bluuebunny/crossref_metadata_2025 huggingface dataset:
Species Sensitivity Distribution Estimation from Uncertain (QSAR-based) Effects Data
Authors: Aldenberg Tom , Rorije Emiel
Published: April 2019

In environmental risk assessment, Species Sensitivity Distributions (SSDs) can be applied to estimate a PNEC (Predicted No-Effect Concentration) for a chemical substance, when sufficient data on species toxicities are available. The European Chemicals Agency (ECHA) recommendation is 10 biological species. The question addressed in this paper, is whether QSAR-predicted toxicities can be included in SSD-based PNEC estimates, and whether any modifications need to be made to account for the uncertainty in the QSAR-model estimates. This problem is addressed from a probabilistic modelling point of view. From classical analysis of variation (ANOVA), we review how the error-in-data SSD problem is similar to separation into between-group and within-group variance. ECHA guidance suggests averaging similar endpoint data for a species, which is consistent with group means, as in ANOVA. This exercise reveals that error-in-data reduces the estimation of the between species variation, i.e. the SSD variance, rather than enlarging it. A Bayesian analysis permits the assessment of the uncertainty of the SSD mean and variance parameters for given values of mean species toxicity error. This requires a hierarchical model. Prototyping this model for an artificial five-species data set seems to suggest that the influence of data error is relatively minor. Moreover, when neglecting this data error, a slightly conservative estimate of the SSD results. Hence, we suggest including (model-predicted) data as model point estimates and handling the SSD as usual. The Bayesian simulation of the error-in-data SSD leads to predictive distributions, being an average of posterior spaghetti plot densities or cumulative distributions. We derive new predictive extrapolation constants with several improvements over previous median uncertainty log10HC5 estimates, in that they are easily calculable from spreadsheet Student -t functions and based on a more realistic uniform prior for the SSD standard deviation. Other advantages are that they are single-number extrapolation constants and they are more sensitive to small sample size.
From Descriptors to Predicted Properties: Experimental Design by Using Applicability Domain Estimation
Authors: Brandmaier Stefan , Novotarskyi Sergii , Sushko Iurii , Tetko Igor V.
Published: April 2019

The importance of reliable methods for representative sub-sampling in terms of experimental design and risk assessment within the European Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system is crucial. We developed experimental design approaches, by utilising predicted properties and the ‘distance to model’ parameter, to estimate the benefits of certain compounds to the quality of a resulting model. A statistical evaluation of four regression data sets and one classification data set showed that the adaptive concept of iteratively refining the representation of the chemical space contributes to a more efficient and more reliable selection in comparison to traditional approaches. The evaluation of compounds with regard to the uncertainty and the correlation of prediction is beneficial, and in particular, for regression data sets of sufficient size, whereas the use of predicted properties to define the chemical space is beneficial for classification models.
Uncertainty in QSAR Predictions
Authors: Sahlin Ullrika
Published: April 2019

It is relevant to consider uncertainty in individual predictions when quantitative structure–activity (or property) relationships (QSARs) are used to support decisions of high societal concern. Successful communication of uncertainty in the integration of QSARs in chemical safety assessment under the EU Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) system can be facilitated by a common understanding of how to define, characterise, assess and evaluate uncertainty in QSAR predictions. A QSAR prediction is, compared to experimental estimates, subject to added uncertainty that comes from the use of a model instead of empirically-based estimates. A framework is provided to aid the distinction between different types of uncertainty in a QSAR prediction: quantitative, i.e. for regressions related to the error in a prediction and characterised by a predictive distribution; and qualitative, by expressing our confidence in the model for predicting a particular compound based on a quantitative measure of predictive reliability. It is possible to assess a quantitative (i.e. probabilistic) predictive distribution, given the supervised learning algorithm, the underlying QSAR data, a probability model for uncertainty and a statistical principle for inference. The integration of QSARs into risk assessment may be facilitated by the inclusion of the assessment of predictive error and predictive reliability into the “unambiguous algorithm”, as outlined in the second OECD principle.
3. Fidelity and Discrimination
Authors: Balls Michael
Published: April 2019

Progress in replacement has been restricted by certain plausible, but untenable, assumptions, summed up as the high fidelity fallacy
Comments and Feedback
Authors: Melissa
Published: April 2019

Will PiLAS be indexed in Medline/PubMed? Also, it looks as if all the articles in PiLAS are just reprints of articles in ATLA — is that going to be typical or is the hope that different articles will be in PiLAS?
Learning to be Humane
Authors: Hendriksen Coenraad , Boumans Iris
Published: April 2019

More effort should be put into reducing severe pain and distress in animal experiments by applying more-humane endpoints
Refinement: Lessons from the 2012 Olympics
Authors: Lilley Elliot , Jennings Maggy
Published: April 2019

Until the day comes when animals are no longer needed for research, refinement needs to be a priority for the scientific community
Balancing Reduction and Refinement
Authors: Fenwick Nicole , Griffin Gilly
Published: April 2019

