ALTEX - Alternatives to animal experimentation <p><strong>The journal ALTEX – Alternatives to Animal Experimentation publishes open access academic articles on the development and implementation of alternatives to the use of animals for scientific purposes and informs on international developments in this field. </strong></p> <p>ALTEX is the official organ of&nbsp;<a href="" target="_blank" rel="noopener">CAAT</a>, <a href="">CAAT-Europe</a>, the Doerenkamp-Zbinden Chairs, <a href="">EUSAAT</a> and <a href="">t<sup>4</sup></a>.</p> ALTEX Edition / Springer Spektrum Springer-Verlag GmbH en-US ALTEX - Alternatives to animal experimentation 1868-596X <p>Articles are distributed under the terms of the Creative Commons Attribution 4.0 International license (, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is appropriately cited (CC-BY). Copyright on any article in ALTEX is retained by the author(s).</p> Editorial Sonja von Aulock ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 U2 U2 Toward Good In Vitro Reporting Standards <p>A good experiment reported badly is worthless. Meaningful contributions to the body of science are made by sharing the full methodology and results so that they can be evaluated and reproduced by peers. Erroneous and incomplete reporting does not do justice to the resources spent on conducting the experiment and the time peers spend reading the article. In theory peer-review should ensure adequate reporting – in practice it does not. Many areas have developed reporting standards and checklists to support the adequate reporting of scientific efforts, but <em>in vitro</em> research still has no generally accepted criteria. It is characterized by a “Wild West” or “anything goes” attitude. Such a culture may undermine trust in the reproducibility of animal-free methods, and thus parallel the “reproducibility crisis” discussed for other life science fields. The increasing data retrieval needs of computational approaches (in extreme as “big data” and artificial intelligence) makes reporting quality even more important so that the scientific community can take full advantage of the results.<br>The first priority of reporting standards is to ensure the completeness and transparency of information provided (data focus). The second tier is a quality of data display that makes information digestible and easy to grasp, compare and further analyze (information focus). This article summarizes a series of initiatives geared towards improving the quality of <em>in vitro</em> work and its reporting. This shall ultimately lead to Good In Vitro Reporting Standards (GIVReSt).</p> Thomas Hartung Rob de Vries Sebastian Hoffmann Helena T. Hogberg Lena Smirnova Katya Tsaioun Paul Whaley Marcel Leist ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 3 17 10.14573/altex.1812191 A three-dimensional model to study human synovial pathology <p>Therapeutic agents that are used by patients with rheumatic and musculoskeletal diseases were originally developed and tested in animal models, and although retrospective studies show that these models have limited predictive value, their use has continued because of a lack of good <em>in vitro </em>alternatives. In this study, we developed a 3-dimensional synovial membrane model made of either human primary synovial cell suspensions or a mix of primary fibroblast-like synoviocytes and CD14+ mononuclear cells. We analyzed the composition of the mature micromasses by immunohistochemical staining and flow cytometry and showed that the outer surface forms a lining layer consisting of fibroblast-like and macrophage-like cells, reflecting the <em>in vivo </em>naïve synovial membrane. To model the affected synovial membrane in rheu­matoid arthritis (RA), micromasses were exposed to the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α), which led to increased pro-inflammatory cytokine expression and production, and to hyperplasia of the membrane. To recreate the synovial membrane in osteoarthritis (OA), the micromasses were exposed to transforming growth factor beta (TGF-β), which led to fibrosis-like changes of the membrane, including increased alpha smooth muscle actin (α-SMA) and increased expression of fibrosis-related genes <em>PLOD2 </em>and <em>COL1A1</em>. Interestingly, the macrophages in the micromasses showed phenotypic plasticity, as prolonged TNF-α or TGF-β stimulation strongly reduced the occurrence of CD163+ M2-like macrophages. We show the plasticity of the micromasses as a synovial model for studying RA and OA pathology and propose that the synovial lining micromass system can be a good alternative for drug testing.</p> Mathijs G. A. Broeren Claire E. J. Waterborg Renske Wiegertjes Rogier M. Thurlings Marije I. Koenders Peter L. E. M. van Lent Peter M. van der Kraan Fons A. J. van de Loo ##submission.copyrightStatement## 2018-10-09 2018-10-09 36 1 18 28 10.14573/altex.1804161 An ex vivo porcine spleen perfusion as a model of bacterial sepsis <p>An <em>ex vivo</em>, porcine spleen perfusion model was established to study the early events occurring in the spleen prior to the onset of bacterial sepsis, using organs retrieved from animals slaughtered for food production. Porcine spleens were harvested from adult pigs and connected to a normothermic extracorporeal perfusion circuit. Constant perfusion of heparinized blood was performed for 6 hours. After injection of <em>Streptococcus pneumoniae </em>to the circuit, serial samples of both blood