Online first

March 11, 2018

Research Article

Testing vaginal irritation with the hen’s egg test-chorioallantoic membrane assay

Rita Palmeira-de-Oliveira, Rita Monteiro Machado, José Martinez-de-Oliveira and Ana Palmeira-de-Oliveira

doi : 10.14573/altex.1710091


Summary

The HET-CAM (Hen’s Egg Test-Chorioallantoic Membrane) assay is an in vitro alternative to the in vivo Draize Rabbit Eye test that mimics vascular changes in the chorioallantoic membrane. This qualitative method assesses the irritancy potential of chemicals. The CAM responds to injury with an inflammatory process similar to that in the rabbit eye’s conjunctival tissue. Regarding topical toxicity assessment of medical devices, ISO 10993-10 states that any skin or eye irritant material shall be directly labelled as a potential vaginal irritant without animal testing, suggesting that the irritation potential for the eye and the vaginal epithelia is similar. The aim of this work was to apply the HET-CAM assay to test the irritancy potential of vaginal formulations. Vaginal semisolid medicines and lubricants currently marketed were tested along with the Universal Placebo formulation that has been shown to be clinically safe. Nonoxynol-9 (N-9), a known vaginal irritant, was enrolled as positive control (concentrations ranging from 0.001 to 100% (v/v)). The assay was conducted according to the ICCVAM - Recommended test method (NIH Publication No. 10-7553 – 2010). Formulations were then classified according to irritation score (IS), using the analysis methods (A) and (B). The studied vaginal formulations showed low potential for irritation. N-9 was classified as a severe irritant at concentrations above 2%, which corroborates clinical data, envisaging a possible in vitro/in vivo correlation. IS (B) was considered a better classification output. Although still requiring further validation, the HET-CAM assay seems an ideal prospect for vaginal irritancy potential in vitro studies.

 

Key words: vaginal irritation, HET-CAM, in vitro


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March 8, 2018

Food for Thought

The US Federal Tox21 Program: A strategic and operational plan for continued leadership

Russell S. Thomas, Richard S. Paules, Anton Simeonov, Suzanne C. Fitzpatrick, Kevin M. Crofton, Warren M. Casey and Donna L. Mendrick

doi: 10.14573/altex.1803011


Summary

The traditional approaches to toxicity testing have posed multiple challenges for evaluating the safety of commercial chemicals, pesticides, food additives/contaminants, and medical products.The challenges include number of chemicals that need to be tested, time and resource intensive nature of traditional toxicity tests, and unexpected adverse effects that occur in pharmaceutical clinical trials despite the extensive toxicological testing.Over a decade ago, the U.S. Environmental Protection Agency (EPA), National Toxicology Program (NTP), National Center for Advancing Translational Sciences (NCATS), and the Food and Drug Administration (FDA) formed a federal consortium for ‘Toxicology in the 21st Century” (Tox21) with a focus on developing and evaluating in vitro high-throughput screening (HTS) methods for hazard identification and providing mechanistic insights.The Tox21 consortium generated data on thousands of pharmaceuticals and datapoor chemicals, developed better understanding of the limits and applications of in vitro methods, and enabled incorporation of HTS data into regulatory decisions. To more broadly address the challenges in toxicology, Tox21 has developed a new strategic and operational plan that expands the focus of its research activities. The new focus areas include developing an expanded portfolio of alternative test systems, addressing technical limitations of in vitrotest systems, curating legacy in vivo toxicity testing data, establishing scientific confidence in the in vitrotest systems, and refining alternative methods for characterizing pharmacokinetics and in vitro assay disposition.The new Tox21 strategic and operational plan addresses key challenges to advance toxicology testing and will benefit both the organizations involved and the toxicology community.


Keywords: Tox21, alternative methods, high-throughput screening, validation, pharmacokinetics


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February 23, 2018, updated February 24, 2018, updated March 5, 2018

t4 Workshop Report

Recommendation on Test Readiness Criteria for New Approach Methods in Toxicology: Exemplified for Developmental Neurotoxicity

Anna Bal-Price, Helena T. Hogberg, Kevin M. Crofton, Mardas Daneshian, Rex E. FitzGerald, Ellen Fritsche, Tuula Heinonen, Susanne Hougaard Bennekou, Stefanie Klima, Aldert H. Piersma, Magdalini Sachana, Timothy J. Shafer, Andrea Terron, Florianne Monnet-Tschudi, Barbara Viviani, Tanja Waldmann, Remco H.S. Westerink, Martin F. Wilks, Hilda Witters, Marie-Gabrielle Zurich and Marcel Leist

doi:10.14573/altex.1712081

Supplementary file (pdf)


Summary

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).


