Online first

November 24, 2016, updated November 28, 2016

Research Article

A human brain microphysiological system derived from induced pluripotent stem cells to study neurological diseases and toxicity

David Pamies, Paula Barreras, Katharina Block, Georgia Makri, Anupama Kumar, Daphne Wiersma, Lena Smirnova, Ce Zhang, Joseph Bressler, Kimberly M. Christian, Georgina Harris, Guo-li Ming, Cindy J. Berlinicke, Kelly Kyro, Hongjun Song, Carlos A. Pardo, Thomas Hartung and Helena T. Hogberg

doi: 10.14573/altex.1609122


Supplementary file1

Supplementary file 2


Summary

Human in-vitro models of brain neurophysiology are needed to investigate molecular and cellular mechanisms associated with neurological disorders and neurotoxicity. We have developed a reproducible iPSC-derived human 3D brain microphysiological system (BMPS), comprised of differentiated mature neurons and glial cells (astrocytes and oligodendrocytes) that reproduce neuronal-glial interactions and connectivity. BMPS mature over eight weeks and show the critical elements of neuronal function: synaptogenesis and neuron-to-neuron (e.g. spontaneous electric field potentials) and neuronal-glial interactions (e.g. myelination), that mimic the microenvironment of the central nervous system, rarely seen in vitro so far. The BMPS shows 40% overall myelination after 8 weeks of differentiation. Myelin was observed by immunohistochemistry and confirmed by confocal microscopy 3D reconstruction and electron microscopy. These findings are of particular relevance since myelin is crucial for proper neuronal function and development. The ability to assess oligodendroglia function and mechanisms associated with myelination in this BMPS model provide an excellent tool for future studies of neurological disorders such as multiple sclerosis and other demyelinating diseases. Thus, the BMPS provides a suitable and reliable model to investigate neuron-neuroglia function in neurotoxicology or other pathogenic mechanisms.

 

Keywords: 3D culture, CNS, myelination, microphysiological system, brain


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November 14, 2016

Consensus Report

Systems biology for organotypic cell cultures

Sonia Grego, Edward R. Dougherty, Francis J. Alexander, Scott S. Auerbach, Brian R. Berridge, Michael L. Bittner, Warren Casey, Philip C. Cooley, Ajit Dash, Stephen S. Ferguson, Timothy R. Fennell, Brian T. Hawkins, Anthony J. Hickey, Andre Kleensang, Michael N. Liebman, Florian Martin, Elizabeth A. Maull, Jason Paragas, Guilin (Gary) Qiao,

Sreenivasa Ramaiahgari, Susan J. Sumner and Miyoung Yoon

doi: 10.14573/altex.1608221


Summary

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, “organotypic” cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics.

On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.


Keywords: systems toxicology, systems pharmacology, multiscale modeling, 3D models, engineered cell cultures


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November 8, 2016

Research Article

Alternative acute oral toxicity assessment under REACH based on sub-acute toxicity values

Andrea Gissi, Kimmo Louekari, Laurence Hoffstadt, Norbert Bornatowicz and Alberto Martin Aparicio

doi: 10.14573/altex.1609121


Supplementary file


Summary

The REACH Regulation requires information on acute oral toxicity for substances produced or imported in quantities greater than one tonne per year. When registering, animal testing should be used as last resort. The standard acute oral toxicity test requires use of animals. Therefore, the European Chemicals Agency examined whether alternative ways exist to generate information on acute oral toxicity. The starting hypothesis was that low acute oral toxicity can be predicted from the results of low toxicity in oral sub-acute toxicity studies. Proving this hypothesis would allow avoiding acute toxicity oral testing whenever a sub-acute oral toxicity study is required or available and indicates low toxicity. ECHA conducted an analysis of the REACH database and found suitable studies on both acute oral and sub-acute oral toxicities for 1,256 substances. 415 of these substances had low toxicity in the sub-acute toxicity study (i.e. NO(A)EL at or above the classification threshold of 1,000 mg/kg). For 98% of these substances, low acute oral toxicity was also reported (i.e. LD50 above the classification threshold of 2,000 mg/kg). On the other hand, no correlation was found between lower NO(A)ELs and LD50. According to the REACH regulation, this approach for predicting acute oral toxicity needs to be considered as part of a weight of evidence analysis. Therefore, additional sources of information to support this approach are presented.

