Women in Alternatives

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Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease

Bewertung und Validierung dreidimensionaler rekonstruierter Modelle der menschlichen Haut als Ersatzmethoden zu Tierversuchen in der Sicherheitstoxikologie

Title page Content Introduction Materials and Methods Results and Discussion - part 1 Results and Discussion - part 2 Results and Discussion - part 3 Results and Discussion - part 4 General Discussion Summary Literature Annex AbbreviationsReconstructed human skin models are one of the most promising in vitro systems that have the potential to replace animal assays in area of topical toxicity testing. However, in regulatory toxicology only highly standardised and commercially available reconstructed human skin models with declared reproducibility within the test batches and over production period are accepted. In addition, tests developed have to be validated to reach the regulatory acceptance. Experiments performed in this thesis demonstrated that the three evaluated reconstructed human skin models (EpiDerm, EPISKIN and SkinEthic) reveal good performance in all investigated parameters and reflect the standards described in OECD TG 431. Still, when developing and validating new in vitro assays, the differences between the reconstructed human skin models must be taken into account. Besides several technical issues related to different storage and culture conditions, attention has to be paid to the different sensitivity to several chemical groups, e.g. surfactants, low concentrated acids, volatile compounds etc. The applicability domain of a new method has to be clearly defined before any validation is performed. An important part of this thesis was the evaluation of the existing reconstructed human skin models for the testing of skin corrosion potential of industrial chemicals. Two models were included in these studies, SkinEthic and EST-1000. The SkinEthic skin corrosion assay was evaluated in a trial between three laboratories, following the general validation principles. The results obtained were highly concordant with previously validated methods (EpiDerm, EPISKIN and TER assay) and classifications given in OECD TG 431. The outcome is currently reviewed by ECVAM Scientific Advisory Committee (ESAC). The EST-1000 provided results comparable to SkinEthic, too, yet the assay variability has still to be assessed in an inter-laboratory study. Another task of this thesis was the optimisation of the EpiDerm skin irritation protocol for an upcoming ECVAM skin irritation validation study. Following the idea of the common protocol concept, the EPISKIN protocol (Portes et al., 2002, and Cotovio et al., 2005), was used as a basis for EpiDerm skin irritation assay. Both reconstructed human skin models provided similar outcome. Moreover, when applying the protocol to the SkinEthic model, almost identical results were obtained. The EpiDerm and EPISKIN skin irritation assays are currently evaluated in an ECVAM validation trial. An ECVAM catch-up validation study with SkinEthic model, based on results presented in this thesis, is in preparation. Finally, outcome of the phototoxicity feasibility study demonstrates that the reconstructed human skin models could be used for the determination of the phototoxic potency of chemicals and, therefore, the estimation of the safe concentrations at which no acute phototoxic effect can be observed. Particularly, in this study, the human data are of great importance as no validated in vivo method exists for the purpose described above. As a next step a validation trial has to be performed with the reconstructed human skin models to reach the regulatory acceptance of the assay. In this thesis, it has been demonstrated that the reconstructed human skin models show excellent capability for the use in almost all domains of topical toxicity testing, e.g. skin corrosion, skin irritation, and phototoxicity. However, a successful validation is the prerequisite for their implementation in regulatory toxicology.Rekonstruierte menschliche Hautmodelle gehören zu den vielversprechenden In- vitro Systemen, welche ein großes Potential für den Ersatz von Tierversuchen zur Feststellung topischer Toxizität besitzen. Für eine breitere Anwendung bei der Klärung sicherheitstoxikologischer Fragestellungen eignen sich jedoch lediglich standardisierte und kommerziell erhältliche Modelle reproduzierbarer Qualität. Zudem müssen solche Testsysteme einen Validierungsprozess durchlaufen haben, um behördliche Akzeptanz zu erlangen. Die Ergebnisse dieser Dissertation zeigen, dass die drei künstlichen Hautmodelle EpiDerm, EPISKIN und SkinEthic die geforderten Standards der OECD Richtlinie 431 erfüllen. Dennoch mussten die individuellen Besonderheiten einzelner Modelle während der Methodenentwicklung und -validierung berücksichtigt werden. Neben verschiedenen technischen Einzelheiten waren Unterschiede in der Empfindlichkeit bei den Reaktionen auf verschiedene chemische Stoffgruppen, wie Tenside, verdünnte Säuren und flüchtige Substanzen, zu beachten. Die Art der Anwendung musste für jede Methode klar definiert sein. Ein wesentlicher Bestandteil dieser Arbeit war die Prüfung der beiden Hautmodelle SkinEthic und EST-1000 auf ihre Eignung zur Untersuchung des ätzenden Potentials von Industriechemikalien. Festgelegten Kriterien zur Validierung folgend wurde das Hautmodell SkinEthic einem Ringversuch unter Beteiligung dreier Laboratorien unterzogen. Die Ergebnisse zeigten eine sehr gute Übereinstimmung mit den zuvor validierten Modellen EpiDerm und EPISKIN sowie dem TER-Test; die Klassifizierungen entsprachen der OECD Richtlinie 431. Diese Ergebnisse werden zur Zeit vom wissenschaftlichen Beirat der ECVAM (ESAC) beurteilt. Obwohl das Modell EST-1000 vergleichbare Ergebnisse zum Modell SkinEthic gezeigt hat, muss die Testvariabilität noch in einem Interlaborvergleich festgestellt und beurteilt werden. Ein weiteres Thema dieser Dissertation war die Optimierung eines Protokolls für die Verwendung des EpiDerm-Modelis in einer ECVAM- Validierungsstudie zur Bestimmung des hautreizenden Potentials in vitro. Gemäß des Konzepts zur Erstellung eines Protokolls, welches das Ziel der gemeinsamen Anwendung bei unterschiedlichen Modellen verfolgt ("Common Protocol Concept"), wurde das Protokoll zur Feststellung der Hautreizung des Modells EPISKIN (Portes et al., 2002, und Cotovio et al., 2005) als Grundlage für das Modell EpiDerm verwendet. Die Untersuchung erbrachte eine gute Vergleichbarkeit beider Modelle. Ferner konnte dieses Protokoll für das Modell SkinEthic angewendet werden. Zur Zeit befinden sich die Protokolle zu den Modellen EpiDerm und EPISKIN in einer ECVAM-Validierungsstudie. Eine zusätzliche "Catch-Up"-Studie für das Modell SkinEthic ist in Vorbereitung. Im letzten Teil der Dissertation wurde ein künstliches Hautmodell auf seine Eignung für die Untersuchung des phototoxischen Potentials chemischer Stoffe durchgeführt, um die Konzentration einer Substanz bestimmen zu können, bei deren topischer Applikation kein akuter phototoxischer Effekt auftritt. Speziell für diese Fragestellung war der Vergleich zu Daten, die beim Menschen gewonnen wurden, von Bedeutung, da keine anerkannte In-vivo Methode existiert. Zur Erlangung behördlicher Akzeptanz muss für die Anwendung der Hautmodelle als In-vitro Phototoxizitätstests eine Validierungsstudie erfolgen. Mit der vorliegenden Arbeit konnte die exzellente Eignung rekonstruierter menschlicher Hautmodelle für ihre Anwendung in den wichtigen Bereichen der topischen Toxizitätsuntersuchung, d.h. Hautätzung, Hautreizung und Phototoxizität, aufgezeigt werden. Dennoch ist eine erfolgreiche Validierung der Modelle eine grundsätzliche Voraussetzung für ihre Aufnahme in die behördlichen Richtlinien zur Sicherheitstoxikologie