Keywords: developmental in vitro neurotoxicity testing, regulatory toxicology, toxicity screening, quality assurance

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January 21, 2018

Research Article

A high-throughput approach to identify specific neurotoxicants/ developmental toxicants in human neuronal cell function assays

Johannes Delp, Simon Gutbier, Stefanie Klima, Lisa Hoelting, Kevin Pinto-Gil, Jui-Hua Hsieh, Michael Aichem, Karsten Klein, Falk Schreiber, Raymond R. Tice, Manuel Pastor, Mamta Behl and Marcel Leist

doi:10.14573/altex.1712182

Supplementary file 1 (pdf)

Supplementary file 2 (xlsx)

 

Summary

The (developmental) neurotoxicity hazard is still unknown for most chemicals. Establishing a test battery covering most of the relevant adverse outcome pathways may close this gap, without requiring a huge animal experimentation program. Ideally, each of the assays would cover multiple mechanisms of toxicity. One candidate test is the human LUHMES cell-based NeuriTox test. To evaluate its readiness for larger-scale testing, a proof of concept library assembled by the U.S. National Toxicology Program (NTP) was screened. Of the 75 unique compounds, seven were defined as specifically neurotoxic after the hit-confirmation phase and additional ten compounds were generally cytotoxic within the concentration range of up to 20 micromolar. As complementary approach, the library was screened in the PeriTox test, which identifies toxicants affecting the human peripheral nervous system. Of the eight PeriTox hits, five were similar to the NeuriTox hits: rotenone, colchicine, diethylstilbestrol, berberine chloride, and valinomycin. The unique NeuriTox hit, methyl-phenylpyridinium (MPP+) is known from in vivo studies to affect only dopaminergic neurons (which LUHMES cells are). Conversely, the known peripheral neurotoxicant acrylamide was picked up in the PeriTox, but not in the NeuriTox assay. All of the five common hits had also been identified in the published neural crest migration (cMINC) assay, while none of them emerged as cardiotoxicant in a previous screen using the same library. These comparative data suggest that complementary in vitro tests can pick up a broad range of toxicants, and that multiple test results might help to predict organ specificity patterns.

 

Keywords: neurite outgrowth inhibition, cytotoxicity, neurotoxicity, high content imaging, developmental toxicity

 

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December 2, 2017

Research Article

RPTEC/TERT1 cells form highly differentiated tubules when cultured in a 3D matrix

Philipp F. Secker, Lisanne Luks, Nadja Schlichenmaier and Daniel R. Dietrich

doi: 10.14573/altex.1710181


Supplementary file (pdf)

 

Summary

The proximal tubule is the primary site for renal solute reabsorption and secretion and thus a main target for drug-induced toxicity. Current nonclinical methods using 2D cell cultures are unable to fully recapitulate clinical drug responses mainly due to limited in vitro functional lifespan. Since extracellular matrices are known to be key regulators of cell development, culturing cells on classic 2D plastic surfaces inevitably results in loss of differentiation. Hence, 3D models of the human proximal tubule that recapitulate the in vivo morphology would allow for improved drug screening and disease modeling. Here, the development and characterization of a 3D proximal tubule model using RPTEC/TERT1 cells is presented. RPTEC/TERT1 cells self-assembled in matrigel to form highly differentiated and stable 3D tubular structures characterized by a branched network of monolayered cells encircling a cell-free lumen thus mimicking the proximal tubule. In vitro tubuli resembled the polarity of a proximal tubule epithelium as indicated by polar expression of Na+/K+- ATPase and ZO-3. Furthermore, 3D cultured RPTEC/TERT1 cells showed overall increased mRNA expression of xenobiotic transporters e.g. OCTs and MATEs and de novo expression of OAT3 when compared to cultures on plastics or membrane inserts. Finally, this model was used to assess delayed cisplatin-induced nephrotoxicity and demonstrated increased sensitivity when compared to 2D culture. Thus, the easy-to-use model described here may prove to be useful for mechanistic investigations, e.g. in discovery of compounds interfering with tubule formation, differentiation and polarization, as well for the detection and understanding of pharmaceutical induced nephrotoxicity.