Ahead of the last REACH registration deadline in 2018, ECHA estimates that registrants of about 550 substances can omit the in vivo acute oral study by using this adaptation.


Keywords: acute toxicity, REACH, weight of evidence, alternative methods, adaptation


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October 24, 2016

Concept Article

Uncertainties of testing methods: What do we (want to) know about carcinogenicity?

Martin Paparella, Annamaria Colacci and Miriam N.Jacobs

doi: 10.14573/altex.1608281

 

Summary

An approach for a systematic description of the uncertainties and complexity of the standard animal testing and assessment approach for carcinogenicity is explored by using a draft OECD Guidance document that was originally developed for reporting defined in vitro approaches to testing and assessment (OECD, 2016). The format is suitable for this re-purposing and it appears that the potential multitude of approaches for integrating and interpreting data from standard animal testing may ultimately be conceptually similar to the challenge of integrating relevant in vitro and in silico data. However this structured approach shall allow 1) fostering interest in developing improved defined in silico and in vitro approaches; 2)the definition of what type of effects should be predicted by the new approach; 3) selection of the most suitable reference data and assessments; 4) definition of the weight that the standard animal reference data should have, compared to human reference data and mechanistic information in the context of assessing the fitness of the new in vitro and in silico approach; 5) definition of a benchmark for the minimum performance of the new approach, based on a conceptual recognition that correlation of alternative assessment results with reference animal results is limited by uncertainties and complexity of the latter. A longer term perspective is indicated for evolving the definition of adversity for classification and regulatory purposes. This work will be further discussed and developed within the OECD expert group on non-genotoxic-carcinogenicity IATA development.

                                                                                  

Keywords: carcinogenicity testing, uncertainty analysis, defined approaches, IATA        

 

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October 21, 2016

Research Article

Human Environmental Disease Network: A computational model to assess toxicology of contaminants

Olivier Taboureau and Karine Audouze

doi: 10.14573/altex.1607201


Supplementary file (.pdf)

 

Summary

During the past decades, many epidemiological, toxicological and biological studies have been performed to assess the role of environmental chemicals as potential toxicants for diverse human disorders. However, the relationships between diseases based on chemical exposure have been rarely studied by computational biology.

We developed a human environmental disease network (EDN) to explore and suggest novel disease-disease and chemical-disease relationships. The presented scored EDN model is built upon the integration on systems biology and chemical toxicology using chemical contaminants information and their disease relationships from the reported TDDB database.

The resulting human EDN takes into consideration the level of evidence of the toxicant-disease relationships allowing including some degrees of significance in the disease-disease associations. Such network can be used to identify uncharacterized connections between diseases. Examples are discussed with type 2 diabetes (T2D).

Additionally, this computational model allows to confirm already know chemical-disease links (e.g. bisphenol A and behavioral disorders) and also to reveal unexpected associations between chemicals and diseases (e.g. chlordane and olfactory alteration), thus predicting which chemicals may be risk factors to human health.

With the proposed human EDN model, it is possible to explore common biological mechanism between two diseases through chemical exposure helping us to gain insight into disease etiology and comorbidity. Such computational approach is an alternative to animal testing supporting the 3R concept.

 

Keywords: computational method, environmental contaminants, predictive toxicology, systems biology, human disease network


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October 21, 2016

Research Article

Keratinocytes improve prediction of sensitization potential and potency of chemicals with THP-1 cells

Jennifer Hennen and Brunhilde Blömeke

doi: 10.14573/altex.1606171

 

Summary

In vitro approaches address key steps of chemical-induced skin sensitization but there is uncertainty how keratinocytes, which play a crucial role not only regarding xenobiotic metabolism but also skin inflammation, impact on chemicals’ potential and potency of dendritic cell activation. We investigated these aspects by coculturing THP-1 cells, as surrogate dendritic cells, with HaCaT keratinocytes. We tested our HaCaT/THP-1 model with a set of 14 sensitizers, containing 7 prohaptens, and 10 non-sensitizers. Compared to THP-1 alone, coculturing resulted in up to 3.1-fold enhanced maximal CD86 and/or CD54 upregulation on THP-1, resulting in improved concentration-dependency. All 14 sensitizers were found positive for CD86 and/or CD54. Based on the current thresholds of Δmean fluorescence intensity (MFI) ≥ 10 and ΔMFI ≥ 50, respectively, only 1 of 10 non-sensitizers was false-positive. Remarkably, coculture with HaCaT keratinocytes improved the rank correlation of concentrations needed to reach the thresholds for positivity with in vivo data on sensitization potency, especially for CD54 (Spearman r = 0.739, p = 0.006; CD86:  r = 0.571, p = 0.041). These promising data suggest that a coculture model has the potential to support the prediction of sensitization potency using in vitro data, helping quantitative risk assessment.

