bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrated Decision-tree Testing Strategies for Skin Corrosion and Irritation with Respect to the Requirements of the EU REACH Legislation
Authors: Grindon Christina , Combes Robert , Cronin Mark T.D. , Roberts David W. , Garrod John F.
Published: April 2019

Liverpool John Moores University and FRAME recently conducted a research project, sponsored by DEFRA, on the status of alternatives to animal testing with regard to the European Union REACH (Registration, Evaluation and Authorisation of Chemicals) system for the safety testing and risk assessment of chemicals. The project covered all the main toxicity endpoints associated with the REACH system. This report focuses on how to maximise the use of alternative methods (both in vitro and in silico) for skin corrosion and irritation testing within a tiered testing strategy. It considers the latest developments in in vitro testing, with particular reference to the reconstituted skin models which have now been now been successfully validated and independently endorsed as suitable for both skin corrosivity and irritancy testing within the EU.
The Skin Compatibility of Distilled Tall Oils: Evaluation with the Bovine Udder Skin In Vitro Model System
Authors: Pittermann Wolfgang , Hopfgarten Fredrik , Kietzmann Manfred
Published: April 2019

Distilled tall oil (DTO) is a natural product, often added as an emulsifying ingredient in cutting fluids used as lubricants and coolants in metal working. The in vitro model used to test the skin compatibility of these substances, was the isolated perfused ex vivo bovine udder skin (BUS) model. After three exposure periods (0.5, 1, and 5 hours), cytotoxic effects were determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and tissue levels of the pre-inflammatory mediator prostaglandin E2 (PGE2) in treated whole skin biopsies were assessed by using an enzyme immunoassay. The BUS standard study design, involving a single application, was previously developed to investigate the skin irritation potential of cosmetics and chemicals. In the current study, four different batches of undiluted DTO, and tall oil fatty acids as a reference compound, were applied both singly and repeatedly (three times), under open conditions which were in line with the potential usage conditions in the work place. Under the standardised single application conditions, no major differences in cytotoxic effects or PGE2 levels between the samples were apparent, so no indication of a skin irritation potential could be concluded. This result is in accordance with prior in vivo studies for acute dermal toxicity. Under repeated application conditions, signs of cytotoxicity were observed after the application of one of the DTO samples, which was known to be derived from different raw materials. Therefore, it was concluded that, generally, the presence of DTO at a concentration of up to 10% in cutting fluids, is not expected to result in any DTO-related deterioration of the skin.
Ammonia-containing Industrial Effluents, Lethal to Rainbow Trout, Induce Vacuolisation and Neutral Red Uptake in the Rainbow Trout Gill Cell Line, RTgill-W1
Authors: Dayeh Vivian R. , Schirmer Kristin , Bols Niels C.
Published: April 2019

Nine samples of whole effluent from the operation of an industrial plant over the course of one year, were tested on rainbow trout for lethality and on the rainbow trout gill cell line, RTgill-W1, for metabolic activity, plasma membrane integrity, and lysosomal activity, as measured by using the alamar Blue (AB), 5-carboxyfluorescein diacetate acetoxymethyl (CFDA-AM), and neutral red (NR) assays, respectively. None of the nine samples caused a loss of plasma membrane integrity, and only two caused a transitory decline in metabolism. Three samples caused massive vacuolisation in RTgill-W1 cells, which was accompanied by increased uptake of NR, and only these three samples were lethal to the rainbow trout. The addition of ammonia to RTgill-W1 cultures also induced vacuolisation and NR uptake, with little change in plasma membrane integrity or metabolism. Subsequently, the effluent source was identified as a nitrogen product producer, and variable levels of ammonia were found in the nine samples. Three of the four samples with the highest non-ionised ammonia levels were those which were toxic to rainbow trout and which caused vacuoles in RTgill-W1 cells. The close correlation between rainbow trout-killing and RTgill-W1 vacuolisation by the effluents, suggests that vacuolisation of RTgill-W1 cells could be used to indicate effluents which would be toxic to rainbow trout as a result of their ammonia content.
In Vitro Skin Irritation Testing: Improving the Sensitivity of the EpiDerm Skin Irritation Test Protocol
Authors: Kandárová Helena , Hayden Patrick , Klausner Mitch , Kubilus Joseph , Kearney Paul , Sheasgreen John
Published: April 2019