and spleen biopsies were collected and analyzed. Functionality of the perfused organs was assessed by monitoring the blood-gas parameters, flow rate and filtering capability of the organ. Interestingly, we observed full clearance of bacteria from the blood and an increase in bacterial counts in the spleen. Classical histology and immunohistochemistry on biopsies also confirmed no major damage in the organ architecture and no changes in the immune cell distribution other than the presence of clusters of pneumococci. A time-course study confirmed that each focus of infection derived from the replication of single pneumococcal cells within splenic macrophages. The model proposed – in line with the 3Rs principles – has utility in the replacement of experimental animals in infection research. Murine models are prevalently used to study pneumococcal infections but are often not predictive for humans due to substantial differences in the immune systems of the two species. This model is designed to overcome these limitations, since porcine immunology, and splenic architecture in particular, closely resemble those of humans.</p> Wen Y. Chung Joseph J. Wanford Rohan Kumar John D. Isherwood Richard D. Haigh Marco R. Oggioni Ashley R. Dennison Giuseppe Ercoli ##submission.copyrightStatement## 2018-08-03 2018-08-03 36 1 29 38 10.14573/altex.1805131 Investigating cell type specific mechanisms contributing to acute oral toxicity <p>The replacement of animals in acute systemic toxicity testing remains a considerable challenge. Only animal data are currently accepted by regulators, including data generated by reduction and refinement methods. The development of integrated approaches to testing and assessment (IATA) is hampered by an insufficient understanding of the numerous toxicity pathways that lead to acute systemic toxicity. Therefore, central to our work has been the collection and evalu­ation of the mechanistic information on eight organs identified as relevant for acute systemic toxicity (nervous system, cardiovascular system, liver, kidney, lung, blood, gastrointestinal system, and immune system). While the nervous and cardiovascular systems are the most frequent targets, no clear relationship emerged between specific mechanisms of target organ toxicity and the level (category) of toxicity. From a list of 114 chemicals with acute oral <em>in vivo </em>and <em>in vitro </em>data, 97 were identified with target organ specific effects, of which 94% (91/97) were predicted as acutely toxic by the 3T3 neutral red uptake cytotoxicity assay and 6% (6/97) as non-toxic. Although specific target organ mechanisms of toxicity could in some cases explain the false negative prediction obtained with the cytotoxicity assay, in general it is difficult to explain <em>in vitro </em>misclassifications only on the basis of mechanistic information. This analysis will help to prioritize the development of adverse outcome pathways for acute oral toxicity, which will support the assessment of chemicals using mechanistically informed IATA.</p> Pilar Prieto Rabea Graepel Kirsten Gerloff Lara Lamon Magdalini Sachana Francesca Pistollato Laura Gribaldo Anna Bal-Price Andrew Worth ##submission.copyrightStatement## 2018-07-12 2018-07-12 36 1 39 64 10.14573/altex.1805181 Retrospective review of anesthetic and analgesic regimens used in animal research proposals <p>Pain has a profound effect on an animal’s wellbeing. In Germany, researchers using animals have been legally required to reduce any possible pain, suffering, distress or lasting harm to an absolute minimum since 1972. To evaluate how these provisions have been implemented in practice, an assessment of refinements to experimental techniques was conducted by retrospectively reviewing 684 surgical interventions described in 506 animal research applications that were sent to the German competent authorities for approval in 2010. This paper focuses on the efficacy of proposed anesthesia and peri- and postoperative analgesia. Postoperative analgesia was not proposed for 30% of surgeries. Following 10% of procedures, animals were to be given pain relieving medication if the investigators decided this was necessary; however, structured assessments to detect pain were absent. Consequences of unalleviated pain and omission of pain assessment techniques are discussed, and some recommendations to improve anesthesia and analgesia are given. The findings of this review highlight the need for improvement, both to fulfil legal requirements and to improve animal welfare. To monitor compliance with animal welfare regulations and ensure good veterinary and scientific practices, education and training need to be intensified. Adherence to the items listed in the PREPARE and ARRIVE guidelines and the Gold Standard Publication Checklist (GSPC) should become legally binding.</p> Kathrin Herrmann Paul Flecknell ##submission.copyrightStatement## 2018-09-14 2018-09-14 36 1 65 80 10.14573/altex.1804011 Botulinum toxin testing on animals is still a Europe-wide issue <p>There have been significant developments in the use of animals to test Botulinum toxin products in Europe in recent years. This paper summarises and discusses these from the perspective of the animal protection organisation. A cell-based assay has been validated by Allergan and is now being used for the replacement of the mouse bioassay for the batch testing of their Botulinum toxin A products. Two further companies (Merz and Ipsen) have recently validated similar cell-based assays to replace animals in their batch testing.