 

Keywords: nephrotoxicity; in vitro; cisplatin; 3D cell culture; kidney


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November 29, 2017

Review Article

Recommendations to improve the EU non-technical summaries of animal experiments
Katy Taylor, Laura Rego and Tilo Weber

doi:10.14573/altex.1708111


Supplementary file (pdf)

 

Summary

Under the new Directive 2010/63/EC, member states have to publish non-technical summaries (NTS) of the projects involving animals that they authorise. These summaries must include information on the objectives of the project including the predicted harm and benefits and the number and types of animals to be used. Summaries should also demonstrate compliance with the 3Rs. The intention was that NTS would help increase the transparency of animal research in the EU. In this article, we review the status of the publication of NTS across member states and give some general observations on publication speed, identification, accessibility and quality. We also review in more detail the quality of reporting in a selection of NTS from Germany and the UK. We consistently found that NTS from Germany and the UK were deficient in their description of what is being done to the animals and what they might experience as a result. Using examples taken from specific NTS we highlight what we view to be good and bad examples to assist member states and researchers in producing better NTS in the future. The NTS can also be an important tool in sharing of best practice in the 3Rs and the avoidance of duplicative animal testing. For this to happen however, member states need to publish timely, ensure that NTS are accurate and, ideally, there needs to be some centralisation of the NTS.

 

Keywords: Directive 2010/63, Europe, transparency, non-technical summaries, 3Rs

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November 23, 2017

Research Article

Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines

Stephanie Kletting, Sarah Barthold, Urska Repnik, Gareth Griffiths, Brigitta Loretz, Nicole Schneider-Daum, Cristiane de Souza Carvalho-Wodarz and Claus‑Michael Lehr
doi: 10.14573/altex.1607191

Supplementary file (pdf)

Summary

The air-blood barrier is mainly composed of alveolar epithelial cells and macrophages. Whereas the epithelium acts as a diffusional barrier, macrophages represent an immunological barrier, in particular for larger molecules and nanoparticles.

This paper describes a new co-culture of human cell lines representing both cell types. Acquiring, culturing and maintaining primary alveolar epithelial cells presents significant logistical and technical difficulties. The recently established human alveolar epithelial lentivirus immortalized cell line, hAELVi, when grown on permeable filters, form monolayers with high functional and morphological resemblance to alveolar type I cells. To model alveolar macrophages, the human cell line THP-1 was seeded on pre-formed hAELVi monolayers.

The co-culture was characterized regarding cellular morphology, viability and barrier function. Macrophages were homogenously distributed on the epithelium and could be kept in co-culture for up to 7 days. Transmission electron microscopy showed loose contact between THP-1 and hAELVi cells. When grown at air liquid interface, both cells were covered with extracellular matrix-like structure, which was absent in THP-1 mono‑culture. In co-culture with macrophages, hAELVi cells displayed similar, sometimes even higher, trans-epithelial electrical resistance than in mono-cultures. When exposed to silver and starch NPs, hAELVi mono-cultures were more tolerant to the particles than THP-1 mono-cultures. The viability in the co-culture was similar to that of hAELVi monocultures. Transport studies with sodium fluorescein in presence/absence of EDTA proved that the co‑culture acts as functional diffusion barrier. These data demonstrate that hAELVi-/THP-1 co-cultures represent a promising model for safety and permeability studies of inhaled chemicals, drugs and nanoparticles.

 

Keywords: In vitro model, air-blood barrier, pulmonary drug delivery, nanoparticles, nanotoxicology


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October 2, 2017

Consensus Report

State-of-the-art and new options to assess T cell activation by skin sensitizers: Cosmetics Europe Workshop

Erwin van Vliet, Jochen Kühnl, Carsten Goebel, Silvia Martinozzi-Teissier, Nathalie Alépée, Takao Ashikaga, Brunhilde Blömeke, Aurelia del Bufalo, Magalie Cluzel, Emanuela Corsini, Nathalie Delrue, Bertrand Desprez, Nichola Gellatly, Christoph Giese, Laura Gribaldo, Sebastian Hoffmann, Martina Klaric, Bernard Maillere, Dean Naisbitt, Marc Pallardy, Marc Vocanson and Dirk Petersohn

doi: 10.14573/altex.1709011

 