By considering reduction before refinement, we risk allowing some animals to experience greater pain and distress. A variety of approaches can help balance reduction and refinement while promoting the welfare of individual animals
5. Refinement
Authors: Balls Michael
Published: April 2019

The freedom of choice of the experimenter is often very much wider than it at first appears. The full use of this freedom is the mark alike of humane and successful experimentation. There is, perhaps, no limit in animal experimentation to the progress of refinement
Ethical Review of Projects Involving Non-human Primates Funded under the European Union's 7th Research Framework Programme
Authors: Sauer Ursula G. , Phillips Barry , Reid Kirsty , Schmit Véronique , Jennings Maggy
Published: April 2019

Internet searches were performed on projects involving non-human primates (‘primates’) funded under the European Union (EU) 7th Research Framework Programme (FP7), to determine how project proposals are assessed from an ethical point of view. Due to the incompleteness of the information publicly available, the types and severity of the experiments could not be determined with certainty, although in some projects the level of harm was considered to be ‘severe’. Information was scarce regarding the numbers of primates, their sourcing, housing, care and fate, or the application of the Three Rs within projects. Project grant holders and the relevant Commission officer were consulted about their experiences with the FP7 ethics review process. Overall, it was seen as meaningful and beneficial, but some concerns were also noted. Ethical follow-up during project performance and upon completion was recognised as a valuable tool in ensuring that animal welfare requirements were adequately addressed. Based upon the outcome of the survey, recommendations are presented on how to strengthen the ethical review process under the upcoming Framework Programme ‘Horizon 2020’, while adequately taking into account the specific requirements of Directive 2010/63/EU, with the aim of limiting the harms inflicted on the animals and the numbers used, and ultimately, replacing the use of primates altogether.
The Three Rs in India
Authors: Sankar Palanisamy , Kandasamy Kannan
Published: April 2019

An overview of the progress being made in implementing alternatives to animal experiments in India
The Current Status of Laboratory Animal Ethics in South Africa
Authors: Mohr Bert
Published: April 2019

An overview of the successes and current challenges in implementing and raising awareness of the Three Rs in South Africa
A Phantom Pig Abdomen as an Alternative for Testing Robotic Surgical Systems: Our Experience
Authors: Ristolainen Asko , Colucci Gianluca , Kruusmaa Maarja
Published: April 2019

The use of animals for testing and validating new medical devices and surgical techniques has raised ethical issues for a long time. Following the introduction of the Three Rs principle, significant efforts have been made to achieve a reduction in the numbers of animals used in testing. Nevertheless, the number of large animals used for testing purposes is still too high. This article describes a potential alternative to the use of large animals in the early phase of the development of surgical equipment — a high-definition phantom pig abdomen. The phantom pig abdomen was developed from computed tomography scans by using affordable materials, and it was used with two different robotic platforms. It permitted the testing of minimally-invasive robotic pancreatic enucleation, with or without intraoperative ultrasound guidance. The phantom pig abdomen has proven to be a realistic tool, with the potential to reduce the cost and time-frame of the experiments.
Validation of the Hepatocyte-like HPCT-1E3 Cell Line as an In Vitro Model for the Prediction of Acute In Vivo Toxicity
Authors: Halwachs Sandra , Lakoma Cathleen , Honscha Walther
Published: April 2019

In a pilot study, we tested 20 randomly-selected chemicals for their cytotoxicity toward the HPCT-1E3 cell model, in order to prove the ability of this in vitro model to predict human acute in vivo toxicity. The study revealed that, in contrast to most other in vitro models, results from the HPCT-1E3 cell-based system show better correlation with the more-relevant human acute lethal doses, whereas results from most other systems have a high predictivity for human lethal serum concentrations. For the prevalidation of the HPCT-1E3 model as a surrogate for regulatory acute in vivo toxicity tests, we have now expanded the list of tested chemicals to 57 substances, and have compared the results with data from the HepG2 cell assay. Again, a better correlation of HPCT-1E3 IC50 values with human oral lethal doses, as compared to correlation with human lethal serum concentrations, was observed after the pooling of all the tested substances ( r2 = 0.53 [ P < 0.001] and r2 = 0.41 [ P = 0.009], respectively). Therefore, the HPCT-1E3 in vitro model may be a valuable tool for prediction of human oral toxicity, and may help to further reduce the number of animals used for in vivo toxicity tests.
7. The Factors Governing Progress
Authors: Balls Michael
Published: April 2019

Progress toward implementation of the Three Rs is affected by a complexity of personality and sociological factors, recognised in The Principles in 1959, but no less effective in the 21st century
New Techniques for Producing Transgenic Animals — a Mixed Blessing from Both the Scientific and Animal Welfare Perspectives
Authors: Hendriksen Coenraad , Spielmann Horst
Published: April 2019