 

Keywords: inflammatory microenvironment, skin sensitization potency, cross talk, THP-1, HaCaT keratinocytes


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October 21, 2016

Research Article

Ex vivo culture platform for assessment of cartilage repair treatment strategies

Andrea Schwab, Annick Meeuwsen, Franziska Ehlicke, Jan Hansmann, Lars Mulder, Anthal Smits, Heike Walles and Linda Kock

doi: 10.14573/altex.1607111

 

Summary

There is a great need for valuable ex vivo models that allow for assessment of cartilage repair strategies to reduce the high number of animal experiments. In this paper we present three studies with our novel ex vivo osteochondral culture platform. It consists of two separated media compartments for cartilage and bone which better represents the in vivo situation and enables supply of specific factors to the different needs of bone and cartilage. We investigated whether separation of the cartilage and bone compartments and/or culture media results in the maintenance of viability, structural and functional properties of cartilage tissue (study A). Next, we evaluated for how long we can preserve cartilage matrix stability of osteochondral explants during long-term culture of 84 days (study B). Finally, we investigated what the optimal defect size is without spontaneous self-healing occurring in this culture system (study C).

It was demonstrated that separated compartments for cartilage and bone in combination with tissue-specific medium allows for long-term culture of osteochondral explants, while maintaining cartilage viability, matrixtissue content, structure and mechanical properties up to at least 56 days. Furthermore, it was shown that we can create critical size cartilage defects of different sizes in the model.

The osteochondral model represents a valuable preclinical ex vivo tool for studying clinically relevant cartilage therapies, such as cartilage biomaterials for their regenerative potential, evaluation of drug and cell therapies or to study mechanisms of cartilage regeneration, which will undoubtedly reduce the number of animals needed for in vivo testing.

 

Keywords: ex vivo model, osteochondral biopsy, cartilage repair, critical size defect, replacement

 

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October 11, 2016

Research Article

Behavior of laboratory dogs before and after rehoming in private homes

Dorothea Döring, Ophelia Nick, Alexander Bauer, Helmut Küchenhoff and Michael H. Erhard

doi: 10.14573/altex.1608171

 

Summary

Although the rehoming of laboratory dogs has gained importance, scientific data that evaluate the process are lacking. Therefore, 145 laboratory beagles were tested before leaving the research facility (Test 1). The new owners were surveyed using a standardized telephone interview 1 week (n = 143) and 12 weeks (n = 126) after adoption. The behavior test was repeated with 68 dogs in their new homes 6 weeks after adoption (Test 2). The predictive power of Test 1 as well as the relevance of various factors was analyzed.

We found no significant differences between Tests 1 and 2 regarding the behavior reactions. In contrast, body language scores and heart rates changed significantly, indicating a more relaxed state of the dogs in their new homes.The interviews revealed a significant change toward desired behavior in most dogs within the 11 week period (p < 0.0001). The main behavior problems included separation problems (28%; n = 126), destroying objects (24%), and not being housebroken (39%).Owners of 9 dogs returned the animals, resulting in a rehoming success rate of 94%. The predictive power of Test 1 was low to moderate. Test 1 revealed a significant age effect (P = 0.0066), with younger and older dogs reaching higher scores than dogs approximately 2 years old. Dogs that had been born and reared in the research facility scored higher than dogs that had originally been acquired from a commercial breeder (P = 0.0257). Altogether, the rehoming of laboratory dogs is a valuable alternative to euthanasia.

