Michael
Published: April 2019

Greater realism and new initiatives promise significant benefits for patients — and for laboratory animals
Training Needs for Toxicity Testing in the 21st Century: A Survey-informed Analysis
Authors: Lapenna Silvia , Gabbert Silke , Worth Andrew
Published: April 2019

Current training needs on the use of alternative methods in predictive toxicology, including new approaches based on mode-of-action (MoA) and adverse outcome pathway (AOP) concepts, are expected to evolve rapidly. In order to gain insight into stakeholder preferences for training, the European Commission's Joint Research Centre (JRC) conducted a single-question survey with twelve experts in regulatory agencies, industry, national research organisations, NGOs and consultancies. Stakeholder responses were evaluated by means of theory-based qualitative data analysis. Overall, a set of training topics were identified that relate both to general background information and to guidance for applying alternative testing methods. In particular, for the use of in silico methods, stakeholders emphasised the need for training on data integration and evaluation, in order to increase confidence in applying these methods for regulatory purposes. Although the survey does not claim to offer an exhaustive overview of the training requirements, its findings support the conclusion that the development of well-targeted and tailor-made training opportunities that inform about the usefulness of alternative methods, in particular those that offer practical experience in the application of in silico methods, deserves more attention. This should be complemented by transparent information and guidance on the interpretation of the results generated by these methods and software tools.
The Declaration on Openness on Animal Research
Authors: Balls Michael
Published: April 2019

Is this a genuine commitment to honest and open dialogue, or just another propaganda exercise?
Trends in Animal Use and Replacement in the ‘Three Is’ of Industry
Authors: Menache André
Published: April 2019

The chemical industry has much to learn from the cosmetics and pharmaceutical industries about the replacement of animal tests
Taking Veterinary Anatomy Online
Authors: Gaitskell-Phillips Gemma , Short Nick , Stanikova Bara
Published: April 2019

The Online Veterinary Anatomy Museum (OVAM) website will help to change anatomy teaching and the way students learn
Alternative Models to Study the Intestinal Barrier Function of Piglets
Authors: De Vos Maartje , Huygelen Véronique , Casteleyn Christophe , Van Cruchten Steven , Van Ginneken Chris
Published: April 2019

Various methods are available for reducing or replacing in vivo studies on intestinal barrier function in piglets
Experimental Assessment of the Environmental Fate and Effects of Triazoles and Benzotriazole
Authors: Durjava Mojca Kos , Kolar Boris , Arnus Lovro , Papa Ester , Kovarich Simona , Sahlin Ullrika , Peijnenburg Willie
Published: April 2019

The environmental fate and effects of triazoles and benzotriazoles are of concern within the context of chemical regulation. As part of an intelligent testing strategy, experimental tests were performed on endpoints that are relevant for risk assessment. The experimental tests included the assessment of ecotoxicity to an alga, a daphnid and zebrafish embryos, and the assessment of ready biodegradability. Triazole and benzotriazole compounds were selected for testing, based on existing toxicity data for vertebrate and invertebrate species, as well as on the principal component analysis of molecular descriptors aimed at selecting the minimum number of test compounds in order to maximise the chemical domain spanned for both compound classes. The experimental results show that variation in the toxicities of triazoles and benzotriazole across species was relatively minor; in general, the largest factor was approximately 20. The study conducted indicated that triazoles are not readily biodegradable.