A further increase in the use of genetically-modified animals will be against the European Union policy to move away from the use of animals in biomedical research
Perceived Barriers to the Adoption of Alternatives to Laboratory Animal Use for Rabies Diagnosis
Authors: Bones Vanessa C. , Clemente Heloísa C. , Weary Daniel M. , Molento Carla F.M.
Published: April 2019

The use of laboratory animals is still common practice, but some uses can be replaced by alternative methods, such as Virus Isolation in Cell Culture (VICC) instead of the Mouse Inoculation Test (MIT) for rabies diagnosis. The objective of this work was to describe current rabies diagnosis methods in Brazil and other countries, and the constraints associated with replacing this use of mice with alternative methods. Nine out of 12 Brazilian and 14 out of 43 non-Brazilian respondents reported that they currently used the MIT. Respondents in countries other than Brazil, male respondents, and those already employing in vitro methods for rabies diagnosis, expressed higher levels of support for the use of alternatives. The most frequently reported constraints associated with the use of alternatives were lack of laboratory facilities, equipment and materials (cited 17 times by respondents), and lack of financial resources (cited 15 times). The results indicate that many laboratories continue to use mice for rabies diagnosis. The proportion of laboratories that use mice appears to be especially high in Brazil, despite animal protection laws and technical guidelines that favour the use of alternatives. The barriers to the adoption of alternative methods identified in the current study provide a basis for facilitating changes in Brazil and elsewhere.
An Analysis of the Use of Animal Models in Predicting Human Toxicology and Drug Safety
Authors: Bailey Jarrod , Thew Michelle , Balls Michael
Published: April 2019

Animal use continues to be central to preclinical drug development, in spite of a lack of its demonstrable validity. The current nadir of new drug approvals and the drying-up of pipelines may be a direct consequence of this. To estimate the evidential weight given by animal data to the probability that a new drug may be toxic to humans, we have calculated Likelihood Ratios (LRs) for an extensive data set of 2,366 drugs, for which both animal and human data are available, including tissue-level effects and MedDRA Level 1–4 biomedical observations. This was done for three preclinical species (rat, mouse and rabbit), to augment our previously-published analysis of canine data. In common with our dog analysis, the resulting LRs show: a) that the absence of toxicity in the animal provides little or virtually no evidential weight that adverse drug reactions (ADRs) will also be absent in humans; and b) that, while the presence of toxicity in these species can add considerable evidential weight for human risk, the LRs are extremely inconsistent, varying by over two orders of magnitude for different classes of compounds and their effects. Therefore, our results for these additional preclinical species have important implications for their use in predicting human toxicity, and suggest that alternative methods are urgently required.
Development of a Training Model for Small Animal Thoracocentesis and Chest Tube Thoracostomy
Authors: Williamson Julie A. , Rito Robin M. Fio
Published: April 2019

Training veterinary students to perform emergency procedures, such as thoracocentesis and chest tube thoracostomy, poses challenges in terms of providing adequate hands-on experience without compromising animal welfare. A small animal thoracocentesis and chest tube thoracostomy model has been developed, that allows repetitive practice in a safe, standardised environment. The model has been incorporated into a clinical skills laboratory, where students work through computerised case studies in small groups, performing thoracocentesis or chest tube thoracostomy where indicated during the case. Student feedback indicated a high degree of satisfaction with the model and the laboratory experience, high perceived value of the case studies in improving learning, and increased confidence to perform the procedures under supervision. This model can replace the use of live animals while students are practising these procedures, improving their technique, and learning the appropriate safeguards used to prevent injuries such as pulmonary trauma.
Missing, Presumed Killed in Action (or the Report that Never Was)
Authors: Combes Robert D.
Published: April 2019

The Home Office Inspectorate's Report, which stems from concerns raised from the undercover BUAV investigation at Imperial College London, has so far not been published. This current lack of transparency by the UK Government is completely unacceptable.
Animal Experimentation and Alternatives: Time to Say Goodbye to the Three Rs and Hello to Humanity?
Authors: Balls Michael
Published: April 2019

The time has come to plan for a future where the Three Rs will have served their purpose, animal experimentation will have been consigned to history, and humane biomedical science in research, testing and education will have become the norm, for the benefit of humans and animals alike
Wider Recommendations for Institutions made in the Brown Report following the BUAV Investigation into the Use of Animals at Imperial College London
Authors: Taylor Katy , Balls Michael
Published: April 2019

In December 2013, a group of experts produced a report on the management of an animal unit at Imperial College London, following a BUAV investigation that found evidence of systematic failures in the care and monitoring of animals used in procedures there. The Brown Report looked at four areas: the animal welfare and ethical review body (AWERB); the operation of the unit; training; and overall culture. The report made 33 recommendations to improve practices at Imperial College, many of which were relevant to other institutions. In this report, we identify the recommendations that are applicable to all animal facilities, and redraft them as a checklist with supporting information to assist those reviewing their animal care policies. We support the Brown Report's recommendation that institutions should have a vision statement and an action plan, as well as a ‘champion’ for the Three Rs. We encourage all institutions that use animals to, as a first step, review the performance of their animal units against this checklist.
A Three Rs Perspective on the Mouse Bioassay in Routine Seafood Safety Testing for Algal Biotoxins — 1: Replacement
Authors: Stewart Ian , McLeod Catherine
Published: April 2019