<br>However, the number of animals being used in batch tests across Europe remains at record levels; an estimated 400,000 animals per year, based on official statistics and non-technical summaries. There are concerns from animal protection organisations about the authorisation of animal testing for Botulinum toxin products that are to be used for aesthetic purposes. Furthermore, should testing for companies that have not yet implemented the alternative method continue to be permitted under EU Directive 2010/63 on the use of animals for scientific purposes? Whilst we are on the cusp of an era where the mouse bioassay has been replaced for the potency testing of Botulinum toxin A for injection, it is important that Europe sees a reduction of animal testing in real terms.</p> Katy Taylor Corina Gericke Laura Rego Alvarez ##submission.copyrightStatement## 2018-10-10 2018-10-10 36 1 81 90 10.14573/altex.1807101 Chemical hazard prediction and hypothesis testing using quantitative adverse outcome pathways <p>Current efforts in chemical safety are focused on utilizing human <em>in vitro </em>or alternatives to animal data in a bio­logical pathway context. However, it remains unclear how biological pathways, and toxicology data developed in that context, can be used to quantitatively facilitate decision-making. The objective of this work is to determine if hypothesis testing using adverse outcome pathways (AOPs) can provide quantitative chemical hazard predictions. Current methods for predicting hazards of chemicals in a biological pathway context were extensively reviewed, spe­cific case studies examined, and computational modeling used to demonstrate quantitative hazard prediction based on an AOP. Since AOPs are chemically agnostic, we propose that AOPs function as hypotheses for how specific chemicals may cause adverse effects via specific pathways. Three broad approaches were identified for testing the hypothesis with AOPs, semi-quantitative weight of evidence, probabilistic, and mechanistic modeling. We then demonstrate how these approaches could be used to test hypotheses using high throughput <em>in vitro </em>data and data from alternatives to animal testing. Finally, we discuss standards in development and documentation that would facilitate use in a regu­latory context. We conclude that quantitative AOPs provide a flexible hypothesis framework for predicting chemical hazards, which accommodates a wide range of approaches that are useful at many stages and build upon one another to become increasingly quantitative.</p> Edward J. Perkins Kalyan Gayen Jason E. Shoemaker Philipp Antczak Lyle Burgoon Francesco Falciani Steve Gutsell Geoff Hodges Aude Kienzler Dries Knapen Mary McBride Catherine Willett Francis J. Doyle III Natàlia Garcia-Reyero ##submission.copyrightStatement## 2018-10-16 2018-10-16 36 1 91 102 10.14573/altex.1808241 Characterizing sources of variability in zebrafish embryo screening protocols <p>There is a need for fast, efficient, and cost-effective hazard identification and characterization of chemical hazards. This need is generating increased interest in the use of zebrafish embryos as both a screening tool and an alternative to mammalian test methods. A Collaborative Workshop on Aquatic Models and 21<sup>st </sup>Century Toxicology identified the lack of appropriate and consistent testing protocols as a challenge to the broader application of the zebrafish embryo model. The National Toxicology Program established the Systematic Evaluation of the Application of Zebrafish in Toxicology (SEAZIT) initiative to address the lack of consistent testing guidelines and identify sources of variability for zebrafish-based assays. This report summarizes initial SEAZIT information-gathering efforts. Investigators in aca­demic, government, and industry laboratories that routinely use zebrafish embryos for chemical toxicity testing were asked about their husbandry practices and standard protocols. Information was collected about protocol components including zebrafish strains, feed, system water, disease surveillance, embryo exposure conditions, and endpoints. Liter­ature was reviewed to assess issues raised by the investigators. Interviews revealed substantial variability across design parameters, data collected, and analysis procedures. The presence of the chorion and renewal of exposure medium (static versus static-renewal) were identified as design parameters that could potentially influence study outcomes and should be investigated further with studies to determine chemical uptake from treatment solution into embryos. The information gathered in this effort provides a basis for future SEAZIT activities to promote more consistent practices among researchers using zebrafish embryos for toxicity evaluation.