A skin irritation test (SIT) utilising a common protocol for two in vitro reconstructed human epidermal (RhE) models, EPISKIN® and EpiDerm™, was developed, optimised and evaluated as a replacement for the in vivo rabbit skin irritation test in an ECVAM-sponsored validation study. In 2007, both RhE models were recognised by an independent peer-review panel and the ECVAM Scientific Advisory Committee (ESAC) as validated for use with the common SIT protocol. The EPISKIN SIT was endorsed as a full replacement of the in vivo rabbit test. Since the EpiDerm SIT proved to be less sensitive than the in vivo test and the EPISKIN SIT, the test was recognised as a validated component of a tiered testing strategy, in which positive results are accepted and negative results require further confirmation. The ESAC, in its April 2007 statement, also recommended increasing the sensitivity of the EpiDerm SIT, in order to gain the full acceptance. Analysis of the EpiDerm and EPISKIN data from the ECVAM validation study indicated that the lower sensitivity of the EpiDerm SIT might be linked to the more robust barrier properties of the EpiDerm model. This hypothesis was also in line with results published previously. To overcome the relatively low sensitivity of the EpiDerm protocol as a hindrance to full regulatory acceptance, a modification of exposure conditions was introduced into the protocol to achieve better agreement with the in vivo rabbit data. In the Modified EpiDerm SIT protocol, the test chemical exposure time was increased from 15 minutes to 60 minutes. In addition, part of the exposure was performed at 37°C. When the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay endpoint was used for classification, a significant increase of sensitivity was obtained (86.1%), whilst maintaining the high specificity of the method (76.3%). With the change to the EU classification system, which now uses higher cut-off for the classification of irritants, the sensitivity of the Modified EpiDerm SIT increased to above 90%. The measurement of interleukin (IL)-1α release did not further contribute to improvement of the method. The results demonstrate that the modified EpiDerm SIT protocol has the required sensitivity and specificity to be accepted as a stand alone method for complete replacement of the in vivo rabbit test.
The Use and Value of In Vitro Technologies in Metabolism Studies
Authors: Doehmer Johannes
Published: April 2019

The detailed investigation of the metabolism of drugs is one of the key issues in drug development. Several in vitro metabolism assays have been developed over the last two decades, to replace time-consuming and expensive animal studies. These have the potential to speed up drug development and increase drug safety, as they can be used to improve the prediction of the effects of drugs on humans. The key factors to be identified in metabolism are: a) the enzymes involved, and b) the metabolites produced by these enzymes. Cytochromes P450 (CYP-450s) are the key enzymes in drug metabolism. Cloning the genes encoding the CYP-450s, and the genetic engineering of suitable cells for heterologous expression, have provided new cell lines for studies on drug metabolism in vitro, under highly defined conditions. The V79 cell line, derived from Chinese hamster lung fibroblasts, was found to be suitable for heterologous expression, as these cells themselves do not express CYP-450s, thus providing a clean background for genetically engineering for the stable expression of any cloned CYP-450. In this way, V79 cell lines were created which specifically express CYP-450s from human, mouse, rat, and fish. These recombinant V79 cells have been applied in several drug metabolism and toxicity studies.
FRAME, Animal Experimentation and the Three Rs: Past, Present and Future
Authors: Balls Michael
Published: April 2019

At the opening of a meeting to celebrate the 50th anniversary of the publication of The Principles of Humane Experimental Technique, by W.M.S. Russell and R.L. Burch, and the 40th anniversary of the establishment of FRAME, some comments on the early days of the Charity are made, with particular reference to the special contributions made by its founder-Chairman, Dorothy Hegarty, and the author's own appointment as a Trustee, and later as Chairman. Reference is made to some key events and successes, and especially to the importance of FRAME's move from London to Nottingham, and the establishment of an ongoing collaboration with the University of Nottingham, including the setting-up of the FRAME Alternatives Laboratory.
Keeping the Archives at the University of Nottingham
Authors: Johnston Dorothy
Published: April 2019

The role of the department of Manuscripts and Special Collections at the University of Nottingham is described, with reference to its early development, growth of collections across all disciplines, and current collection strengths. The professional activities of acquisition, cataloguing and providing access are set in the wider perspective of national standards and expectations of delivery. The recent establishment of the W.M.S. (Bill) and Claire Russell Archive is discussed in this context, with some account of the challenges and opportunities which the archive offers for both curators and researchers.
Experience with the HET-CAM Method in the Routine Testing of a Broad Variety of Chemicals and Formulations
Authors: Schrage Arnhild , Gamer Armin O. , van Ravenzwaay Ben , Landsiedel Robert
Published: April 2019

Data on eye irritation are generally needed for the hazard identification of chemicals. For the routine testing of a broad variety of chemicals and formulations, we have used the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method. In the course of a tiered-testing strategy, and due to the lack of global regulatory acceptance of the HET-CAM method, we have also performed the Rabbit Eye Irritation test according to OECD Test Guideline 405. Of the 145 substances tested, 76% were classified as non-irritant/mild irritant and 13% were identified as irritant in vivo, according to the EU classification system (61% and 28%, respectively, with the GHS classification). The remaining 11% were severe irritants in vivo, based on the irreversibility of the effects and not due to sufficiently high irritation scores in the three days following application. The retrospective analysis revealed that the overall accuracy of the HET-CAM assay was 65% and the overall rates of false-negatives (FN) and false-positives (FP) were 50% and 33%, respectively. The HET-CAM assay was sufficiently specific (few FP) for water-soluble substances, but failed to identify nearly all the severe irritants within this group. In contrast, it was highly sensitive (no FN) for non-soluble and oil-soluble substances, but the specificity for this group was rather low. Therefore, we conclude that the HET-CAM assay is not useful in our tiered-testing strategy for eye irritation testing. However, for water-insoluble substances, it might be applicable in combination with another in vitro method, provided that regulatory acceptance is gained.
An Estimation of the Extent of Animal Use in Research in Brazil, as Determined by Bibliographic Sampling from Journals Published in the State of Paraná
Authors: Silla Vanessa Carli Bones , de Oliveira Sans Elaine Cristina , Molento Carla Forte Maiolino
Published: April 2019

