Online first

June 21, 2016

Consensus Report

Advancing toxicology research using in vivo high throughput toxicology with small fish models

Antonio Planchart, Carolyn J. Mattingly, David Allen, Patricia Ceger, Warren Casey, David Hinton, Jyotshna Kanungo, Seth W. Kullman, Tamara Tal, Maria Bondesson, Shawn M. Burgess, Con Sullivan, Carol Kim, Mamta Behl, Stephanie Padilla, David M. Reif, Robert L. Tanguay and Jon Hamm

doi: 10.14573/altex.1601281



Small freshwater fish models, especially zebrafish,offer advantages over traditional rodent models, including low maintenance and husbandry costs, high fecundity, genetic diversity, physiology similar to that of traditional biomedical models, and reduced animal welfare concerns. The Collaborative Workshop on Aquatic Models and 21st Century Toxicology was held at North Carolina State University on May 5–6, 2014, in Raleigh, North Carolina, USA. Participants discussed the ways in which small fish are being used as models to screen toxicants and understand mechanisms of toxicity. Workshop participants agreed that the lack of standardized protocols is an impediment to broader acceptance of these models, whereas development of standardized protocols, validation, and subsequent regulatory acceptance would facilitate greater usage. Given the advantages and increasing application of small fish models, there was widespread interest in follow-up workshops to review and discuss developments in their use. In this article, we summarize the recommendations formulated by workshop participants to enhance the utility of small fish species in toxicology studies, as well as many of the advances in the field of toxicology that resulted from using small fish species, including advances in developmental toxicology, cardiovascular toxicology, neurotoxicology, and immunotoxicology. We alsoreview many emerging issues that will benefit from using small fish species, especially zebrafish, and new technologies that will enable using these organisms to yield results unprecedented in their information content to better understand how toxicants affect development and health.


Keywords: aquatic models, 21st century toxicology, alternatives



June 2, 2016

Short Communication

Bridging the gap between regulatory acceptance and industry use of non-animal methods

Amy J. Clippinger, Erin Hill, Rodger Curren and Patricia Bishop

doi: 10.14573/altex.1601311



Collaboration between industry and regulators resulted in the development of a decision tree approach using in vitro or ex vivo assays to replace animal tests when determining the eye irritation potential of antimicrobial cleaning products (AMCPs) under the United States Environmental Protection Agency (EPA) Office of Pesticide Programs’ hazard classification and labeling system. A policy document issued by the EPA in 2013 and updated in 2015 describes the alternate testing framework that industry could apply to new registrations of AMCPs and, on a case-by-case basis, to conventional pesticide products. Despite the collaborative effort, the availability of relevant non-animal methods, and the EPA’s change in policy, only a limited number of AMCPs have been registered using the framework. Companies continue to conduct animal tests when registering AMCPs due to various challenges surrounding adoption of the new testing framework; however, recent discussions between industry, regulators, and other interested parties have identified ways these challenges may be overcome. In this article we explore how use of the alternate framework could be expanded through efforts such as increasing international harmonization, more proactively publicizing the framework, and enhancing the training of regulatory reviewers. Not only can these strategies help to increase use of the EPA alternate eye irritation framework, they can also be applied to facilitate the uptake of other alternative approaches to animal testing in the future.

Keywords: non-animal testing strategy, eye hazard classification, EPA, antimicrobial cleaning products, pesticides



May 19, 2016

Research Article

MUTZ-3 Langerhans Cell maturation and CXCL12 independent migration in reconstructed human gingiva

