in vitro

/Tag:in vitro

In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products: Workshop Proceedings, Conclusions and Paths Forward for In Vitro Model Use

Holger Behrsing, Erin Hill, Hans Raabe, Raymond Tice, Suzanne Fitzpatrick, Robert Devlin, Kent Pinkerton, Günter Oberdörster, Chris Wright, Roman Wieczorek, Michaela Aufderheide, Sandro Steiner, Tobias Krebs, Bahman Asgharian, Richard Corley, Michael Oldham, Jason Adamson, Xiang Li, Irfan Rahman, Sonia Grego, Pei-Hsuan Chu, Shaun McCullough and Rodger Curren

In 2009, the passing of the Family Smoking Prevention and Tobacco Control Act facilitated the establishment of the FDA Center for Tobacco Products (CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 4–6 April 2016, the Institute for In Vitro Sciences, Inc. (IIVS) convened a workshop conference entitled, In Vitro Exposure Systems and Dosimetry Assessment Tools for Inhaled Tobacco Products, to bring together stakeholders representing regulatory agencies, academia and industry to address the research priorities articulated by the FDA CTP. Specific topics were covered to assess the status of current in vitro smoke and aerosol/vapour exposure systems, as well as the various approaches and challenges to quantifying the complex exposures in in vitro pulmonary models developed for evaluating adverse pulmonary events resulting from tobacco product exposures. The four core topics covered were: a) Tobacco Smoke and E-Cigarette Aerosols&#59; b) Air–Liquid Interface-In Vitro Exposure Systems&#59; c) Dosimetry Approaches for Particles and Vapours/In Vitro Dosimetry Determinations&#59; and d) Exposure Microenvironment/Physiology of Cells. The 2.5-day workshop included presentations from 20 expert speakers, poster sessions, networking discussions, and breakout sessions which identified key findings and provided recommendations to advance these technologies. Here, we will report on the proceedings, recommendations, and outcome of the April 2016 technical workshop, including paths forward for developing and validating non-animal test methods for tobacco product smoke and next generation tobacco product aerosol/vapour exposures. With the recent FDA publication of the final deeming rule for the governance of tobacco products, there is an unprecedented necessity to evaluate a very large number of tobacco-based products and ingredients. The questionable relevance, high cost, and ethical considerations for the use of in vivo testing methods highlight the necessity of robust in vitro approaches to elucidate tobacco-based exposures and how they may lead to pulmonary diseases that contribute to lung exposure-induced mortality worldwide.
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In Vitro Assessment of Skin Irritation Potential of Surfactant-based Formulations by Using a 3-D Skin Reconstructed Tissue Model and Cytokine Response

Russel M. Walters, Lisa Gandolfi, M. Catherine Mack, Michael Fevola, Katharine Martin, Mathew T. Hamilton, Allison Hilberer, Nicole Barnes, Nathan Wilt, Jennifer R. Nash, Hans A. Raabe and Gertrude-Emilia Costin

The personal care industry is focused on developing safe, more efficacious, and increasingly milder products, that are routinely undergoing preclinical and clinical testing before becoming available for consumer use on skin. In vitro systems based on skin reconstructed equivalents are now established for the preclinical assessment of product irritation potential and as alternative testing methods to the classic Draize rabbit skin irritation test. We have used the 3-D EpiDerm™ model system to evaluate tissue viability and primary cytokine interleukin-1α release as a way to evaluate the potential dermal irritation of 224 non-ionic, amphoteric and/or anionic surfactant-containing formulations, or individual raw materials. As part of our testing programme, two representative benchmark materials with known clinical skin irritation potential were qualified through repeated testing, for use as references for the skin irritation evaluation of formulations containing new surfactant ingredients. We have established a correlation between the in vitro screening approach and clinical testing, and are continually expanding our database to enhance this correlation. This testing programme integrates the efforts of global manufacturers of personal care products that focus on the development of increasingly milder formulations to be applied to the skin, without the use of animal testing.

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The Adverse Outcome Pathway Concept: A Basis for Developing Regulatory Decision-making Tools

Nathalie Delrue, Magdalini Sachana, Yuki Sakuratani, Anne Gourmelon, Eeva Leinala, Robert Diderich