Animal use in research is an issue of increasing ethical concern. The objective of this work was to investigate animal use in research described in the papers appearing in 18 journals published in the State of Paraná in 2006. The fields used in the bibliographic sampling were: agrarian science, biological science, biological and health science, environmental science, food technology, and health science. Of the 865 papers analysed, 41% involved the use of animals — a total of 3,497,653 animals, of which 216,223 were vertebrates. Procedures which were classified as A or B for degree of invasiveness were involved in 67% of the papers; 571 fish were employed in procedures classified as E. Only 11% of the journals required certificates from Animal Use Ethics Committees. These results suggest that Brazil is important in the context of worldwide animal use for research, in terms of both the total numbers of animals and the numbers of vertebrates used. Bibliographical sampling is a useful method for estimating the extent of animal use in experiments in Brazil. However, there are limitations to this approach, resulting from the geographical distribution of the authors, the existence of papers presenting insufficient information, and the exclusive inclusion of animal experimentation that actually reaches publication. Thus, the introduction of a formal system to record and control laboratory animal use in Brazil, is urgently required.
Reporting the Implementation of the Three Rs in European Primate and Mouse Research Papers: Are We Making Progress?
Authors: Taylor Katy
Published: April 2019

It is now more than 20 years since both Council of Europe Convention ETS123 and EU Directive 86/609?EEC were introduced, to promote the implementation of the Three Rs in animal experimentation and to provide guidance on animal housing and care. It might therefore be expected that reports of the implementation of the Three Rs in animal research papers would have increased during this period. In order to test this hypothesis, a literature survey of animal-based research was conducted. A randomly-selected sample from 16 high-profile medical journals, of original research papers arising from European institutions that featured experiments which involved either mice or primates, were identified for the years 1986 and 2006 (Total sample = 250 papers). Each paper was scored out of 10 for the incidence of reporting on the implementation of Three Rs-related factors corresponding to Replacement (justification of non-use of non-animal methods), Reduction (statistical analysis of the number of animals needed) and Refinement (housing aspects, i.e. increased cage size, social housing, enrichment of cage environment and food; and procedural aspects, i.e. the use of anaesthesia, analgesia, humane endpoints, and training for procedures with positive reinforcement). There was no significant increase in overall reporting score over time, for either mouse or primate research. By 2006, mouse research papers scored an average of 0 out of a possible 10, and primate research papers scored an average of 1.5. This review provides systematic evidence that animal research is still not properly reported, and supports the call within the scientific community for action to be taken by journals to update their policies.
Development of In Vitro Models for a Better Understanding of the Early Pathogenesis of Batrachochytrium dendrobatidis Infections in Amphibians
Authors: Van Rooij Pascale , Martel An , Brutyn Melanie , Maes Sofie , Chiers Koen , Van Waeyenberghe Lieven , Croubels Siska , Haesebrouck Freddy , Pasmans Frank
Published: April 2019

Batrachochytrium dendrobatidis, the causal agent of chytridiomycosis, is implicated in the global decline of amphibians. This chytrid fungus invades keratinised epithelial cells, and infection is mainly associated with epidermal hyperplasia and hyperkeratosis. Since little is known about the pathogenesis of chytridiomycosis, this study was designed to optimise the conditions under which primary keratinocytes and epidermal explants of amphibian skin could be maintained ex vivo for several days. The usefulness of the following set-ups for pathogenesis studies was investigated: a) cultures of primary keratinocytes; b) stripped epidermal (SE) explants; c) full-thickness epidermal (FTE) explants on Matrigel™; d) FTE explants in cell culture inserts; and e) FTE explants in Ussing chambers. SE explants proved most suitable for short-term studies, since adherence of fluorescently-labelled zoospores to the superficial epidermis could be observed within one hour of infection. FTE explants in an Ussing chamber set-up are most suitable for the study of the later developmental stages of B. dendrobatidis in amphibian skin up to five days post-infection. These models provide a good alternative for in vivo experiments, and reduce the number of experimental animals needed.
The Latest Statistics of Scientific Procedures on Living Animals Reveal Little Three Rs Progress in Great Britain in 2010
Authors: Hudson Michelle
Published: April 2019

The 2010 Statistics of Scientific Procedures on Living Animals showed that the level of animal experimentation in Great Britain continues to rise, with just over 3.7 million procedures being conducted. This is almost exclusively due to the sustained increase in the breeding and utilisation of genetically-altered animals. Here, the general trends in the species used and the numbers and types of procedures are reviewed. In addition, the impact of the recent Government announcement to ban testing of household products on animals is discussed, along with the implications of the fish becoming the second most-frequently used animal in scientific research. Finally, concerns about primate use, the REACH System, cosmetics testing and the new EU Directive on the protection of laboratory animals are explained.
Willingness to Spare Animals in Undergraduate Medical Education in Southern India: A Preliminary Questionnaire-based Investigation
Authors: Shehnaz Syed Ilyas , Sreedharan Jayadevan , Mathew Elsheba , Gomathi Kadayam Guruswami , Khan Nelofer Sami
Published: April 2019