Summary

Significant progress has been made in the development and validation of non-animal test methods for skin sensitization assessment. At present, three of the four key events of the Adverse Outcome Pathway (AOP) are assessable by OECD-accepted in vitro methods. The fourth key event describes the immunological response in the draining lymph node where activated dendritic cells present major histocompatibility complex-bound chemically modified peptides to naive T cells, thereby priming the proliferation of antigen-specific T cells. Despite substantial efforts, modelling and assessing this adaptive immune response to sensitizers with in vitro T cell assays still represents a challenge. The Cosmetics Europe Skin Tolerance Task Force organized a workshop, bringing together academic researchers, method developers, industry representatives and regulatory stakeholders to review the scientific status of T cell-based assays, foster a mutual scientific understanding and conceive new options to assess T cell activation. Participants agreed that current T cell assays have come a long way in predicting immunogenicity, but that further investment and collaboration is required to simplify assays, optimize their sensitivity, better define human donor-to-donor variability and evaluate their value to predict sensitizer potency. Furthermore, the potential role of T cell assays in AOP-based testing strategies and subsequent safety assessment concepts for cosmetic ingredients was discussed. It was agreed that it is currently difficult to anticipate uses of T cell assay data for safety assessment and concluded that experience from case studies on real-life risk assessment scenarios is needed to further consider the usefulness of assessing the fourth AOP key event.

 

Keywords: skin sensitization, adverse outcome pathway, key events, T cell assays, safety assessment


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September 18, 2017

Research Article

Integrated strategy for mutagenicity prediction applied to food contact chemicals

Serena Manganelli, Benoît Schilter, Emilio Benfenati, Alberto Manganaro and Elena Lo Piparo

doi:10.14573/altex.1707171


Supplementary file 1 (pdf)

Supplementary file 2 (.xlsx)


Summary

Food contamination due to unintentional leakage of chemicals from food contact materials (FCM) is a source of increasing concern. Since for many of these substances, only limited or no toxicological data are available, the development of alternative methodologies to establish rapidly and cost-efficiently level of safety concern is critical to ensure adequate consumer protection. Computational toxicology methods are considered the most promising solutions to cope with this data gap. In particular, mutagenicity assessment has a particular relevance and is a mandatory requirement for all substances released from plastic FCM, regardless how low migration and exposure are. In the present work, a strategy integrating a number of (Quantitative) Structure Activity Relationship ((Q)SAR) models for Ames mutagenicity predictions is proposed. A list of chemicals representing likely migrating moieties from FCM was selected to test the value of the newly defined strategy and the possibility to combine predictions given by the different algorithms was evaluated. In particular, a scheme to integrate mutagenicity estimations into a single final assessment was developed resulting in an increased domain of applicability. In most cases, a deeper analysis of experimental data, where available, allowed fixing misclassification errors, highlighting the importance of data curation in the development, validation and application of in silico methods. The high accuracy of the strategy provided the rationales for its application for toxicologically uncharacterized chemicals. Finally, the overall strategy of integration will be automated through its implementation into a freely available software application.

 

Keywords: FCM migrating substances, integration, mutagenicity, in silico models, (Q)SAR


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September 8, 2017

Research Article

Ex vivo model unravelling cell distribution effect in hydrogels for cartilage repair

Vivian H. M. Mouser, Noёl M. M. Dautzenberg, Riccardo Levato, Mattie H. P. van Rijen, Wouter J. A. Dhert, Jos Malda and Debby Gawlitta

doi: 10.14573/altex.1704171


Supplementary file (pdf)


Summary

The implantation of chondrocyte-laden hydrogels is a promising cartilage repair strategy. Chondrocytes can be spatially positioned in hydrogels and thus in defects, while current clinical cell-therapies introduce chondrocytes in the defect depth. The main aim of this study was to evaluate the effect of spatial chondrocyte distribution on the reparative process. To reduce animal experiments, an ex vivo osteochondral plug model was used and evaluated. Finally, the role of the delivered and endogenous cells in the repair process was investigated. Full thickness cartilage defects were created in equine osteochondral plugs. Defects were filled with (A) chondrocytes at the bottom of the defect, covered with a cell-free hydrogel, (B) chondrocytes homogeneously encapsulated in a hydrogel, and (C, D) combinations of A and B with different cell densities. Plugs were cultured up to 57 days, after which the cartilage and repair tissues were characterized and compared to baseline samples. Additionally, at day 21, the origin of cells in the repair tissue was evaluated. Best outcomes were obtained with conditions C and D, which resulted in well-integrated cartilage-like tissue that completely filled the defect, regardless of the initial cell density. A critical role of the spatial chondrocyte distribution in the repair process was observed. Moreover, the osteochondral plugs stimulated cartilage formation in the hydrogels when cultured in the defects. Finally, the resulting repair tissue originated from the delivered cells. These findings confirm the potential of the osteochondral plug model for the optimization of the composition of cartilage implants and for studying repair mechanisms.