The Adverse Outcome Pathway (AOP) concept is expected to guide risk assessors in their work to use all existing information on the effects of chemicals on humans and wildlife, and to target the generation of additional information to the regulatory objective. AOPs will therefore be used in the Organisation for Economic Co-operation and Development (OECD) chemical safety programme, as underlying scientific rationales for the development of alternative methods for hazard assessment, such as read-across, in vitro test methods or the development of integrated testing strategies that have the potential to replace animal tests. As a proof-of-concept, the OECD has developed an AOP for skin sensitisation, and as a follow-up has: a) implemented the AOP into the OECD QSAR Toolbox, so that information related to the Key Events (KEs) in the AOP can be used to group chemicals that are expected to act by the same mechanism and hence have the same skin sensitisation potential; b) developed alternative test methods for the KEs, so that ultimately chemicals can be tested for skin sensitisation without the use of animal tests. The development of integrated testing strategies based on the AOP is ongoing. Building on this proof-of-concept, the OECD has launched an AOP development programme with a first batch of AOPs published in 2016. A number of IT tools, which together form an AOP Knowledge Base, are at various stages of development, and support the construction of AOPs and their use in the development of integrated approaches for testing and assessment. Following the publication of the first batch of AOPs, OECD member countries will decide on priorities for their use in supporting the development of tools for regulatory use.
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Autologous Co-culture of Primary Human Alveolar Macrophages and Epithelial Cells for Investigating Aerosol Medicines. Part II: Evaluation of IL-10-loaded Microparticles for the Treatment of Lung Inflammation

Marius Hittinger, Nico Alexander Mell, Hanno Huwer, Brigitta Loretz, Nicole Schneider-Daum and Claus-Michael Lehr

Acute respiratory distress syndrome is linked to inflammatory processes in the human lung. The aim of this study was to mimic in vitro the treatment of lung inflammation by using a cell-based human autologous co-culture model. As a potential trial medication, we developed a pulmonary dry powder formulation loaded with interleukin-10 (IL-10), a potent anti-inflammatory cytokine. The inflammatory immune response was stimulated by lipopolysaccharide. The co-culture was combined with the Pharmaceutical Aerosol Deposition Device on Cell Cultures )PADDOCC), to deposit the IL-10-loaded microparticles on the inflamed co-culture model at the air-liquid interface. This treatment significantly reduced the secretion of interleukin-6 and tumour necrosis factor, as compared to the deposition of placebo (unloaded) particles. Our results show that the alveolar co-culture model, in combination with a deposition device such as the PADDOCC, may serve as a powerful tool for testing the safety and efficacy of dry powder formulations for pulmonary drug delivery.

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Evaluation of an In Vitro Cell Culture Assay for the Potency Assessment of Recombinant Human Erythropoietin

Francine T. Machado, Fernanda P.S. Maldaner, Rafaela F. Perobelli, Bruna Xavier, Francielle S. da Silva, Guilherme W. de Freitas, Paolo Bartolini, Maria Tereza C.P. Ribela and Sérgio L. Dalmora

Recombinant human erythropoietin is a sialoglycoprotein that stimulates erythropoiesis. To assess potency of human erythropoietin produced by recombinant technology, we investigated an in vitro TF-1 cell proliferation assay, which was applied in conjunction with a reversed-phase liquid chromatography method for the determination of the content of sialic acids. The results obtained, which were higher than 126.8ng/μg, were compared with those obtained with the in vivo normocythaemic mouse bioassay. The in vitro assay resulted in a non-significant lower mean difference of the estimated potencies (0.61% ± 0.026, p > 0.05). The use of this combination of methods represents an advance toward the establishment of alternative in vitro approaches, in the context of the Three Rs, for the potency assessment of biotechnology-derived medicines.

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Assessment of In Vitro COPD Models for Tobacco Regulatory Science: Workshop Proceedings, Conclusions and Paths Forward for In Vitro Model Use

Holger Behrsing, Hans Raabe, Joseph Manuppello, Betsy Bombick, Rodger Curren, Kristie Sullivan, Sanjay Sethi, Richard Phipps, Yohannes Tesfaigzi, Sherwin Yan, Carl D’Ruiz, Robert Tarran, Samuel Constant, Gary Phillips, Marianna Gaça, Patrick Hayden, Xuefei Cao, Carole Mathis, Julia Hoeng, Armin Braun and Erin Hill

The Family Smoking Prevention and Tobacco Control Act of 2009 established the Food and Drug Administration Center for Tobacco Products (FDA-CTP), and gave it regulatory authority over the marketing, manufacture and distribution of tobacco products, including those termed 'modified risk'. On 8-10 December 2014, IIVS organised a workshop conference, entitled Assessment of In Vitro COPD Models for Tobacco Regulatory Science, to bring together stakeholders representing regulatory agencies, academia, industry and animal protection, to address the research priorities articulated by the FDA-CTP. Specific topics were covered to assess the status of current in vitro technologies as they are applied to understanding the adverse pulmonary events resulting from tobacco product exposure, and in particular, the progression of chronic obstructive pulmonary disease (COPD). The four topics covered were: a) Inflammation and Oxidative Stress; b) Ciliary Dysfunction and Ion Transport; c) Goblet Cell Hyperplasia and Mucus Production; and d) Parenchymal/Bronchial Tissue Destruction and Remodelling. The 2.5 day workshop included 18 expert speakers, plus poster sessions, networking and breakout sessions, which identified key findings and provided recommendations to advance the in vitro technologies and assays used to evaluate tobacco-induced disease etiologies. The workshop summary was reported at the 2015 Society of Toxicology Annual Meeting, and the recommendations led to an IIVS-organised technical workshop in June 2015, entitled Goblet Cell Hyperplasia, Mucus Production, and Ciliary Beating Assays, to assess these assays and to conduct a proof-of-principle multi-laboratory exercise to determine their suitability for standardisation. Here, we report on the proceedings, recommendations and outcomes of the December 2014 workshop, including paths forward to continue the development of non-animal methods to evaluate tissue responses that model the disease processes that may lead to COPD, a major cause of mortality worldwide.