Animal experiments continue to play an integral role in Indian undergraduate medical education, even though alternatives are becoming increasingly available. In this context, this study aimed to assess the perceptions of pharmacology faculty members from medical colleges in southern India regarding the use of animals and alternatives in experimental pharmacology, and to determine the association between these perceptions and the socio-demographic characteristics of the participants. Data were collected from 59 faculty members of 15 medical colleges in southern India. The response rate was 84.3%. A 30-statement, five-domain questionnaire was used, with a global score of 120. The mean ± SD global score was 60.9 ± 17.3. Significant differences were observed in domain scores and individual statement scores with respect to the extent of teaching experience. There were no statistically significant differences in perceptions with respect to age, gender or educational qualifications. All the participating colleges were conducting at least 3–8 animal experiments per year on the rabbit, rat, mouse and frog/toad. The pharmacology faculty members in the southern India medical colleges included in the study (especially the more experienced teachers) supported animal use in undergraduate medical education, in spite of being aware of the drawbacks of animal experiments and the availability of alternatives.
The Rational Use of Animals in Drug Development: Contribution of the Innovative Medicines Initiative
Authors: Gunn Magda , Vaudano Elisabetta , Goldman Michel
Published: April 2019

Animal models are still widely used to assess the efficacy or safety of new pharmaceutical products. Since their limitations in predicting actions of drugs in humans are becoming more and more apparent, there is an urgent need to revisit the use of animals in pharmaceutical research. Herein, we review how the Innovative Medicines Initiative (IMI), the largest public–private partnership in the life sciences, is reducing, refining and replacing the use of animals in the context of its global mission, namely, to boost research and the development of new medicines across the European Union.
Searching Effectively for Three Rs Information
Authors: Hudson-Shore Michelle
Published: April 2019

Resources are available to facilitate the search for information on the Three Rs and their implementation; guidance is needed on their use
The Use of Genetically-engineered Animals in Science: Perspectives of Canadian Animal Care Committee Members
Authors: Ormandy Elisabeth H. , Dale Julie , Griffin Gilly
Published: April 2019

The genetic engineering of animals for their use in science challenges the implementation of refinement and reduction in several areas, including the invasiveness of the procedures involved, unanticipated welfare concerns, and the numbers of animals required. Additionally, the creation of genetically-engineered animals raises problems with the Canadian system of reporting animal numbers per Category of Invasiveness, as well as raising issues of whether ethical limits can, or should, be placed on genetic engineering. A workshop was held with the aim of bringing together Canadian animal care committee members to discuss these issues, to reflect on progress that has been made in addressing them, and to propose ways of overcoming any challenges. Although previous literature has made recommendations with regard to refinement and reduction when creating new genetically-engineered animals, the perception of the workshop participants was that some key opportunities are being missed. The participants identified the main roadblocks to the implementation of refinement and reduction alternatives as confidentiality, cost and competition. If the scientific community is to make progress concerning the implementation of refinement and reduction, particularly in the creation and use of genetically-engineered animals, addressing these roadblocks needs to be a priority.
Cigarette Smoke-induced Morphological Transformation of Bhas 42 Cells In Vitro
Authors: Weisensee Dirk , Poth Albrecht , Roemer Ewald , Conroy Lynda L. , Schlage Walter K.
Published: April 2019

In vitro cell transformation assays detect transformed cells that have acquired the distinct characteristics of malignant cells and thus model one stage of in vivo carcinogenesis. These assays have been proposed as surrogate models for predicting the non-genotoxic carcinogenic potential of chemicals. The Bhas 42 cell transformation assay, a short-term assay that uses v-Ha- ras-transfected Balb/c 3T3 cells, can detect the tumour promoter-like activities of chemicals, but has not previously been used with cigarette smoke. The particulate phase of cigarette smoke (total particulate matter [TPM]) is known to induce tumours in vivo in the mouse skin painting assay. Therefore, we investigated the ability of this Bhas cell assay to form morphologically transformed foci in vitro when repeatedly challenged with TPM from a standard research cigarette. TPM induced a dose-dependent increase in Type III foci, and a significant increase (up to 20-fold) in focus formation at moderately toxic concentrations between 5 and 60μg TPM/ml, with a peak at 20μg/ml. Three batches of TPM were tested in three independent experiments. Precision (repeatability and reproducibility) was calculated by using 0, 5, 10, and 20μg TPM/ml. Repeatability and reproducibility, expressed as the relative standard deviation obtained from the normalised slopes of the dose–response curves, were 17.2% and 19.6%, respectively; the slopes were 0.7402 ± 0.1247, 0.9347 ± 0.1316, and 0.8772 ± 0.1767 (increase factor*ml/mg TPM; mean ± SD); and the goodness of fit ( r2) of the mean slopes, each derived from n = 6 repeats, was 0.9449, 0.8198, and 0.8344, respectively. This in vitro assay with Bhas 42 cells, which are regarded as already initiated in the two-stage paradigm of carcinogenesis (initiation and promotion), is able to detect cell transformation induced by cigarette smoke in a dose-dependent manner with a high sensitivity and good precision. Because this assay is fast and yields reliable results, it may be useful in product assessment, as well as for further investigation of the non-geno-toxic carcinogenic activity of tobacco smoke-related test substances.
4. Reduction
Authors: Balls Michael
Published: April 2019