Ilona J. Kosten, Sander W. Spiekstra, Tanja D. de Gruijl and Susan Gibbs

doi: 10.14573/altex.1510301



Here we describe a reconstructed full thickness human oral mucosa (gingiva) equivalent with integrated Langerhans Cells (GE-LC) and use it to compare LC activation and migration from oral versus skin epithelium. The physiologically representative models consist of differentiated reconstructed epithelium (keratinocytes and Langerhans-like cells derived from the MUTZ-3 cell line) on a fibroblast-populated collagen hydrogel which serves as a lamina propria for gingiva and dermis for skin. Topical exposure of GE-LC and the skin equivalent (SE-LC) to sub-toxic concentrations of the allergens cinnamaldehyde, resorcinol and nickel sulphate, resulted in LC migration out of the epithelia. Neutralizing antibody to CXCL12 blocked allergen-induced LC migration in SE-LC but not in GE-LC. Also, gingival fibroblasts secreted very low amounts of CXCL12 compared to skin fibroblasts even when stimulated with rhTNFα or rhIL-1α. Surprisingly, cinnamaldehyde exposure of GE-LC resulted in an increase in MUTZ-3 LC and CD83 mRNA in the hydrogel but did not result in an increase in CD1a+ cells in the collagen hydrogel (as was observed for SE-LC. These results indicate that in gingiva, upon allergen exposure, MUTZ-3 LC migrate in a CXCL12 independent manner from epithelium-to-lamina propria and in doing so mature become CD1a- and increase CD83+ mRNA. These physiologically relevant in vitro models which not only are human but which also resemble specific tissues, may aid in the identification of factors regulating immune stimulation which in turn will aid the development of therapeutic interventions for allergy and inflammation, anti-cancer vaccines as well as improving diagnostics for skin and oral allergy.


Keywords: dendritic cell, skin equivalent, gingiva equivalent, in vitro, allergen



May 15, 2016

Research Article

A first vascularized skin equivalent as an alternative to animal experimentation

Florian Groeber, Lisa Engelhardt, Julia Lange, Szymon Kurdyn, Freia F. Schmid, Christoph Rücker, Stephan Mielke, Heike Walles and Jan Hansmann

doi: 10.14573/altex.1604041


Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.


Keywords: skin equivalents, alternative to animal testing, vascularization, tissue engineering


May 15, 2016

t4 Workshop Report

Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing

Uwe Marx, Tommy B. Andersson, Anthony Bahinski, Mario Beilmann, Sonja Beken, Flemming R. Cassee, Murat Cirit, Mardas Daneshian, Susan Fitzpatrick, Olivier Frey, Claudia Gaertner, Christoph Giese, Linda Griffith, Thomas Hartung, Minne B. Heringa, Julia Hoeng, Wim H. de Jong, Hajime Kojima, Jochen Kuehnl, Marcel Leist, Andreas Luch, Ilka Maschmeyer, Dmitry Sakharov, Adrienne J. A. M. Sips, Thomas Steger-Hartmann, Danilo A. Tagle, Alexander Tonevitsky, Tewes Tralau, Sergej Tsyb, Anja van de Stolpe, Rob Vandebriel, Paul Vulto, Jufeng Wang, Joachim Wiest, Marleen Rodenburg and Adrian Roth

doi: 10.14573/altex.1603161



The recent advent of microphysiological systems – microfluidic biomimetic devices that aspire to emulate the biology of human tissues, organs and circulation in vitro – is envisaged to enable a global paradigm shift in drug development. An extraordinary US governmental initiative and various dedicated research programs in Europe and Asia have led recently to the first cutting-edge achievements of human single-organ and multi-organ engineering based on microphysiological systems. The expectation is that test systems established on this basis would model various disease stages, and predict toxicity, immunogenicity, ADME profiles and treatment efficacy prior to clinical testing. Consequently, this technology could significantly affect the way drug substances are developed in the future. Furthermore, microphysiological system-based assays may revolutionize our current global programs of prioritization of hazard characterization for any new substances to be used, for example, in agriculture, food, ecosystems or cosmetics, thus, replacing laboratory animal models used currently. Thirty-six experts from academia, industry and regulatory bodies present here the results of an intensive workshop (held in June 2015, Berlin, Germany). They review the status quo of microphysiological systems available today against industry needs, and assess the broad variety of approaches with fit-for-purpose potential in the drug development cycle. Feasible technical solutions to reach the next levels of human biology in vitro are proposed. Furthermore, key organ-on-a-chip case studies, as well as various national and international programs are highlighted. Finally, a roadmap into the future is outlined, to allow for more predictive and regulatory-accepted substance testing on a global scale.