 

Keywords: adoption; behavior problem; behavior test; laboratory dog; rehoming

 

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October 11, 2016, updated October 27, 2016

Research Article

In vitro chemotaxis and tissue remodeling assays quantitatively characterize foreign body reaction

Maren Jannasch, Tobias Weigel, Lisa Engelhardt, Judith Wiezoreck, Sabine Gaetzner, Heike Walles, Tobias Schmitz and Jan Hansmann

doi: 10.14573/altex.1610071

 

Summary

Surgical implantation of a biomaterial triggers foreign-body-induced fibrous encapsulation. Two major mechanisms of this complex physiological process are (I) chemotaxis of fibroblasts from surrounding tissue to the implant region, followed by (II) tissue remodeling. As an alternative to animal studies, we here propose an process-aligned in vitro test platform to investigate the material dependency of fibroblast chemotaxis and tissue remodeling, mediated by material-resident macrophages.

Embedded in a biomimetic three-dimensional collagen hydrogel, chemotaxis of fibroblasts in direction of macrophage-material-incubated cell culture supernatant was analysed by live-cell imaging. A combination of statistical analysis with a complementary parameterized random walk model allowed quantitative and qualitative characterization of the cellular walk process. We thereby identified an increasing macrophage-mediated chemotactic potential ranking from tested biomaterials glass over polytetrafluorethylene to titanium. To address long-term effects of biomaterial-resident macrophages on fibroblasts in a three-dimensional microenvironment, we further studied tissue remodeling by applying macrophage-material-incubated medium on fibrous in vitro tissue models. A high correlation to state of the art in vivo studies to the proposed in vitro tissue model was found. Titanium exhibited significantly lower tissue remodeling capacity compared to polytetrafluorethylene. By this approach we identified a material dependency for both processes chemotaxis and tissue remodeling, strengthening their specific contribution to foreign body reaction.

 

Keywords: Foreign body reaction, fibroblast chemotaxis, tissue remodeling, in vitro, quantitative characterization

 

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September 30, 2016

Research Article
Hepatotoxicity prediction by systems biology modeling of disturbed metabolic pathways using gene expression data

Pablo Carbonell, Oriol Lopez, Alexander Amberg, Manuel Pastor and Ferran Sanz

doi: 10.14573/altex.1602071


Supplementary file (.zip)


Summary

The present study applies a systems biology approach for the in silico predictive modeling of drug toxicity on the basis of high-quality preclinical drug toxicity data with the aim of increasing the mechanistic understanding of toxic effects of compounds at different levels (pathway, cell, tissue, organ). The model development has been carried out using 77 compounds for which gene expression data are available in the LINCS database for primary human hepatocytes treated with the compounds, as well as rodent in vivo hepatotoxicity information is available in the eTOX database. The data from LINCS were used in a systems biology approach to determine the type and number of pathways disturbed by each compound, and to estimate the extent of disturbance (network perturbation elasticity), analyzing the correspondence with the in vivo information from eTOX. Predictive models were developed through this integrative analysis, and their specificity and sensitivity were assessed. The quality of the predictions was determined on the basis of the area under the curve (AUC) of plots of true positive vs. false positive rates (ROC curves). The ROC AUC reached values of up to 0.9 (out of 1.0) for some hepatotoxicity endpoints. Moreover, the most frequently disturbed metabolic pathways were determined across the studied toxicants. They included e.g. mitochondrial beta-oxidation of fatty acids and amino acid metabolism. The process was exemplified by successful predictions on various statins. In conclusion, an entirely new approach linking gene expression alterations to the prediction of complex organ toxicity has been developed and evaluated.

 

Keywords: systems biology, predictive modeling, drug toxicity, hepatotoxicity, gene regulation


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September 29, 2016

Research Article

Toward achieving harmonization in a nano-cytotoxicity assay measurement through an interlaboratory comparison study

John T. Elliott, Matthias Rösslein, Nam Woong Song, Blaza Toman, Agnieszka Kinsner-Ovaskainen, Rawiwan Maniratanachote, Marc L. Salit, Elijah J. Petersen, Fatima Sequeira, Erica L. Romsos, Soo Jin Kim, Jieun Lee, Nadia R. Von Moos, François Rossi, Cordula Hirsch, Harald F. Krug, Wongsakorn Suchaoin and Peter Wick

doi: 10.14573/altex.1605021


Supplementary file (.pdf)

 