Reduction should be the easiest of the Three Rs to achieve, but it will not happen without a revolution in education and training on the design and analysis of animal experiments
Comparative Studies of Apoptosis in Xenopus laevis and Mouse Thymoma Cell Lines
Authors: Johnson Rachel O. , Tittle Thomas V. , Sefchick Maria P.M. , Zettergren Leslie D. , Ruben Laurens N. , Clothier Richard H. , Balls Michael
Published: April 2019

With the use of in vitro methods and cell lines, functional aspects of apoptosis in the Xenopus laevis B3/B7 and mouse EL4 thymoma cell lines are revealed. Moreover, by using information gleaned from digital imaging and immunocytochemistry, changes in locations of key proteins implicated in apoptotic anti-cancer responses, e.g. p53 and Mdm2, are shown. Suggestions are offered as to what these results might mean with respect to the evolutionary conservation of the function and structure of these two molecules and to cancer resistance in amphibians. Finally, studies are described on resveratrol as an anti-cancer therapeutic reagent in the two thymoma cell lines and in normal X. laevis thymocytes.
Development of a Vessel Organ Culture System: Characterisation of the Method and Implications for the Reduction of Animal Experiments
Authors: Zaniboni Andrea , Zannoni Augusta , Bernardini Chiara , De Cecco Marco , Bombardi Cristiano , Seren Eraldo , Forni Monica , Bacci Maria L.
Published: April 2019

In the field of cardiovascular research, the pig is considered to be an excellent animal model of human diseases. It is well-known that primary cultures of endothelial cells (ECs) are a powerful tool for the study of vascular physiology and pathology, and, according to the principles of the Three Rs, their use results in a substantial reduction in the numbers of experimental animals required. However, a limitation of EC culture is that the cells are not in their physiological context. Here, we describe and characterise a method for the culture of porcine vessels that overcomes the limitation of EC cultures, with the advantage of reducing the number of animals used for research purposes. The organ cultures were set-up by using an aortic cylinder obtained from the arteries of control pigs sacrificed for other experimental purposes. In order to characterise the method, vascular endothelial growth factor (VEGF) secretion, matrix metalloproteinase (MMP) activation and the vessel's structural features were evaluated during organ culture. These analyses confirm that the culture of aortic cylinder lumen, in a medium specific for ECs, results in a stable system in terms of VEGF and MMP secretion. The ECs do not undergo cell division during the organ culture, which is also the case in vivo, if no stimulation occurs. Overall, we show that this novel system closely resembles the in vivo context. Importantly, porcine aortas can be collected from either veterinary surgeries or slaughterhouses, without having to sacrifice animals specifically for the purposes of this type of research.
Statistics of Scientific Procedures on Living Animals 2012: Another Increase in Experimentation — Genetically-altered Animals Dominate Again
Authors: Hudson-Shore Michelle
Published: April 2019

The Annual Statistics of Scientific Procedures on Living Animals Great Britain 2012 reveal that the level of animal experimentation in Great Britain continues to rise, with just over 4.1 million procedures being started in that year. Despite the previous year's indication that the dominance of the production and use of genetically-altered (GA, i.e. genetically-modified animals plus animals with harmful genetic defects) animal might be abating, it returned with a vengeance in 2012. Breeding increased from 43% to 48% of all procedures, and GA animals were involved in 59% of all the procedures. Indeed, if the breeding of these animals were removed from the statistics, the total number of procedures would actually decline by 2%. In order to honour their pledge to reduce animal use in science, the Coalition Government will have to address this issue. The general trends in the species used, and the numbers and types of procedures, are also reviewed. Finally, forthcoming changes to the statistics are discussed.
Laboratory Animal Ethics Course Planning in Iran
Authors: Aldavood Seyed Javid , Fard Ramin Mazaheri Nezhad , Naderynezhad Fataneh
Published: April 2019

A laboratory animal ethics course based on Islamic rules could be more-readily and more-easily accepted in Islamic countries
A Tutorial for Analysing the Cost-effectiveness of Alternative Methods for Assessing Chemical Toxicity: The Case of Acute Oral Toxicity Prediction
Authors: Norlen Hedvig , Worth Andrew P. , Gabbert Silke
Published: April 2019