Open letter: Selection of a new Executive Director of the European Chemicals Agency (ECHA) provides an opportunity for the EU to lead in the field of chemicals management and implementation of innovative science

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Two Novel Prediction Models Improve Predictions of Skin Corrosive Sub-categories by Test Methods of OECD Test Guideline No. 431

Alternative test methods often use prediction models (PMs) for converting endpoint measurements into predictions. Two PMs are used in the OECD Test Guideline No. 431 (TG431) on skin corrosion. One is specific to EpiSkin™ test method, whereas EpiDerm™, SkinEthic™RHE and epiCS® share a common PM. These four methods are Reconstructed human Epidermis models wherein several cell viability values are measured. Their PMs allow translating those values into sub-categories of corrosive chemicals, Category 1A (Cat1A) and a combination of Categories 1B/1C (Cat1BC), and identifying non-corrosive chemicals. EpiSkin™'s PM already results in sufficiently accurate predictions. The common PM of the three others accurately identifies all corrosive chemicals but, regarding their sub-categorization, an important fraction of Cat1BC chemicals (40-50%) is over-predicted as Cat1A. This paper presents a post-hoc analysis of validation data on a consistent set of n=80 chemicals. It investigates (i) why this common PM causes these over-predictions and (ii) how two novel PMs that we developed (PMvar1 and PMvar2) improve the predictive capacity of these methods. PMvar1 is based on a two-step approach; PMvar2 is based on a single composite indicator of cell viability. Both showed a marked greater capacity to predict Cat1BC, and Cat1A correct predictions remain at least at the same level of EpiSkin™. We suggest revising TG431, and its performance standards, to include the novel PMs in view of improving the predictive capacity of its in vitro skin corrosion methods.JRC.I.5-Systems Toxicolog