 

Keywords: cartilage repair, regeneration, osteochondral plug, GelMA/gellan gum


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August 17, 2017

Research Article

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition

Jorid B. Sørli, Yishi Huang, Emilie Da Silva, Jitka S. Hansen, Yi Y. Zuo, Marie Frederiksen, Asger W. Nørgaard, Niels E. Ebbehøj, Søren T. Larsen and Karin S. Hougaard

doi: 10.14573/altex.1705181


Supplementary file (pdf)


Summary

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if they inhibited LS function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e. the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e. the in vitro method found three false positive of the 21 tested products. Six of the products have been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

 

Keywords: impregnation product, lung surfactant, constrained drop surfactometer, acute inhalation toxicity, OECD TG 403 and 436

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August 9, 2017; updated August 12, 2017; updated October 13, 2017

Consensus Report

Fetal Bovine Serum (FBS): Past – Present – Future

Jan van der Valk, Karen Bieback, Christiane Buta, Brett Cochrane, Wilhelm G. Dirks, Jianan Fu, James J. Hickman, Christiane Hohensee, Roman Kolar, Manfred Liebsch, Francesca Pistollato, Markus Schulz, Daniel Thieme, Tilo Weber, Joachim Wiest, Stefan Winkler and Gerhard Gstraunthaler

doi: 10.14573/altex.1705101


Summary

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as ‘fetal calf serum’) is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where (a) regulatory aspects, (b) the serum dilemma, (c) alternatives to FBS, (d) case-studies of serum-free in vitro applications, and (e) the establishment of serum-free databases, were discussed.

The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated, despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with above mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.   

 

Keywords: serum-free, cell culture, databases, 3Rs, replacement


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July 24, 2017

Research Article

Use of high-throughput in vitro toxicity screening data in cancer hazard evaluations by IARC Monograph Working Groups

Weihsueh A. Chiu, Kathryn Z. Guyton, Matthew T. Martin, David M. Reif and Ivan Rusyn

doi: 10.14573/altex.1703231

 

Supplementary file (pdf)

 

Summary

Evidence regarding carcinogenic mechanisms serves a critical role in International Agency for Research on Cancer (IARC) Monograph evaluations. Three recent IARC Working Groups pioneered inclusion of the US Environmental Protection Agency (EPA) ToxCast program high-throughput screening (HTS) data to supplement other mechanistic evidence. In Monograph V110, HTS profiles were compared between perfluorooctanoic acid (PFOA) and prototypical activators across multiple nuclear receptors. For Monograph V112 -113, HTS assays were mapped to 10 key characteristics of carcinogens identified by an IARC expert group, and systematically considered as an additional mechanistic data stream. Both individual assay results and ToxPi-based rankings informed mechanistic evaluations. Activation of multiple nuclear receptors in HTS assays showed that PFOA targets peroxisome proliferator activated and other receptors. ToxCast assays substantially covered 5 of 10 key characteristics, corroborating literature evidence of “induces oxidative stress” and “alters cell proliferation, cell death or nutrient supply” and filling gaps for “modulates receptor-mediated effects.” Thus, ToxCast HTS data were useful both in evaluating specific mechanistic hypotheses and in the overall evaluation of mechanistic evidence. However, additional HTS assays are needed to provide more comprehensive coverage of the 10 key characteristics of carcinogens that form the basis of current IARC mechanistic evaluations.