Countries that have discontinued routine mouse bioassay testing might gain a market advantage in domestic and export seafood products over countries that continue to use the mouse test
A Step Forward in the Quality Control Testing of Inactivated Rabies Vaccines — Extensive Evaluation of European Vaccines by Using Alternative Methods to the In Vivo Potency Tests
Authors: Servat Alexandre , Kempff Sébastien , Brogat Valère , Litaize Estelle , Schereffer Jean-Luc , Cliquet Florence
Published: April 2019

The mouse challenge test still remains the reference method for the potency determination of human and animal inactivated rabies vaccines, and it is still widely used throughout the world. This test suffers from many disadvantages — it is expensive and time consuming, uses a large number of mice, causes significant animal distress, and suffers from high variability. Recently, the European Pharmacopoeia has recognised the use of a serological potency assay (SPA) as an alternative method to the challenge test. This new test is based on the determination of rabies neutralising antibody titres in vaccinated mice, by using the modified Rapid Fluorescent Focus Inhibition Test (mRFFIT). With the objective of adopting this new method for the batch release of inactivated rabies vaccines, we evaluated its performance on a large collection of rabies vaccines currently assessed in our laboratory. The Fluorescent Antibody Virus Neutralisation test (FAVNt) was used in parallel with the mRFFIT, and the results were compared to the mouse challenge test. Our results demonstrate that the SPA is capable of estimating the potency of vaccines formulated with a potency margin well above the minimum of 1IU/dose. For low potency vaccines, this new method demonstrated some limitations, due to the recurrent invalidation of the assay. We have also demonstrated the superior sensitivity of the FAVNt when compared to the mRFFIT, and the importance of minimising the risk of detecting non-responders in vaccinated mice.
The Cost of Standing Strong for Replacement
Authors: Brown Katy
Published: April 2019

The testimonies of these individuals largely speak for themselves. The responses point to the importance of specific institutions or research groups that focus on the development and use of alternatives, and these should, of course, be better supported. Those who find themselves outside such institutions or teams, are more likely to feel stranded and isolated. Then again, Liz did have the support of a research group dedicated to replacement, but she has still had a significant struggle to find funding. The interviews with some of these particular young researchers indeed pointed toward a tangible ‘cost’ in terms of having to steer their career on the often difficult path toward the use of non-animal based methods.
To be Credible, Success in “Reducing the use of Animals in Scientific Research” Must Involve the use of Fewer Animals
Authors: Balls Michael
Published: April 2019

Much worthy effort on the Three Rs is under way in the UK, but its promise of a reduction in laboratory animal use will only be credible and laudable when the number of procedures and animals are progressively and permanently reduced
Optimisation of the Bovine Whole In Vitro Embryo System as a Sentinel for Toxicity Screening: A Cadmium Challenge
Authors: Jorssen Ellen P.A. , Vergauwen Lucia , Goossens Karen , Hagenaars An , Van Poucke Mario , Petro Evi , Peelman Luc , Knapen Dries , Leroy Jo L.M.R. , Bols Peter E.J.
Published: April 2019

Developmental toxicity testing could greatly benefit from the availability of an in vitro alternative model based on the use of animal embryos that have better human-like physiology than the currently-used alternative models. These current models are insufficient, as extrapolation of the results can be challenging. Therefore, an in vitro bovine embryo culture system was used to expose individual morulae to test substances, and to study developmental characteristics up to the blastocyst stage. Cadmium was chosen as the reference toxicant to investigate the sensitivity of the bovine morulae to various concentrations and exposure times. Oocytes from slaughterhouse-obtained bovine ovaries, were maturated, fertilised and cultured up until the morula stage. Morulae were exposed to different cadmium concentrations for 18 or 70 hours, and developmental competence, embryo quality and the expression of cadmium exposure-related genes were evaluated. Cadmium exposure hampered embryonic developmental competence and quality. Compared with the 18-hour exposure, the 70-hour exposure induced a 20-fold higher toxic response with regard to developmental competence and a more ‘cadmium-typical’ transcript expression. The bovine morula might be a promising tool for toxicity testing as, following exposure, the embryos reacted in a sensitive and ‘cadmium-typical’ manner to our reference toxicant.
How Long Must They Suffer? Success and Failure of our Efforts to End the Animal Tragedy in Laboratories
Authors: Kolar Roman
Published: April 2019