Compared with traditional animal methods for toxicity testing, in vitro and in silico methods are widely considered to permit a more cost-effective assessment of chemicals. However, how to assess the cost-effectiveness of alternative methods has remained unclear. This paper offers a user-oriented tutorial for applying cost-effectiveness analysis (CEA) to alternative (non-animal) methods. The purpose is to illustrate how CEA facilitates the identification of the alternative method, or the combination of methods, that offers the highest information gain per unit of cost. We illustrate how information gains and costs of single methods and method combinations can be assessed. By using acute oral toxicity as an example, we apply CEA to a set of four in silico methods (ToxSuite, TOPKAT, TEST, ADMET Predictor), one in vitro method (the 3T3 Neutral Red Uptake cytotoxicity assay), and various combinations of these methods. Our results underline that in silico tools are more cost-effective than the in vitro test. Battery combinations of alternative methods, however, do not necessarily outperform single methods, because additional information gains from the battery are easily outweighed by additional costs.
Autologous Co-culture of Primary Human Alveolar Macrophages and Epithelial Cells for Investigating Aerosol Medicines. Part I: Model Characterisation
Authors: Hittinger Marius , Janke Julia , Huwer Hanno , Scherließ Regina , Schneider-Daum Nicole , Lehr Claus-Michael
Published: April 2019

The development of new formulations for pulmonary drug delivery is a challenge on its own. New in vitro models which address the lung are aimed at predicting and optimising the quality, efficacy and safety of inhaled drugs, to facilitate the more rapid translation of such products into the clinic. Reducing the complexity of the in vivo situation requires that such models reproducibly reflect essential physiological factors in vitro. The choice of cell types, culture conditions and the experimental set-up, can affect the outcome and the relevance of a study. In the alveolar space of the lung, epithelial cells and alveolar macrophages are the most important cell types, forming an efficient cellular barrier to aerosols. Our aim was to mimic this barrier with primary human alveolar cells. Cell densities of alveolar macrophages and epithelial cells, isolated from the same human donor, were optimised, with a focus on barrier properties. The combination of 300,000 epithelial cells/cm2 together with 100,000 macrophages/cm2 showed a functional barrier (transepithelial electrical resistance > 500ω.cm2). This cell model was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures. The functionality of the in vitro system was investigated with spray-dried fluorescently labelled poly(lactic-co-glycolic) acid particles loaded with ovalbumin as a model drug.
Pain in Laboratory Animals: A Possible Confounding Factor?
Authors: Sneddon Lynne U.
Published: April 2019

Reducing pain in animals is an ethical and sometimes legal requirement, but how do we assess pain and does it confound data collection?
Does the Stress Inherent to Laboratory Life and Experimentation on Animals Adversely Affect Research Data?
Authors: Bailey Jarrod
Published: April 2019

Stress and distress in laboratory animals is often inherent and unavoidable. The effect of these factors on the reliability and relevance of experimental data is not sufficiently appreciated. Greater awareness, debate and discussion of this issue are urgently required
The Use of Gatekeeping Procedures in the Statistical Planning of Animal Experiments
Authors: Mayer Benjamin , Stahl Vicky , Kron Martina
Published: April 2019

Statistical sample size calculation is essential when planning animal experiments in basic medical research. Usually, such trials involve the testing of multiple hypotheses, and interpreting them in a confirmative manner would require the appropriate adjustment of the Type 1 error. This has to be taken into account as early as possible during sample size estimation — otherwise, all the results obtained would be exploratory, i.e. without cogency. In this paper, the concept of gatekeeping is introduced, along with alternative approaches for Type 1 error adjustment. The application of gatekeeping to the calculation of sample size is demonstrated by using data sets from case studies. Overall, the evaluation of these examples showed that gatekeeping is able to keep the required number of animals comparatively small. In contrast to exploratory planning, which led to the lowest sample sizes, gatekeeping suggested a mean increase of 12% in sample size, while conservative Bonferroni adjustment raised the sample size by 34% on average. Gatekeeping is a prominent strategy for handling the multiple testing problem, and has been proven to keep the required sample sizes in animal studies comparatively low. Therefore, it is a suitable approach to a compromise between the Three Rs principle of reduction and the appropriate handling of the multiplicity issue in animal trials with a confirmative focus.
Fetal Bovine Serum (FBS) — A Pain in the Dish?
Authors: van der Valk Jan , Gstraunthaler Gerhard
Published: April 2019

The use of Fetal Bovine Serum in replacement alternative methods is associated with serious animal welfare concerns, as well as worrying reproducibility issues
The Myth of Replacement and the Legal Reality
Authors: Bowles Edwina
Published: April 2019

Despite EU law being in force, animals are often used where alternative methods already exist and are readily available
A Survey to Understand Public Opinion regarding Animal use in Medical Training
Authors: Merkley Ryan , Pippin John J. , Joffe Ari R.
Published: April 2019