Keywords: microphysiological systems, organ-on-a-chip, in vitro models, predictive toxicology, drug testing


May 9, 2016

Short Communication

Organotypic retinal explant cultures as in vitro alternative for diabetic retinopathy studies

Joaquín Valdés, Laura Trachsel-Moncho, Ayse Sahaboglu, Dragana Trifunović, María Miranda, Marius Ueffing, François Paquet-Durand and Oliver Schmachtenberg

doi: 10.14573/altex.1603111


Diabetic retinopathy (DR) is a major cause of vision loss and one of the most common and debilitating complications of diabetes. Research to prevent DR is hindered by a lack of experimental model systems that faithfully reproduce the disease pathology, in particular for type 2 diabetes, which requires prolonged disease progression in animals to develop some hallmarks of DR. Here, we introduce an alternative in vitro model system for DR, based on serum-free, organotypic rodent retinal explant cultures, which allow physiological and pharmacological manipulation of the retina for up to two weeks under tightly controlled conditions. Retinal explant cultures have the advantage of isolating direct neuronal consequences of diabetic conditions from indirect systemic effects mediated via the retinal vasculature or the immune system. Exposed to conditions emulating type 1 or type 2 diabetes, retinal explants displayed elevated cell death rates among inner retinal neurons as well as photoreceptors, with a particularly strong loss of cone photoreceptors. Our results support a direct impact of diabetic conditions on retinal neurons and may help explain color vision defects observed in DR patients. This serum-free in vitro DR model avoids the animal suffering of established DR models and reduces the overall number of animals needed for such research. It should prove useful to study the mechanisms of neuronal cell death caused by DR and to screen for potential future DR treatments.

Keywords: retina, diabetes, animal models, photoreceptors, cell death


May 8, 2016

Research Article

Ex vivo assessment of testicular toxicity induced by carbendazim and iprodione, alone or in a mixture

Cédric Pisani, Sébastien Voisin, Karim Arafah, Philippe Durand, Marie-Hélène Perrard, Marie-Roberte Guichaoua, Philippe Bulet and Odette Prat

doi: 10.14573/altex.1601253


Supplementary file 1 (.xlsx)

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Supplementary file 5 (.xlsx)


To measure the testicular toxicity of two fungicides (carbendazim and iprodione), alone or in a mixture, we used a rat ex vivo model of seminiferous tubules, greatly reducing the number of rodents used, in accordance with the 3R rule (Replacement, Reduction, and Refinement). This model allows the representation of puberty, a critical life period with regard to endocrine disruptors. The cellular modifications were followed for three weeks through transcriptomic and proteomic profiling analysis. A quantitative and comparative method was developed to estimate how known pathways were disturbed by each substance. This pathway-driven analysis revealed a strong alteration of steroidogenesis and an impairment of meiosis in all cases, albeit the initial molecular events were different for both substances. The ex vivo cytogenetic analysis confirmed that both fungicides alter the course of the first meiotic prophase. In addition, the mixture of both substances triggered effects greater than the sum of their cumulative effects and compromised future sperm motility after a shorter time of exposure compared with the fungicides tested separately. The alliance of an ex vivo culture with “omics” strategies complemented with a physiological examination is a powerful combination of tools for testing substances, separately or in a mixture, for their testicular toxicity. In particular, proteomics allowed the identification of systematically differentially expressed proteins in the secretomes of exposed cultures, such as FUCO and PEBP1, two proteins linked with the motility and fertilizing ability of spermatozoa, respectively. These proteins may be potential biomarkers of testicular dysfunction and infertility.