Scientific findings have revealed how much we have dramatically underestimated the intellectual, social and emotional capabilities of non-human animals, including their levels of self-consciousness and ability to suffer from psychological stress. In the 21st century, the field of animal ethics has evolved as a serious scientific discipline, and nowadays largely advocates that the way we treat animals, both legally and in practice, is morally wrong. Politics and legislation have reacted to these facts, to some extent, but neither current legislation nor current practice reflect the scientific and moral state-of-the-art. Too often, the will to change things is watered down in the decision-making process, e.g. in the drafting of legislation. In the field of animal experimentation there have been many genuine efforts by various players, to advance and apply the principles behind the Three Rs. However, the fundamental problem, i.e. the overall number of animals sacrificed for scientific purposes, has increased. Clearly, if we are serious about our will to regard animal experimentation as an ethical and societal problem, we have to put much more emphasis on addressing the question of how to avoid the use of animals in science. To achieve this goal, certain issues need to be considered: a) the present system of ethical evaluation of animal experiments, including testing for regulatory purposes, needs to be reformed and applied effectively to meet the legal and moral requirements; b) animal testing must be avoided in future legislation, and existing legislation has to be revised in that regard; c) resources from animal-based research have to re-allocated toward alternatives; and d) the academic curricula must be reformed to foster and integrate ethical and animal welfare issues.
In Vitro Methodologies in Ecotoxicological Hazard Assessment: The Case of Bioaccumulation Testing for Fish
Authors: Segner Helmut
Published: April 2019

The concerted research efforts undertaken in recent years have highlighted the potential of in vitro approaches, as part of an integrated testing strategy, to replace or reduce in vivo bioaccumulation testing in fish
16. The Three Rs: The Way Forward
Authors: Balls Michael
Published: April 2019

It is now 20 years since Russell and Burch last met to discuss, with others, the way forward for the Three Rs concept
Peer Review and the Three Rs: A Challenge to Integrity?
Authors: Balls Michael
Published: April 2019

Peer review is undoubtedly an imperfect process, but there is no other acceptable way of attempting to ensure the highest standards in research and the publication of opinions and results
Summary and Analysis of the Currently Existing Literature Data on Metal-based Nanoparticles Published for Selected Aquatic Organisms: Applicability for Toxicity Prediction by (Q)SARs
Authors: Chen Guangchao , Vijver Martina G. , Peijnenburg Willie J.G.M.
Published: April 2019

This review establishes an inventory of existing toxicity data on nanoparticles (NPs) with the purpose of developing (Quantitative) Structure–Activity Relationships for NPs (nano-[Q]SARs), and also of maximising the use of scientific sources for NP risk assessment. From a data search carried out on 27 February 2014, a total of 910 publications were retrieved from the Web of Science™ Core Collection, and a database comprising 886 records of toxicity endpoints, based on these publications, was built. The test organisms mainly comprised bacteria, algae, yeast, protozoa, nematoda, crustacea and fish. The NPs consisted mostly of metals, metal oxides, nanocomposites and quantum dots. The data were analysed further, in order to: a) categorise each toxicity endpoint and the biological effects triggered by the NPs; b) survey the characterisation of the NPs used; and c) assess whether the data were suitable for nano-(Q)SAR development. Despite the efforts of numerous scientific programmes on nanomaterial safety and design, our study concluded that lack of data consistency prevents the use of experimental data in developing and validating nano-(Q)SARs. Finally, an outlook on the future of nano-(Q)SAR development is provided.
The Use of 3-D Models as Alternatives to Animal Testing
Authors: Kojima Hajime
Published: April 2019

A number of three-dimensional in vitro models are now available, but significant further developments are needed before their routine and widespread use as alternatives to animal testing will be possible
Development and Validation of a Low-fidelity Simulator to Suture a Laparotomy in Rabbits
Authors: Pérez-Rivero Juan J. , Batalla-Vera Tonantzin , Rendón-Franco Emilio
Published: April 2019

An easily constructed, low-cost simulator is assessed for its efficacy in the surgical training of veterinary science undergraduates
18. Rationalisation and Intellectualisation
Authors: Balls Michael
Published: April 2019

Russell and Burch saw failure to accept the correlation between humanity and efficacy as an example of rationalisation, a psychological defence mechanism
Do E-cigarettes Pose a Risk to Human Health?
Authors: Kenna Gerry
Published: April 2019

The claim by Public Health England that e-cigarettes have no concerning adverse health effects is flawed, because it is not evidence based
Predicting Human Drug Toxicity and Safety via Animal Tests: Can Any One Species Predict Drug Toxicity in Any Other, and Do Monkeys Help?
Authors: Bailey Jarrod , Thew Michelle , Balls Michael
Published: April 2019