A random survey was performed by ORC International Telephone CARAVAN®, on 24–27 March 2016, by trained interviewers. The aim of this survey was to gain further understanding of public perceptions in the United States of laboratory animal use, specifically for the purposes of medical training. Five statements were read in random order to the participants, who were then asked whether they agreed or disagreed with the statement. Survey responses were obtained from 1011 participants. For the combined statements: “If effective non-animal methods are available to train a) medical students and physicians, b) emergency physicians and paramedics, and c) paediatricians, those methods should be used instead of live animals”, most respondents (82–83%) agreed. For the statement: “You want your doctor to be trained by using methods that replicate human anatomy instead of live animals”, most respondents (84%) agreed. For the statement: “If effective non-animal methods are available, it is morally wrong or unethical to use live animals to train medical students, physicians and paramedics”, 67% of respondents agreed. Responses were similar among the 15 pre-specified demographic subgroups. Given that effective non-animal training methods are readily available, the survey suggests that a substantial majority of the public wants the use of animals in medical training to cease.
EDIT: A New International Multicentre Programme to Develop and Evaluate Batteries of In Vitro Tests for Acute and Chronic Systemic Toxicity
Authors: Ekwall Björn , Clemedson Cecilia , Ekwall Barbro , Ring Patrik , Romert Lennart
Published: April 2019

The Multicenter Evaluation of In Vitro Cytotoxicity (MEIC) programme provided a battery of three basal cytotoxicity tests with a good (R2= 0.77) prediction of human acute lethal blood concentrations. The predictive power of this battery would be considerably improved by the addition of new supplementary in vitro tests. The development of these new tests will be facilitated by a close coupling of test development to evaluation. The Cytotoxicology Laboratory, Uppsala (CTLU), is therefore inviting all interested in vitro toxicologists to take part in the Evaluation-guided Development of In Vitro Toxicity and Toxicokinetic Tests (EDIT). All EDIT activities (subprojects) will be designed on a case-by-case basis, but will follow a common pattern. The CTLU will use the accumulated MEIC/EDIT data, and its experience from the previous MEIC evaluation, to suggest priority areas, i.e. the need for certain in vitro toxicity data/tests as supplements to existing in vitro models/batteries on human systemic toxicity. Detailed research programmes corresponding to these areas will be published on the Internet. The CTLU will also try to raise funds for some projects and will coordinate multilaboratory studies. Interested laboratories developing or already using priority tests are encouraged to join the subprojects and to test specific sets of substances (usually sets of MEIC reference chemicals) in their new assays. The CTLU will provide adequate human reference data and will also evaluate results as single components of complex models, together with the laboratory conducting the test. At present, ten priority areas have been identified: a) repeat dose toxicity in vitro; b) urgent mechanistic information from in vitro studies of protein denaturation, morphology of cell injury, differential toxicity between various rapidly measured end-points (10–60 minutes) and 24-hour cytotoxicity, toxicity to aerobic cells, and discrimination between rapid and slow cytotoxic mechanisms; c) in vitro tests on vitally important, specific receptor toxicity in humans; d) excitatory cytotoxicity; e) reversibility of cell toxicity; f) in vitro tests on passage across the blood–brain barrier; g) in vitro tests on absorption in the gut; h) protein binding in vitro; i) in vitro tests on distribution volumes (Vd); and j) in vitro tests on bio-transformation to more-toxic metabolites (hepatocytes plus target cells). This paper gives a short presentation of the rationale for each subproject and reports on ongoing activities.
Effects of Chromatin Function Inhibitors on Yeast Whole Cells and Spheroplasts
Authors: Tiligada Ekaterini , Stavrinidis Elias , Manolis Christos , Delitheos Andreas
Published: April 2019

Saccharomyces cerevisiae has long been used as an alternative experimental model in the study of cancer and anticancer drug action. Although this simple eukaryote has provided useful information, its value as an experimental model is often controversial due to the presence of the cell wall — a cellular structure which is absent in higher eukaryotes. The aim of this study was to investigate the possible involvement of the cell wall in the ineffectiveness of some anticancer drugs in yeast, by enzymatic removal of the cell wall. The effects of exposing whole-cell cultures and spheroplasts to chromatin function inhibitors for 22 hours were investigated. Vinblastine, etoposide and paclitaxel had no cytotoxic effects on whole-cell cultures either with or without the addition of verapamil. The growth profiles of yeast spheroplasts following drug exposure were similar to those observed in whole cells. These data demonstrate that the resistance of the lower eukaryote to these drugs was not overcome by the enzymatic removal of the cell wall.
Characterizing white matter fiber orientation effects on multi-parametric quantitative BOLD assessment of oxygen extraction fraction
Authors: Kaczmarz Stephan , Göttler Jens , Zimmer Claus , Hyder Fahmeed , Preibisch Christine
Published: April 2019