 

Keywords: carcinogenicity, high throughput screening, in vitro, mechanisms


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June 26, 2017

Research Article

A validated algorithm for selecting non-toxic chemical concentrations

Julita Stadnicka-Michalak, Melanie Knöbel, Anže Županič and Kristin Schirmer

doi: 10.14573/altex.1701231


Supplementary file 1 (pdf)

Supplementary file 2 (7z, zip) updated December 1, 2017


Summary

The maximal chemical concentration that causes an acceptably small or no effect in an organism or isolated cells is an often-sought-after value in toxicology. Existing approaches to derive this value have raised several concerns; thus, it is often chosen case-by-case based on personal experience. To overcome this ambiguity, we propose an approach for choosing the non-toxic concentration (NtC) of a chemical in a rational, tractable way. We developed an algorithm that identifies the highest chemical concentration which causes no more than 10% effect (≤EC10) including the modeled 95% confidence intervals and considering each of the measured biological replicates; and whose toxicity is not significantly different from no effect.The developed algorithm was validated in two steps: by comparing its results with measured and modeled data for 91 dose-response experiments with fish cell lines and/or zebrafish embryos; and by measuring actual effects caused by NtCs in a separate set of experiments using a fish cell line and zebrafish embryos. The algorithm provided an NtC that is more protective than NOEC (No-Observed-Effect-Concentration), NEC (modeled No-Effect Concentration), EC10 and Benchmark Dose (BMD). Despite focusing on small scale bioassays here, this study indicates that the NtC algorithm could be used in various systems. Its application on the survival of zebrafish embryos and on metabolic activity in cell lines showed that NtCs can be applied to different effect measurements, time points and levels of biological organization. The algorithm is available as Matlab and R code, and as a free, user friendly online application.

 

Keywords: fish cell lines and embryos, in vitro, bioassays, toxicology, animal testing alternatives


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May 25, 2017; updated May 29, 2017

t4 Workshop Report

From in vivo to in vitro: The medical device testing paradigm shift

Dayna Kerecman Myers, Alan M. Goldberg, Albrecht Poth, Michael F. Wolf, Joseph Carraway, James McKim, Kelly P. Coleman, Richard Hutchinson, Ronald Brown, Harald F. Krug, Anthony Bahinski and Thomas Hartung

doi: 10.14573/altex.1608081

 

Summary

Amid growing efforts to advance the replacement, reduction, and refinement of the use of animals in research, there is a growing recognition that in vitro testing of medical devices can be more effective, both in terms of cost and time, and also more reliable than in vivo testing. Although the technological landscape has evolved rapidly in support of these concepts, regulatory acceptance of alternative testing methods has not kept pace. Despite the acceptance by regulators of some in vitro tests (cytotoxicity, gene toxicity, and some hemocompatibility assays), many toxicity tests still rely on animals (irritation, sensitization, acute toxicity, reproductive/developmental toxicity), even where other industrial sectors have already abandoned them. Bringing about change will require a paradigm shift in current approaches to testing - and a concerted effort to generate better data on risks to human health from exposure to leachable chemicals from medical devices, and to boost confidence in devices and alternative testing methods. To help advance these ideas, stir debate about best practices, and coalesce around a roadmap forward, the JHU-Center for Alternatives to Animal Testing (CAAT) hosted a symposium in Baltimore, Maryland, in December 2013 - believed to be the first gathering dedicated to the topic of in vitro testing of medical devices. Industry representatives, academics, and regulators in attendance presented evidence to support the unique strengths and challenges associated with the approaches currently in use as well as new methods under development, and drew next steps to push the field forward from their presentations and discussion.


Keywords: in vitro testing, refinement, skin sensitization, Threshold of Toxicological Concern (TTC), nanotoxicology


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May 3, 2017

Review Article

Extracorporeal perfusion of isolated organs of large animals – Bridging the gap between in vitro and in vivo studies

Carola R. Daniel, Raphael Labens, David Argyle and Theresia F. Licka

doi: 10.14573/altex.1611291


Summary

Since the early 20th century extracorporeal perfusion of large animal organs has been used to investigate a broad variety of research questions, thereby overcoming common draw backs of in vitro studies without suffering from ethical concerns associated with live animal research. The technique is in accordance with the 3R principles and represents an excellent opportunity to investigate in detail the physiology of organs under standardised conditions. It is also suitable for the translation of basic pre-clinical research into a more relevant arena prior to or avoiding altogether live animal research. Furthermore, organ perfusion has also been an important tool in developing methods of organ preservation for transplantation surgery. Yet due to the nature of the experiments only short term observations can be made and while cells are still exposed to their regional secretome, the whole organ itself is isolated from the body and correlations between organ-systems cannot be taken into consideration. This review gives an overview over the history of extracorporeal perfusion of large animal organs and limbs highlighting major achievements in the field and discussing different experimental set-ups. Advantages and limitations of this technique are presented. Prospective future research perspectives, which might include tracking of specific cell types and study of their distinct behaviour towards different stimuli, are given to illustrate the relevance of this method.