Animals are still widely used in drug development and safety tests, despite evidence for their lack of predictive value. In this regard, we recently showed, by producing Likelihood Ratios (LRs) for an extensive data set of over 3,000 drugs with both animal and human data, that the absence of toxicity in animals provides little or virtually no evidential weight that adverse drug reactions will also be absent in humans. While our analyses suggest that the presence of toxicity in one species may sometimes add evidential weight for risk of toxicity in another, the LRs are extremely inconsistent, varying substantially for different classes of drugs. Here, we present further data from analyses of other species pairs, including nonhuman primates (NHPs), which support our previous conclusions, and also show in particular that test results inferring an absence of toxicity in one species provide no evidential weight with regard to toxicity in any other species, even when data from NHPs and humans are compared. Our results for species including humans, NHPs, dogs, mice, rabbits, and rats, have major implications for the value of animal tests in predicting human toxicity, and demand that human-focused alternative methods are adopted in their place as a matter of urgency.
Scandinavia and the Replacement of in Vivo Toxicity Tests: Some Personal Reflections
Authors: Balls Michael
Published: April 2019

A personal, and therefore unavoidably biased, review is given, of the significance of the contributions made by selected Scandinavian individuals, organisations and events, to the development of in vitro toxicology procedures as potential replacements for toxicity tests in laboratory animals. In addition to their wider significance, these contributions had a profound effect on whatever contributions I have been able to make, myself. Nevertheless, while there has been much progress in the last 35 years or so, and many lessons have been learned, there is still much to be done, especially as animal tests remain entrenched as the preferred methods which set the gold standards and make regulators feel comfortable. Many of the clues to dealing with the questions and concerns which plague hazard prediction and risk assessment have long been available, but they have been ignored, largely for reasons which have little to do with the science of toxicology and the need to maintain the highest scientific standards. I have little doubt that Björn Ekwall, whose memory I feel privileged to honour, would have agreed with that last statement.
Young Researchers — The Ethical Challenge
Authors: Ram Rebecca
Published: April 2019

Never before has there been a better opportunity for young researchers to focus on replacement alternatives, not only to save animals from unnecessary pain and suffering, but also to pave the way for a career in cutting-edge innovation
A Comparative Evaluation of Six Principal IgY Antibody Extraction Methods
Authors: Ren Hao , Yang Wenjing , Thirumalai Diraviyam , Zhang Xiaoying , Schade Rüdiger
Published: April 2019

Egg yolk has been considered a promising source of antibodies. Our study was designed to compare six principal IgY extraction methods (water dilution, polyethylene glycol [PEG] precipitation, caprylic acid extraction, chloroform extraction, phenol extraction, and carrageenan extraction), and to assess their relative extraction efficiencies and the purity of the resulting antibodies. The results showed that the organic solvents (chloroform or phenol) minimised the lipid ratio in the egg yolk. The water dilution, PEG precipitation and caprylic acid extraction methods resulted in high yields, and antibodies purified with PEG and carrageenan exhibited high purity. Our results indicate that phenol extraction would be more suitable for preparing high concentrations of IgY for non-therapeutic usage, while the water dilution and carrageenan extraction methods would be more appropriate for use in the preparation of IgY for oral administration.
The Use of In Vivo, Ex Vivo, In Vitro, Computational Models and Volunteer Studies in Vision Research and Therapy, and Their Contribution to the Three Rs
Authors: Combes Robert D. , Shah Atul B.
Published: April 2019

Much is known about mammalian vision, and considerable progress has been achieved in treating many vision disorders, especially those due to changes in the eye, by using various therapeutic methods, including stem cell and gene therapy. While cells and tissues from the main parts of the eye and the visual cortex (VC) can be maintained in culture, and many computer models exist, the current non-animal approaches are severely limiting in the study of visual perception and retinotopic imaging. Some of the early studies with cats and non-human primates (NHPs) are controversial for animal welfare reasons and are of questionable clinical relevance, particularly with respect to the treatment of amblyopia. More recently, the UK Home Office records have shown that attention is now more focused on rodents, especially the mouse. This is likely to be due to the perceived need for genetically-altered animals, rather than to knowledge of the similarities and differences of vision in cats, NHPs and rodents, and the fact that the same techniques can be used for all of the species. We discuss the advantages and limitations of animal and non-animal methods for vision research, and assess their relative contributions to basic knowledge and clinical practice, as well as outlining the opportunities they offer for implementing the principles of the Three Rs ( Replacement, Reduction and Refinement).
More than 70 Years of Pyrogen Detection: Current State and Future Perspectives
Authors: Fennrich Stefan , Hennig Ulrike , Toliashvili Leila , Schlensak Christian , Wendel Hans Peter , Stoppelkamp Sandra
Published: April 2019