Summary

Design and development of reliable cell-based nanotoxicology assays are important for evaluation of potentially hazardous engineered nanomaterials. Challenges to producing a reliable assay protocol include working with nanoparticle dispersions and living cell lines, and the potential for nano-related interference effects. Here we demonstrate the use of a 96-well plate design with several measurement controls and an interlaboratory comparison study involving five laboratories to characterize the robustness of a nano-cytotoxicity MTS cell viability assay. The consensus EC50 values were 22.1 mg/l (95 % confidence intervals 16.9 mg/l to 27.2 mg/l) and 52.6 mg/l (44.1 mg/l to 62.6 mg/l) for the A549 cell line from ATCC for positively charged polystyrene nanoparticles for the serum free and serum conditions, respectively, and were 49.7 µmol/l (47.5 µmol/l to 51.5 µmol/l) and 77.0 µmol/l (54.3 µmol/l to 99.4 µmol/l) for positive chemical control cadmium sulfate for the serum free and serum conditions, respectively. Results from the measurement controls can be used to evaluate the sources of variability and their relative magnitudes within and between laboratories. This information revealed steps of the protocol that may need to be modified to improve the overall robustness and precision. The results suggest that protocol details such as cell line ID, media exchange, cell handling, and nanoparticle dispersion are critical to ensure protocol robustness and comparability of nano-cytotoxicity assay results. The combination of system control measurements and interlaboratory comparison data yielded insights that would not have been available by either approach by itself.

 

Keywords: nanotoxicology, MTS assay, interlaboratory comparison, polystyrene nanoparticles, alternatives to animal testing

 

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September 26, 2016

Review Article

Replacing, reducing and refining the use of animals in tuberculosis vaccine research

Rachel Tanner and Helen McShane

doi: 10.14573/altex.1607281

 

Summary

Tuberculosis (TB) remains a serious global health threat and an improved vaccine is urgently needed. New candidate TB vaccines are tested using preclinical animal models such as mice, guinea pigs, cattle and non-human primates. Animals are routinely infected with virulent Mycobacterium tuberculosis (M.tb) in challenge experiments to evaluate protective efficacy, raising ethical issues regarding the procedure of infection itself, symptoms of disease and humane end-points. We summarise the importance and limitations of animal models in TB vaccine research and review current alternatives and modifications in the context of the NC3Rs framework for replacing, reducing and refining the use of animals for scientific purposes.

 

Keywords: tuberculosis, vaccine, animal models, M.tb challenge, 3Rs


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August 23, 2016; updated October 5, 2016

t4 Workshop Report

Good Cell Culture Practice for stem cells and stem-cell-derived models

David Pamies, Anna Bal-Price, Anton Simeonov, Danilo Tagle, Dave Allen, David Gerhold, Dezhong Yin, Francesca Pistollato, Takashi Inutsuka, Kristie Sullivan, Glyn Stacey, Harry Salem, Marcel Leist, Mardas Daneshian, Mohan C. Vemuri, Richard McFarland, Sandra Coecke, Suzanne C. Fitzpatrick, Uma Lakshmipathy, Amanda Mack, Wen Bo Wang, Yamazaki Daiju, Yuko Sekino, Yasunari Kanda, Lena Smirnova and Thomas Hartung

doi: 10.14573/altex.1607121


Summary

The first guidance on Good Cell Culture Practice dates back to 2005. This document expands this to aspects of quality assurance for in vitro cell culture focusing on the increasingly diverse cell types and culture formats used in research, product development, testing and manufacture of biotechnology products and cell-based medicines. It provides a set of basic principles of best practice which can be used in training new personnel, reviewing and improving local procedures, and helping to assure standard practices and conditions for the comparison of data between laboratories and experimentation performed at different times. This includes recommendations for the documentation and reporting of culture conditions. It is intended as guidance, which will facilitate the generation of reliable data from cell culture systems, but is not intended to conflict with local or higher level legislation or regulatory requirements. It may not be possible to meet all recommendations in this guidance for practical, legal or other reasons. However, when necessary to divert from the principles of GCCP, the risk of decreasing the quality of work and safety of laboratory staff should be addressed and any conclusions or alternative approaches justified. This workshop report is considered as a first step toward a revised GCCP 2.0.


Keywords: Good Cell Culture Practices, in vitro methods, alternatives to animals, induced pluripotent stem cells


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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)

 

Summary

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

 

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July 25, 2016; updated September 28, 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 H. Westerink, Bastian Zimmer and Pamela J. Lein

doi: 10.14573/altex.1604201

 

Summary

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

 

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July 21, 2016; updated September 27, 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


Summary

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         

 

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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)


Summary

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

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