Relative oxygen extraction fraction (rOEF) is a fundamental indicator of cerebral metabolic function. An easily applicable method for magnetic resonance imaging (MRI) based rOEF mapping is the multi-parametric quantitative blood oxygenation level dependent (mq-BOLD) approach with separate acquisitions of transverse relaxation times [Formula: see text] and T2 and dynamic susceptibility contrast (DSC) based relative cerebral blood volume (rCBV). Given that transverse relaxation and rCBV in white matter (WM) strongly depend on nerve fiber orientation, mq-BOLD derived rOEF is expected to be affected as well. To investigate fiber orientation related rOEF artefacts, we present a methodological study characterizing anisotropy effects of WM as measured by diffusion tensor imaging (DTI) on mq-BOLD in 30 healthy volunteers. Using a 3T clinical MRI-scanner, we performed a comprehensive correlation of all parameters ([Formula: see text], T2, [Formula: see text], rCBV, rOEF, where [Formula: see text]=1/[Formula: see text]–1/T2) with DTI-derived fiber orientation towards the main magnetic field (B0). Our results confirm strong dependencies of transverse relaxation and rCBV on the nerve fiber orientation towards B0, with anisotropy-driven variations up to 37%. Comparably weak orientation-dependent variations of mq-BOLD derived rOEF (3.8%) demonstrate partially counteracting influences of [Formula: see text] and rCBV effects, possibly suggesting applicability of rOEF as an oxygenation sensitive biomarker. However, unresolved issues warrant caution when applying mq-BOLD to WM.
Systemic inflammation in hemorrhagic strokes – A novel neurological sign and therapeutic target?
Authors: Saand Aisha R , Yu Fang , Chen Jun , Chou Sherry H-Y
Published: April 2019

Growing evidences suggest that stroke is a systemic disease affecting many organ systems beyond the brain. Stroke-related systemic inflammatory response and immune dysregulations may play an important role in brain injury, recovery, and stroke outcome. The two main phenomena in stroke-related peripheral immune dysregulations are systemic inflammation and post-stroke immunosuppression. There is emerging evidence suggesting that the spleen contracts following ischemic stroke, activates peripheral immune response and this may further potentiate brain injury. Whether similar brain–immune crosstalk occurs in hemorrhagic strokes such as intracerebral hemorrhage (ICH) and subarachnoid hemorrhage (SAH) is not established. In this review, we systematically examined animal and human evidence to date on peripheral immune responses associated with hemorrhagic strokes. Specifically, we reviewed the impact of clinical systemic inflammatory response syndrome (SIRS), inflammation- and immune-associated biomarkers, the brain–spleen interaction, and cellular mediators of peripheral immune responses to ICH and SAH including regulatory T cells (Tregs). While there is growing data suggesting that peripheral immune dysregulation following hemorrhagic strokes may be important in brain injury pathogenesis and outcome, details of this brain-immune system cross-talk remain insufficiently understood. This is an important unmet scientific need that may lead to novel therapeutic strategies in this highly morbid condition.
Registration with a small number of sparse measurements
Authors: Arun Srivatsan Rangaprasad , Zevallos Nicolas , Vagdargi Prasad , Choset Howie
Published: April 2019

This work introduces a method for performing robust registration given the geometric model of an object and a small number (less than 20) of sparse point and surface normal measurements of the object’s surface. Such a method is of critical importance in applications such as probing-based surgical registration, contact-based localization, manipulating objects devoid of visual features, etc. Our approach for sparse point and normal registration (SPNR) is iterative in nature. In each iteration, the current best pose estimate is perturbed to generate several candidate poses. Among the generated poses, one pose is selected as the best, by evaluating an inexpensive cost function. This pose is used as the initial condition to estimate the locally optimum registration. This process is repeated until the registration estimate converges within a tolerance bound. Two variants are developed: deterministic (dSPNR) and probabilistic (pSPNR). The dSPNR is faster than pSPNR in converging to the local optimum, but the pSPNR requires fewer parameters to be tuned. The pSPNR also provides pose-uncertainty information in addition to the registration estimate. Both approaches were evaluated in simulation using various standard datasets and then compared with results obtained using state-of-the-art methods. Upon comparison with other methods, both dSPNR and pSPNR were found to be robust to initial pose errors as well as noise in measurements. The effectiveness of the approaches are also demonstrated with robot experiments for the application of probing-based registration.
Hoist on its Own Media
Authors: Elam Peter
Published: April 2019

The struggle over the state-run Hungarian Radio and Television (HR TV) has continued to dominate political debates. Last-ditch attempts by the government to fill the airwaves with propaganda in order to avert a humiliating electoral defeat in the general elections in May backfired.
The Power of Being There for Each Other: The Importance of Self-Awareness, Identifying Stress and Burnout, and Proactive Self-Care Strategies for Sex-Offender Treatment Providers
Authors: Parsonson Karen , Alquicira Luz
Published: April 2019

This was an exploratory study which hypothesized that there would be significant individual differences in feelings, perceptions, experiences, self-care strategies, and barriers to practicing self-care between sex offender treatment professionals. Nine sex offender treatment providers, all female, received structured interviews. Experience with the population varied from 3 to 15 years. Qualitative data were evaluated for themes in perceived context and nature of their work. The focus was upon conceptualization of self-care and implications, both professionally and personally. Three areas of self-care strategies emerged: personal, professional, and organizational. Perspectives differed with years of experience, with differential impacts on each area. Barriers were found to practicing self-care at all levels. To maintain well-functioning, it is suggested that there is an individual goodness of fit. Connection to others, however, appears to be extremely important, as does work–life balance. Recommendations include practicing regular self-care, as well as education about self-awareness and self-care during training.