Keywords: pesticides, toxicogenomics, spermatogenesis, endocrine disruption, biomarker


April 27, 2016

Concept Article

International regulatory needs for development of an IATA for non-genotoxic carcinogenic chemical substances

Miriam N. Jacobs, Annamaria Colacci, Kimmo Louekari, Mirjam Luijten, Betty C. Hakkert, Martin Paparella and Paule Vasseur

doi: 10.14573/altex.1601201



Although regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests. If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, which allows the detection of non-genotoxic carcinogens (NGTxC). However, under most chemical regulations the cancer bioassay is rarely requested, specific requests to obtain information on non-genotoxic mechanisms of carcinogenicity are few, and there are no OECD approved screening methods. When the in vitro genotoxicity battery is negative, usually no further carcinogenicity testing is requested. Consequently NGTxC might remain unidentified and therefore the risks they may pose to human health will not be managed. In contrast to genotoxic carcinogens NGTxCact through a large variety of specific mechanisms, and a panel of tests covering multiple biological traits will be needed.

The development of an Integrated Approach to Testing and Assessment (IATA) of NGTxC could assist regulatory decision makers. We examine what NGTxC are and discuss chemical regulatory requirements and limitations. With a strong drive to reduce animal testing and costs in mind, it is essential that proper and robust alternatives for animal testing (3Rs) methods for addressing non-genotoxic modes of action are developed and used. Therefore relevant in vitro mechanisms and assays are described and tentatively organized in levels of information, indicating both a possible structure of the future IATA for NGTxC and associated OECD Test Guideline development priorities.


Keywords: non-genotoxic carcinogens, integrated approaches to testing and assessment of chemicals


March 24, 2016

Research Article

Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons?

Anke M. Tukker, Martje W.G.D.M. de Groot, Fiona M.J. Wijnolts, Emma E.J. Kasteel, Laura Hondebrink and Remco H.S. Westerink

doi: 10.14573/altex.1510091


Current neurotoxicity testing heavily relies on expensive, time consuming and ethically debated in vivo animal experiments that are unsuitable for screening large number of chemicals. Consequently, there is a clear need for (high-throughput) in vitro test strategies, preferably using human cells as this increases relevance and eliminates the need for interspecies translation. However, human stem cell-derived neurons used to date are not well characterised, require prolonged differentiation and are potentially subject to batch-to-batch variation, ethical concerns and country-specific legislations. Recently, a number of human induced pluripotent stem cell (iPSC)-derived neurons became commercially available that may circumvent these concerns.

We therefore used immunofluorescent stainings to demonstrate that human iPSC-derived neurons from various suppliers form mixed neuronal cultures, consisting of different types of (excitatory and inhibitory) neurons. Using multi-well microelectrode array (mwMEA) recordings, we demonstrate that these human iPSC-derived cultures develop spontaneous neuronal activity over time, which can be modulated by different physiological, toxicological and pharmacological compounds. Additional single cell calcium imaging illustrates the presence of functional GABA, glutamate, and acetylcholine receptors as well as voltage-gated calcium channels.

While human iPSC-derived neuronal cultures appear not yet suitable to fully replace the rat primary cortical model, our data indicate that these rapidly differentiating, commercially available human iPSC-derived neuronal cultures are already suitable for in vitro prioritisation and effect screening studies. Further characterisation and toxicological validation is now required to facilitate acceptance and large-scale implementation of these animal-free, physiologically-relevant human iPSC-based modelsfor future neurotoxicity testing.

Keywords: In vitro neurotoxicity screening, multi-electrode array (MEA), human induced pluripotent stem cell-derived neurons, mixed neuronal cultures, alternatives to animal testing



March 17, 2016

Research Article

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier

Anna Kuehn, Stephanie Kletting, Cristiane de Souza Carvalho-Wodarz, Urska Repnik, Gareth Griffiths, Ulrike Fischer, Eckart Meese, Hanno Huwer, Dagmar Wirth, Tobias May, Nicole Schneider-Daum and Claus-Michael Lehr

doi: 10.14573/altex.1511131



This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.