Open letter: Selection of a new Executive Director of the European Chemicals Agency (ECHA) provides an opportunity for the EU to lead in the field of chemicals management and implementation of innovative science

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Pre-validation of an in vitro skin irritation test for medical devices using the reconstructed human tissue model EpiDerm™.

Assessment of dermal irritation is an essential component of the safety evaluation of medical devices. Reconstructed human epidermis (RhE) models have replaced rabbit skin irritation testing for neat chemicals and their mixtures (OECD Test Guideline 439). However, this guideline cannot be directly applied to the area of medical devices (MD) since their non-toxicity assessment is largely based on the testing of MD extracts that may have very low irritation potential. Therefore, the RhE-methods previously validated with neat chemicals needed to be modified to reflect the needs for detection of low levels of potential irritants. A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers (Casas et al., 2013, TIV). In 2014 and 2015 MatTek In Vitro Life Science Laboratories (IVLSL) and RIVM assessed the transferability of the assay. After the successful transfer and standardization of the protocol, 17 laboratories were trained in the use of the protocol in the preparation for the validation. Laboratories produced data with 98% agreement of predictions for the selected references and controls. We conclude that a modified RhE skin irritation test has the potential to address the skin irritation potential of the medical devices. Standardization and focus on the technical issues is essential for accurate prediction

The ECVAM International Validation Study on In Vitro Tests for Acute Skin Irritation: Report on the Validity of the EPISKIN and EpiDerm Assays and on the Skin Integrity Function Test

ECVAM sponsored a formal validation study on three in vitro tests for skin irritation, two employing reconstituted human epidermis models (EPISKIN, EpiDerm) and the skin integrity function test (SIFT), which employs ex vivo mouse skin. The goal of the study was to assess whether the in vitro tests would predict in vivo classifications according to the EU classification scheme, R 38 and No-label (i.e. non-irritant). 58 chemicals (25 irritants and 33 non-irritants) were tested, having been selected to give broad coverage of physico-chemical properties, and an adequate distribution of irritancy scores derived from in vivo rabbit skin irritation tests. In Phase 1, 20 of these chemicals (9 irritants and 11 non-irritants) were tested with coded identity by a single (lead) laboratory for each of the methods, to confirm the suitability of the protocol improvements introduced after a prevalidation phase. When cell viability (evaluated by the MTT reduction test) was used as the endpoint, the predictive ability of both EpiDerm and EPISKIN was considered sufficient to justify their progression to Phase 2, while the predictive ability of the SIFT was inadequate. Since both the skin models provided false predictions around the in vivo classification border (rabbit Draize test score 2), the release of the cytokine, interleukin-1a (IL-1¿), was also determined. In Phase 2, each human skin model was tested in three laboratories, with 58 chemicals. The main endpoint measured for both EpiDerm and EPISKIN was cell viability. In samples from chemicals which gave MTT assay results above the threshold of 50% viability, IL-1a release was also measured, to determine whether the additional endpoint would improve the predictive ability of the tests. For EPISKIN, the sensitivity was 75% and the specificity was 81% (MTT assay only); assessed with the combination of the MTT and IL-1a assays, the sensitivity increased to 91%, with a specificity of 79%. For EpiDerm, the sensitivity was 57% and the specificity was 85% (MTT assay only), while the predictive capacity of EpiDerm was not improved by the measurement of IL-1a release. Following independent peer review, in April 2007 the ECVAM Scientific Advisory Committee endorsed the scientific validity of the EPISKIN test as a replacement for the rabbit skin irritation method, and of the EpiDerm method for identifying skin irritants as part of a tiered testing strategy. This new alternative approach will probably be the first in vitro toxicity test to replace the Draize rabbit skin irritation test in Europe and internationally, since, in the very near future, new EU and OECD Test Guidelines will be proposed for regulatory acceptance.JRC.I.2-Validation of biomedical testing method