Online first

July 27, 2016

Research Article

Design of a high-throughput human neural crest cell migration assay to indicate potential developmental toxicants

Johanna Nyffeler, Christiaan Karreman, Heidrun Leisner, Yong Jun Kim, Gabsang Lee, Tanja Waldmann and Marcel Leist

doi: 10.14573/altex.1605031

Supplementary file (.pdf)



Migration of neural crest cells (NCCs) is one of the pivotal processes of human fetal development. Malformations arise, if NCC migration and differentiation are impaired genetically or by toxicants. In the currently available test systems for migration inhibition of NCC (MINC), the manual generation of a cell-free space results in extreme operator dependencies, and limits throughput. Therefore, a new test format was established here. The assay avoids scratching by plating cells around a commercially available circular stopper. Removal of this barrier after cell attachment initiates migration. This microwell-based circular migration zone NCC function assay (cMINC) was further optimized for toxicological testing of human pluripotent stem cell (hPSC)-derived NCCs. The challenge of automated image processing to obtain data on viability and migration was addressed by development of a software made generally available for downloading. To optimize the biological system, data on cell proliferation were obtained by labelling of replicating cells, and by careful assessment of cell viability for each experimental sample. The role of cell proliferation as experimental confounder was tested experimentally by performance of the cMINC in the presence of the proliferation-inhibiting drug cytosine arabinoside (AraC), and by a careful evaluation of mitotic events over time. Data from these studies led to an adaptation of the test protocol, so that toxicant exposure was limited to 24 h. Under these conditions, a prediction model was developed that allowed classification of toxicants as either being inactive, leading to unspecific cytotoxicity or specifically inhibiting NC migration at non-cytotoxic concentrations.


Keywords: cell tracking, cell proliferation, high content imaging, developmental toxicity, human stem cells



July 25, 2016

t4 Workshop Report

Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use

Michael Aschner, Sandra Ceccatelli, Mardas Daneshian, Ellen Fritsche, Nina Hasiwa, Thomas Hartung, Helena T. Hogberg, Marcel Leist, Abby Li, William R. Mundy, Stephanie Padilla, Aldert H. Piersma, Anna Bal-Price, Andrea Seiler, Remco Westerink, Bastian Zimmer and Pamela Lein

doi: 10.14573/altex.1604201



There is a paucity of information concerning the developmental neurotoxicity (DNT) hazard posed by industrial and environmental chemicals. New testing approaches will most likely be based on batteries of alternative and complementary (non-animal) tests. As DNT is assumed to result from the modulation of fundamental neurodevelopmental processes (such as neuronal differentiation, precursor cell migration or neuronal network formation) by chemicals, the first generation of alternative DNT tests target these processes. The advantage of such types of assays is that they capture toxicants with multiple targets and modes-of-action. Moreover, the processes modelled by the assays can be linked to toxicity endophenotypes, i.e. alterations in neural connectivity that form the basis for neurofunctional deficits in man. The authors of this review convened in a workshop to define criteria for the selection of positive/negative controls, to prepare recommendations on their use, and to initiate the setup of a directory of reference chemicals. For initial technical optimization of tests, a set of >50 endpoint-specific control compounds was identified. For further test development, an additional "test" set of 33 chemicals considered to act directly as bona fide DNT toxicantsis proposed, and each chemical is annotated to the extent it fulfills these criteria. A tabular compilation of the original literature used to select the test set chemicals provides information on statistical procedures, and toxic/non-toxic doses (both for pups and dams). Suggestions are provided on how to use the >100 compounds (including negative controls) compiled here to address specificity, adversity and use of alternative test systems.


Keywords: neurotoxicity, specificity, test development, AOP, validation



July 21, 2016

Research Article

Assigning ethical weights to clinical signs observed during toxicity testing

Joakim Ringblom, Elin Törnqvist, Sven Ove Hansson, Christina Rudén and Mattias Öberg

doi: 10.14573/altex.1512211


Reducing the number of laboratory animals used and refining experimental procedures to enhance animal welfare are fundamental questions to be considered in connection with animal experimentation. Here, we explored the use of cardinal ethical weights for clinical signs and symptoms in rodents by conducting Trade-Off interviews with members of Swedish Animal Ethics Committees in order to derive such weights for nine typical clinical signs of toxicity. The participants interviewed represent researchers, politically nominated laypersons and representatives of animal welfare organizations. We observed no statistically significant differences between these groups with respect to the magnitude of the ethical weights assigned, even though the political nominees tended to assign lower weights. Hunched posture was considered the most severe clinical sign and body weight loss the least severe. The ethical weights assigned varied considerably between individuals, from none to infinite value, indicating discrepancies in prioritization of reduction and refinement. Cardinal ethical weights may be utilized to include both animal welfare refinement and reduction of animal use in designing as well as in retrospective assessments of animal experiments. Such weights may also be used to estimate ethical costs of animal experiments.  


Keywords: 3Rs, animal ethics, ethical weights, ethical committees, toxicity testing         



July 7, 2016; updated July 11, 2016

Research Article

3-D nasal cultures: Systems toxicological assessment of a candidate modified-risk tobacco product

Anita R. Iskandar, Carole Mathis, Florian Martin, Patrice Leroy, Alain Sewer, Shoaib Majeed, Diana Kuehn, Keyur Trivedi, Davide Grandolfo, Maciej Cabanski, Emmanuel Guedj, Celine Merg, Stefan Frentzel, Nikolai V. Ivanov, Manuel C. Peitsch and Julia Hoeng

doi: 10.14573/altex.1605041


Supplementary file (.pdf)


In vitro toxicology approaches have evolved, from a focus on molecular changes within a cell to understanding of toxicity-related mechanisms in systems that can mimic the in vivo environment. The recent development of three dimensional (3-D) organotypic nasal epithelial culture models offers a physiologically robust system for studying the effects of exposure through inhalation. Exposure to cigarette smoke (CS) is associated with nasal inflammation; thus the nasal epithelium is relevant for evaluating the pathophysiological impact of CS exposure. The present study investigated further the application of in vitro human 3-D nasal epithelial culture models for toxicological assessment of inhalation exposure. Aligned with 3Rs strategy, this study aimed to explore the relevance of a human 3-D nasal culture model to assess the toxicological impact of aerosols generated from a candidate modified risk tobacco product (cMRTP), the Tobacco Heating System (THS)2.2, as compared with smoke generated from reference cigarette 3R4F. A series of experimental repetitions where multiple concentrations of THS2.2 aerosol and 3R4F smoke were applied, were conducted to obtain reproducible measurements to understandthe cellular/molecular changes that occur following exposure. In agreement with the Vision and Strategy of the Toxicity Testing in the 21st Century, this study implemented a systems toxicology approach and found that for all tested concentrations, the impact of 3R4F smoke was substantially greater than that of THS2.2 aerosol in terms of cytotoxicity levels, alterations in the tissue morphology, secretion of pro-inflammatory mediators, impaired ciliary function, and increased perturbed transcriptomes and miRNA expression profiles.


Keywords: air-liquid interface, organotypic culture, cigarette smoke, modified risk tobacco product, systems toxicology


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 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)

Supplementary file 2 (.xlsx)

Supplementary file 3 (.xlsx)

Supplementary file 4 (.xlsx)

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


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