In the quality assurance of medical products, tests for sterility are essential. For parenteral pharmaceuticals, avoiding the presence of pyrogens is crucial. These fever-inducing substances (endotoxins and non-endotoxins) are not eliminated by standard sterilisation processes, and are biologically active once in the bloodstream, causing risks to human health, ranging from mild reactions (e.g. fever) to septic shock and death. Therefore, for injectable formulations, pyrogen testing is mandatory. Over the years, various pyrogen testing methods have been introduced, namely: in the 1940s, the rabbit pyrogen test, which is an in vivo test that measures the fever reaction as an endpoint; in the 1970s, the Limulus Amoebocyte Lysate (LAL) test, which is an in vitro test (with the haemolymph of the horseshoe crab) that specifically detects endotoxin; and in 2010, the Monocyte-Activation Test (MAT), which is a non-animal based in vitro pyrogen test that represents a full replacement of the rabbit test. Due to the ubiquity and biological significance of pyrogens, we are currently further developing the MAT so that it can be used for other applications. More specifically, our focus is on the detection of pyrogenic contamination on medical devices, as well as on the measurement of air quality. In addition, further improvements to permit the use of cryopreserved blood in the MAT, to overcome the limitations in the availability of freshly-drawn blood from human donors, are ongoing.
Animal Experimentation: Oh, when will they ever learn?
Authors: Balls Michael
Published: April 2019

Public support for animal research currently depends on misunderstandings of its cost (to animals) and benefits (to humans)
The European Citizens’ Stop Vivisection Initiative and the Revision of Directive 2010/63/EU
Authors: Menache Andre
Published: April 2019

Animal experimentation is presented to the public as an ongoing debate between research scientists on one hand, and the animal protection community on the other. An opportunity to break out of this mindset presented itself in the form of a European Citizens’ Initiative, Stop Vivisection, which challenged Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals for scientific purposes. The manifesto of the initiative called upon the European Commission to replace the existing Directive with a new proposal that does away with animal experimentation, and instead makes compulsory the use of human data as a predictive modality for the study of human diseases and responses to drugs. Although the Initiative succeeded in gathering the required one million signatures, the European Commission ultimately rejected the proposal. However, some of the lessons learned from the Initiative may well be relevant to the revision of Directive 2010/63/EU, due to take place by 2017.
Skin Sensitisation, Adverse Outcome Pathways and Alternatives
Authors: Basketter David
Published: April 2019

For toxicologists who are in any way associated with skin sensitisation, the last two decades have seen a series of fundamental changes. We have migrated from old-style guinea-pig assays, via the refined and reduced Local Lymph Node Assay (LLNA), to witness the imminent dominance of in vitro and in silico methods. Yet, over the same period, the use of the output data for human safety assurance has evolved from ‘black box’ risk assessment, via the quantitative risk assessment enabled by the LLNA measurement of potency, to a new period of relative uncertainty. This short review will endeavour to address these topics, all the while keeping a focus on three essential principles: a) that skin sensitisation potential is intrinsic in the molecular structure of the chemical; b) that test methods should have a mechanistic foundation; and finally c) that the only reason for undertaking any skin sensitisation work has to be the protection of human health.
The Use of Peptide Reactivity Assays for Skin Sensitisation Hazard Identification and Risk Assessment
Authors: Gerberick G. Frank
Published: April 2019

Over the past 20 years or more, investigators have been developing non-animal test methods for use in assessing the skin sensitisation potential of chemicals. In parallel with this effort, the key biological events of skin sensitisation have been well-characterised in an Adverse Outcome Pathway (AOP) proposed by the Organisation for Economic Co-operation and Development (OECD). The key molecular initiating event of this AOP is haptenation or covalent modification of epidermal proteins. In this review, the strengths and limitations of the Direct Peptide Reactivity Assay (DPRA) are described, and the more recently developed Peroxidase Peptide Reactivity Assay (PPRA). The DPRA has been formally validated and incorporated into an OECD Test Guideline (TG442C). The DPRA shows promise for assisting in hazard identification as well as for assessing skin sensitisation potency when used in an integrated testing strategy.
White Paper and Colourful Language: Toward a Realistic View of Animal Research
Authors: Grimm Herwig , Eggel Matthias
Published: April 2019

Ostensibly high scientific standards and the promise of short-term benefits are significant challenges for animal research
Human Aortic Endothelial Cells Respond to Shear Flow in Well-Plate Microfluidic Devices
Authors: Makwana Om , Flockton Hannah , Watters Gary P. , Nisar Rizwan , Smith Gina A. , Fields Wanda , Bombick Betsy
Published: April 2019

Although chronic progressive cardiovascular diseases such as atherosclerosis are often challenging to fully model in vitro, it has been shown that certain in vitro methods can effectively evaluate some aspects of disease progression. This has been demonstrated in in vitro and in vivo studies of endothelial cells that have illustrated the effects of nitric oxide (NO) production, filamentous actin (F-actin) formation, and cell and actin angle alignment on vascular function and homeostasis. Systems utilising shear flow have been established, in order to create a physiologically relevant environment for cells that require shear flow for homeostasis. Here, we investigated the use of a well-plate microfluidic system and associated devices (0–20dyn/cm2) to demonstrate applied shear effects on primary Human Aortic Endothelial Cells (HAECs). Changes in cell and actin alignment in the direction of flow, real-time production of NO and gross cell membrane shape changes in response to physiological shear flow were observed. These commercial systems have a range of potential applications, including within the consumer and pharmaceutical industries, thereby reducing the dependency on animal testing for regulatory safety assessments.
Use of the Bovine Udder Skin Model to Evaluate the Tolerability of Mesem Cosmetic Cream
Authors: Raak Christa , Molsberger Friedrich , Pittermann Wolfgang , Bertram Mathias , Robens Sibylle , Ostermann Thomas
Published: April 2019