Keywords: Lentivirus immortalization, ATI cells, pulmonary drug delivery, inhalation toxicology


March 9, 2016

Research Article

Contribution of animal models to contemporary understanding of Attention Deficit Hyperactivity Disorder

Constança Carvalho, Mariana Vieira Crespo, Luisa Ferreira Bastos, Andrew Knight and Luís Vicente

doi: 10.14573/altex.1507311


Attention Deficit Hyperactivity Disorder (ADHD) is a poorly understood neurodevelopmental disorder of multifactorial origin. Animal-based research has been used to investigate ADHD aetiology, pathogenesis and treatment, but the efficacy of this research for patientshas not yet been systematically evaluated. However, such evaluation is important, given the resource consumption and ethical concerns incurred by animal use. Accordingly, we used the citation tracking facility within Web of Science to locate original research performed on animal models related to ADHD, prior to 2010.Human medical papers citing those animal studies were carefully analyzed by two independent raters to evaluate the contribution of the animal to the human studies. 211 publications describing relevantanimal studies were located. Approximately half (3,342) of their 6,406 citations were by other animal studies. 446 human medical papers cited 121 of these 211 animal studies, a total of 500 times. 254 of these 446 papers were human studies of ADHD. However, only eight animal papers (cited 10 times) were relevant to the hypothesis of the human medical study in question. Three of these eight papers described results from both human and animal studies, but their citations solely referred to the human data. Five animal research papers were relevant to the hypotheses of the applicable human medical papers. Citation analysis indicates that animal research has contributed very little to contemporary understanding of ADHD. To ensure optimal allocation of Research & Development funds targeting this disease the contribution of other research methods should be similarly evaluated.


Keywords: ADHD, animal models, citation analysis


January 29, 2016

Research Article

The Elnady Technique: An innovative, new method for tissue preservation

Fawzy A. Elnady

doi: 10.14573/altex.1511091



At the Faculty of Veterinary Medicine, Cairo University, there is an increasing number of students but a limited availability of animal cadavers used for dissection, and student exposure to formalin is a known hazard. In order to address these challenges, a new method for tissue preservation was developed, the “Elnady Technique.” This method is a modified form of plastination, where the chemicals used are not patented, are inexpensive and locally available, and the process is performed at room temperature. The produced specimens are realistic, durable, have no offensive odor, and are dry, soft and flexible. They can be used to replace the use of animals killed for teaching basic anatomy, embryology, pathology, parasitology and forensic medicine. They have great potential to support training in clinical skills and surgery, including for clinical examination, endoscopy, surgical sutures, and obstetrics simulation.


Keywords: veterinary anatomy, alternatives, plastination, preservation


December 22, 2015

Research Article

Bitter tastant responses in the amoeba Dictyostelium correlate with rat and human taste assays­

Marco Cocorocchio, Robert Ives, David Clapham, Paul L. R. Andrews and Robin S. B. Williams

doi: 10.14573/altex.1509011

Supplementary file (.avi)


Treatment compliance is reduced when pharmaceutical compounds have a bitter taste and this is particularly marked for paediatric medications. Identification of bitter taste liability during drug discovery utilises the rat in vivo brief access taste aversion (BATA) test which apart from animal use is time consuming with limited throughput. We investigated the suitability of using a simple, non-animal model, the amoeba Dictyostelium discoideum to investigate taste-related responses and particularly identification of compounds with a bitter taste liability. The effect of taste-related compounds on Dictyostelium behaviour following acute exposure (15 minutes) was monitored. Dictyostelium did not respond to salty, sour, umami or sweet tasting compounds, however, cells rapidly responded to bitter tastants. Using time-lapse photography and computer-generated quantification to monitor changes in cell membrane movement, we developed an assay to assess the response of Dictyostelium to a wide range of structurally diverse known bitter compounds and blinded compounds. Dictyostelium showed varying responses to the bitter tastants, with IC50 values providing a rank order of potency. Comparison of Dictyostelium IC50 values to those observed in response to a similar range of compounds in the rat in vivo brief access taste aversion test showed a significant (p = 0.0172) positive correlation between the two models, and additionally a similar response to that provided by a human sensory panel assessment test. These experiments demonstrate that Dictyostelium may provide a suitable model for early prediction of bitterness for novel tastants and drugs. Interestingly, a response to bitter tastants appears conserved from single-celled amoebae to humans. 

Keywords: BATA test, bitter tastants, Dictyostelium discoideum, taste aversion, replacement


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