Keywords: Extracorporeal perfusion, large animal, research model, isolated perfused organ

 

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April 12, 2017, updated April 23, 2017

Research Article

The GARD platform for potency assessment of skin sensitizing chemicals

Kathrin S. Zeller, Andy Forreryd, Tim Lindberg, Robin Gradin, Aakash Chawade and Malin Lindstedt

doi: 10.14573/altex.1701101

 

Summary

Contact allergy induced by certain chemicals is a common health concern, and several alternative methods have been developed to fulfill the requirements of European legislation with regard to hazard assessment of potential skin sensitizers. However, validated methods, which provide information about the potency of skin sensitizers, are still lacking. The cell-based assay Genomic Allergen Rapid Detection (GARD), targeting key event 3, dendritic cell activation, of the skin sensitizer AOP, uses gene expression profiling and a machine learning approach for the prediction of chemicals as sensitizers or non-sensitizers. Based on the GARD platform, we here expanded the assay to predict three sensitizer potency classes according to the European Classification, Labelling and Packaging (CLP) Regulation, targeting categories 1A (strong), 1B (weak) and no cat (non-sensitizer). Using arandom forest approach and 70 training samples, a potential biomarker signature of 52 transcripts was identified. The resulting model could predict an independent test set consisting of 18 chemicals, six from each CLP category and all previously unseen to the model, with an overall accuracy of 78%. Importantly, the model was shown to be conservative and only underestimated the class label of one chemical. Furthermore, an association of defined chemical protein reactivity with distinct biological pathways illustrates that transcriptional approach can reveal information contributing to the understanding of underlying mechanisms in sensitization.

 

Keywords: In vitro assay, sensitization, potency, biomarkers, random forest


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April 12, 2017, updated June 8, 2017

Review Article

Non-animal approaches for toxicokinetics in risk evaluations of food chemicals

Ans Punt, Ad A.C.M. Peijnenburg, Ron L.A.P. Hoogenboom and Hans Bouwmeester

doi: 10.14573/altex.1702211


Summary

The objective of the present work was to review the availability and predictive value of non-animal toxicokinetic approaches and to evaluate their current use in European risk evaluations of food contaminants, additives and food contact materials, as well as pesticides and medicines. Results revealed little use of quantitative animal or human kinetic data in risk evaluations of food chemicals, compared with pesticides and medicines. Risk evaluations of medicines provided sufficient in vivo kinetic data from different species to evaluate the predictive value of animal kinetic data for humans. These data showed a relatively poor correlation between the in vivo bioavailability in rats and dogs vs that in humans. In contrast, in vitro (human) kinetic data have been demonstrated to provide adequate predictions of the fate of compounds in humans, using appropriate in vitro-in vivo scalers and by integration of in vitro kinetic data with in silico kinetic modelling. Even though in vitro kinetic data were found to be occasionally included within risk evaluations of food chemicals, particularly results from Caco-2 absorption experiments and in vitro data on gut-microbial conversions, only a minor use of in vitro methods for metabolism and quantitative in vitro-in vivo extrapolation methods was identified. Yet, such quantitative predictions are essential in the development of alternatives to animal testing as well as to increase human relevance of toxicological risk evaluations. Future research should aim at further improving and validating quantitative alternative methods for kinetics thereby increasing regulatory acceptance of non-animal kinetic data.

 

Keywords: In vitro kinetics, alternatives to animal testing, PBPK, regulatory acceptance


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March 16, 2017

Short Communication

Adaptation of the KeratinoSensTM skin sensitisation test to animal-product-free cell culture

Nathalie Belot, Bushra Sim, Christopher Longmore, Lottie Roscoe and Carol Treasure

doi: 10.14573/altex.1701311

 

Summary

Skin sensitisation is the process by which a substance leads to an allergic reaction following skin contact. The process has been described as an adverse outcome pathway (AOP), including several key events, from skin penetration and covalent protein binding, to keratinocyte activation, dendritic cell activation and T-lymphocyte proliferation. The in vitro assay KeratinoSensTM measures the activation of keratinocytes. It is fully accepted at a regulatory level (OECD TG 442d) and appropriate for compliance with a range of legislation including the EU Cosmetics Regulation, REACH, and the CLP Regulation.