</p> Jon T. Hamm Patricia Ceger David Allen Matt Stout Elizabeth A. Maull Greg Baker Amy Zmarowski Stephanie Padilla Edward Perkins Antonio Planchart Donald Stedman Tamara Tal Robert L. Tanguay David C. Volz Mitch S. Wilbanks Nigel J. Walker ##submission.copyrightStatement## 2018-11-10 2018-11-10 36 1 103 120 10.14573/altex.1804162 Preclinical alternative model for analysis of porous scaffold biocompatibility in bone tissue engineering <p>Porous scaffolds represent a potential approach to repair critical-size bone defects. Vascularization is essential for bone formation and healing. This study establishes a method to monitor angiogenesis within porous biopolymer scaffolds made on the basis of polyhydroxybutyrate and chitosan. We used the chick and quail chorioallantoic membrane (CAM) assay as an alternative <em>in vivo </em>model to study the formation of new blood vessels inside the scaffold structure. The chemical properties of the biopolymer scaffold matrix surface were characterized as well as the tissue reaction of the CAM. Placing a piece of polymer scaffold on the CAM resulted in a vascular reaction documented visually and by ultrasound biomicroscopy. Histological analysis showed a myofibroblast reaction (smooth muscle actin-positive cells) without excessive collagen deposition. Cell invasion into the implant was observed and the presence of a vascular network was confirmed by identifying hemangioblasts and endothelial cells of quail origin using the QH1 marker. The CAM assay is a rapid and easy way to test biocompatibility and vasculogenic potential of new candidate scaffolds for bone tissue bioengineering while respecting the 3Rs.</p> <p>&nbsp;</p> <p>&nbsp;</p> Eva Petrovova Maria Giretova Alena Kvasilova Oldrich Benada Jan Danko Lubomir Medvecky David Sedmera ##submission.copyrightStatement## 2018-11-23 2018-11-23 36 1 121 130 10.14573/altex.1807241 Zebrafish embryo and acute fish toxicity test show similar sensitivity for narcotic compounds <p>The zebrafish embryo test has been discussed as an alternative test system to provide data on acute fish toxicity required by diverse regulations. A meta-analysis of zebrafish embryo acute toxicity (ZFET) data has revealed conflicting evidence that narcotic compounds (i.e., compounds with baseline toxicity) may exhibit weaker sensitivity in the ZFET compared to the acute (adult) fish toxicity test (AFT). Therefore, six compounds with presumably narcotic or unknown mode of action, and for which a previous meta-analysis had indicated weaker sensitivity, were experimentally analyzed for their fish embryo acute toxicity and exposure concentrations were monitored. The data indicated that ZFET and AFT for the selected compounds had similar sensitivity and differences were in the range of species differences of the AFT.</p> Anita Birke Stefan Scholz ##submission.copyrightStatement## 2018-10-29 2018-10-29 36 1 131 135 10.14573/altex.1808101 The current status of alternative methods for cosmetics safety assessment in China Fei-ya Luo Zhe Su Jing Wu Feng-lan Zhang Shu-xia Xing Gang-li Wang Yong Lu ##submission.copyrightStatement## 2018-11-19 2018-11-19 36 1 136 139 10.14573/altex.1808021 Science instead of animal experiments Corina Gericke Silke Strittmatter ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 140 141 10.14573/altex.1811291 Alternative methods to replace or reduce animal models in biomedical research Florenza Lüder Ripoli Manuela Buettner André Bleich ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 141 142 10.14573/altex.1812171 Is there an end in sight for animal testing? Sonja von Aulock ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 142 144 10.14573/altex.1812121 Advanced in vitro models analysis Katarzyna S. Kopanska Markus Rimann Sandra Latenser Michael Raghunath ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 144 147 10.14573/altex.1812131 Corners Sonja von Aulock ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 148 153 Chemical concentrations in cell culture compartments (C5) – concentration definitions <p>Some laboratory issues are taken for granted as they seem to be simple and not worth much thought. This applies to “concentrations of a chemical tested for bioactivity/toxicity”. Can there be any issue about weighing a compound, diluting it in culture medium and calculating the final mass (or particle number)-to-volume ratio? We discuss here some basic concepts about concentrations and their units, addressing also differences between “dose” and “concentration”. The problem of calculated nominal concentrations not necessarily corresponding to local concentrations (relevant for biological effects of a chemical) is highlighted. We present and exemplify different concentration measures, for instance those relying on weight, volume, or particle number of the test compound in a given volume; we also include normalizations to the mass, protein content, or cell number of the reference system. Interconversion is discussed as a major, often unresolved, issue. We put this into the context of the overall objective of defining concentrations, i.e., the determination of threshold values of bioactivity (e.g., an EC50). As standard approach for data display, the negative decadic logarithm of the molar concentrations (–log(M)) is recommended here, but arguments are also presented for exceptions from such a rule. These basic definitions are meant as a foundation for follow-up articles that examine the concepts of nominal, free, and intracellular concentrations to provide guidance on how to relate <em>in vitro </em>concentrations to <em>in vivo </em>doses by <em>in vitro</em>-to-<em>in vivo </em>extrapolation (IVIVE) in order to advance the use of new approach methods (NAM) in regulatory decision making.</p> Jaffar Kisitu Susanne Hougaard Bennekou Marcel Leist ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 154 160 10.14573/altex.1901031 Imprint Sonja von Aulock ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 U3 U3 EUSAAT 2019 Helmut Appl ##submission.copyrightStatement## 2019-01-08 2019-01-08 36 1 U4 U4