Observational studies of Mesem cream (based on Mesembryanthemum crystallinum L. plant extract) found that it had positive effects on skin hydration and smoothing of the skin. However, some patients reported skin irritation effects. The current study evaluated the skin tolerability of Mesem cream, as compared to the carrier cream (without the active ingredient), by using the isolated perfused bovine udder skin model. The primary outcomes investigated were cytotoxicity (i.e. cell viability), assessed with the MTT assay, and irritancy and inflammation, assessed by measuring PGE2 tissue levels. A total reaction score was calculated by combining the results for each parameter. In the case of a single topical application, significant differences were found between the carrier cream and the Mesem cream. While the application of carrier cream resulted in low cytotoxicity (–8.4% change in viability, as compared to the untreated control), the Mesem cream was more cytotoxic (–18.7% change). In addition, one hour after application, PGE2 levels were higher in Mesem cream-treated skin, as compared to carrier cream-treated skin (16.6% versus 11.3%). Further experiments (tape-stripped skin and repeated application) also found significant differences between the two creams in the results obtained. Evaluation of the effectiveness, safety and tolerability of phyto-cosmetic products is important. Our results confirmed the findings of two previous human observational studies (the human patch test and open application study). Future experiments to understand the underlying principles of its effectiveness, safety and tolerability should include extracts of M. crystallinum L. juice, as well as the Mesem cream itself.
2016 Lush Science Prize
Authors: McCann Jenny , McCann Terry
Published: April 2019

The Lush Prize supports animal free testing by awarding monetary prizes totalling £250,000 to the most effective projects and individuals who have been working toward the goal of replacing animals in product or ingredient safety testing. Prizes are awarded for developments in five strategic areas: Science; Lobbying; Training; Public Awareness; and Young Researchers. In the event of a major breakthrough leading to the replacement of animal tests in the area of 21st Century Toxicology, a Black Box Prize (equivalent to the entire annual fund of £250,000) is awarded. The Science Prize is awarded to the researchers whose work the judging panel believe has made the most significant contribution to the replacement of animal testing in the preceding year. This Background Paper outlines the research projects that were shortlisted and presented to the judging panel as potential candidates for the 2016 Lush Science Prize. This process involved reviewing recent work of the relevant scientific institutions and projects in this area, such as the OECD, CAAT, The Hamner Institutes, ECVAM, UK NC3Rs, and the US Tox21 Programme. Recent developments in toxicity testing research were also identified by searching for relevant published papers in the literature, and analysing abstracts from conferences focusing on animal replacement in toxicity testing that had been held in the preceding 12 months — for example the EUSAAT-Linz, Society of Toxicology, and SEURAT-1 conferences.
A Comparison of Scaffold-free and Scaffold-based Reconstructed Human Skin Models as Alternatives to Animal Use
Authors: Kinikoglu Beste
Published: April 2019

Tissue engineered full-thickness human skin substitutes have various applications in the clinic and in the laboratory, such as in the treatment of burns or deep skin defects, and as reconstructed human skin models in the safety testing of drugs and cosmetics and in the fundamental study of skin biology and pathology. So far, different approaches have been proposed for the generation of reconstructed skin, each with its own advantages and disadvantages. Here, the classic tissue engineering approach, based on cell-seeded polymeric scaffolds, is compared with the less-studied cell self-assembly approach, where the cells are coaxed to synthesise their own extracellular matrix (ECM). The resulting full-thickness human skin substitutes were analysed by means of histological and immunohistochemical analyses. It was found that both the scaffold-free and the scaffold-based skin equivalents successfully mimicked the functionality and morphology of native skin, with complete epidermal differentiation (as determined by the expression of filaggrin), the presence of a continuous basement membrane expressing collagen VII, and new ECM deposition by dermal fibroblasts. On the other hand, the scaffold-free model had a thicker epidermis and a significantly higher number of Ki67-positive proliferative cells, indicating a higher capacity for self-renewal, as compared to the scaffold-based model.
Why are Validated Alternatives Not Being used to Replace Animal Tests?
Authors: Balls Michael
Published: April 2019

The stipulation in Directive 2010/63/EU that animal tests are replaced when validated alternative methods are available has no value, unless, as with all laws and regulations, this requirement is rigorously enforced, and unless governments, scientists and industries show greater respect for the scientific method