Currently, many in vitro methods use animal-derived components in the cell culture systems. Many stakeholders in the cosmetics industry have both scientific and ethical concerns relating to this issue and have stated a strong preference for fully human in vitro test systems. We have adapted the KeratinoSensTM method to animal product-free conditions, and carried out an in-house validation with 21 reference substances, including those listed in the Performance Standards associated with OECD TG442d. The modified method was shown to be totally equivalent to the Validated Reference Method (VRM), with comparable values for accuracy (85.7%), sensitivity (84.6%) and specificity (87.5%), and all acceptance criteria being met. In Europe, data generated by the adapted method may be used in REACH submissions, and we are now seeking approval to list the adaptation in OECD TG 442d, enabling formal compliance with a range of global regulations.

 

Keywords: skin sensitization, KeratinoSensTM, alternatives to animal testing, 3R, in vitro

 

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February 23, 2017

Research Article

The borderline range of toxicological methods: Quantification and implications for evaluating precision

Maria Leontaridou, Daniel Urbisch, Susanne N. Kolle, Katharina Ott, Denis S. Mulliner, Silke Gabbert and Robert Landsiedel

doi: 10.14573/altex.1606271


Summary

Testing methods to assess the skin sensitisation potential of a substance usually use threshold criteria to dichotomise continuous experimental read-outs into yes/no conclusions. The threshold criteria are prescribed in the respective OECD test guidelines and the conclusion is used for regulatory hazard assessment, i.e. classification and labelling of the substance. We can identify a borderline range (BR) around the classification threshold within which test results are non-conclusive due to a testing method’s biological and technical variability. We quantify BRs in the prediction models of the non-animal testing methods DPRA, LuSens and h-CLAT, and of the animal test LLNA, respectively. Depending on the size of the BR we find that between 6% and 28% of the substances in the sets tested with these methods were considered borderline. If the results of individual non-animal test methods are combined into integrated testing strategies (ITS), borderline test results of individual tests can also affect the overall assessment of the skin sensitisation potential of the testing strategy. This was analysed for the ‘2-out-of-3’ ITS: Four out of 40 substances (10%) were considered borderline. Based on our findings we propose expanding the standard binary classification of substances into ‘positive’/’negative’ or ‘hazardous’/’non-hazardous’ by adding a ‘borderline’ or ‘non-conclusive’ alert for cases where test results fall within the borderline range.


Keywords: non-animal methods, variability, borderline range, skin sensitisation.

 

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February 17, 2017

Research Article

Evaluation of the GARD assay in a blind Cosmetics Europe study

Henrik Johansson, Robin Gradin, Andy Forreryd, Maria Agemark, Kathrin Zeller, Angelica Johansson, Olivia Larne, Erwin van Vliet, Carl Borrebaeck and Malin Lindstedt

doi: 10.14573/altex.1701121

 

Summary

Chemical hypersensitivity is an immunological response towards foreign substances, commonly referred to as sensitizers, which gives rise primarily to the clinical symptoms known as allergic contact dermatitis. For the purpose of mitigating risks associated with consumer products, chemicals are screened for sensitizing effects. Historically, such predictive screenings have been performed using animal models. However, due to industrial and regulatory demand, animal models for the purpose of sensitization assessment are being replaced by animal-free testing methods, a global trend that is spreading across industries and market segments. To meet this demand, the Genomic Allergen Rapid Detection (GARD) assay was developed. GARD is a novel, cell-based assay that utilizes the innate recognition of xenobiotic substances by dendritic cells, as measured by a multivariate readout of genomic biomarkers. Following cellular stimulation, chemicals are classified as sensitizers or non-sensitizers based on induced transcriptional profiles. Recently, a number of animal-free methods were comparatively evaluated by Cosmetic Europe, using a coherent and blinded test panel of reference chemicals with human and local lymph node assay data, comprising a wide range of sensitizers and non-sensitizers. In this paper, the outcome of the GARD assay is presented. It was demonstrated that GARD is a highly functional assay with a predictive performance of 83% in this Cosmetics Europe dataset. The average accumulated predictive accuracy of GARD across independent datasets was 86%, for skin sensitization hazard.

 

Keywords: GARD, sensitization, in vitro, predictive accuracy, alternative methods

 

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