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The Björn Ekwall Memorial Award 2015

Introduction by Ada Kolman & lecture text by Michael Balls

A personal, and therefore unavoidably biased, review is given, of the significance of the contributions made by selected Scandinavian individuals, organisations and events, to the development of in vitro toxicology procedures as potential replacements for toxicity tests in laboratory animals. In addition to their wider significance, these contributions had a profound effect on whatever contributions I have been able to make, myself. Nevertheless, while there has been much progress in the last 35 years or so, and many lessons have been learned, there is still much to be done, especially as animal tests remain entrenched as the preferred methods which set the gold standards and make regulators feel comfortable. Many of the clues to dealing with the questions and concerns which plague hazard prediction and risk assessment have long been available, but they have been ignored, largely for reasons which have little to do with the science of toxicology and the need to maintain the highest scientific standards. I have little doubt that Björn Ekwall, whose memory I feel privileged to honour, would have agreed with that last statement.
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Barriers to the Uptake of Human-based Test Methods, and How to Overcome Them

Kathy Archibald, Tamara Drake and Robert Coleman

Although there is growing concern as to the questionable value of animal-based methods for determining the safety and efficacy of new medicines, which has in turn led to many groups developing innovative human-based methods, there are many barriers to their adoption for regulatory submissions.
The reasons for this are various, and include a lack of confidence that the available human-based methods, be they in vivo, in silico or in vitro, can be sufficiently predictive of clinical outcomes. However, this is not the only problem: the issue of validation presents a serious impediment to progress, a particularly frustrating situation, in view of the fact that the existing animal-based methods have never themselves been formally validated. Superimposed upon this is the issue of regulatory requirements, where, although regulators may be willing to accept non-animal approaches in place of particular animal tests, nowhere is this explicitly stated in their guidelines. Such problems are far from trivial, and represent major hurdles to be overcome. In addition, there are a range of other barriers, real or self-imposed, that are hindering a more-predictive approach to establishing a new drug’s clinical safety and efficacy profiles. Some of these barriers are identified, and ways forward are suggested.
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The Ex Vivo Eye Irritation Test as an Alternative Test Method for Serious Eye Damage/Eye Irritation

Felix Spöler, Oya Kray, Stefan Kray, Claudia Panfil, and Norbert F. Schrage

Ocular irritation testing is a common requirement for the classification, labelling and packaging of chemicals (substances and mixtures). The in vivo Draize rabbit eye test (OECD Test Guideline 405) is considered to be the regulatory reference method for the classification of chemicals according to their potential to induce eye injury. In the Draize test, chemicals are applied to rabbit eyes in vivo, and changes are monitored over time. If no damage is observed, the chemical is not categorised. Otherwise, the classification depends on the severity and reversibility of the damage. Alternative test methods have to be designed to match the classifications from the in vivo reference method. However, observation of damage reversibility is usually not possible in vitro. Within the present study, a new organotypic method based on rabbit corneas obtained from food production is demonstrated to close this gap. The Ex Vivo Eye Irritation Test (EVEIT) retains the full biochemical activity of the corneal epithelium, epithelial stem cells and endothelium. This permits the in-depth analysis of ocular chemical trauma beyond that achievable by using established in vitro methods. In particular, the EVEIT is the first test to permit the direct monitoring of recovery of all corneal layers after damage. To develop a prediction model for the EVEIT that is comparable to the GHS system, 37 reference chemicals were analysed. The experimental data were used to derive a three-level potency ranking of eye irritation and corrosion that best fits the GHS categorisation. In vivo data available in the literature were used for comparison. When compared with GHS classification predictions, the overall accuracy of the three-level potency ranking was 78%. The classification of chemicals as irritating versus non-irritating resulted in 96% sensitivity, 91% specificity and 95% accuracy.
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Non-animal Replacements for Acute Toxicity Testing

Carol Barker-Treasure, Kevin Coll, Nathalie Belot, Chris Longmore, Karl Bygrave, Suzanne Avey and Richard Clothier

Current approaches to predicting adverse effects in humans from acute toxic exposure to cosmetic ingredients still heavily necessitate the use of animals under EU legislation, particularly in the context of the REACH system, when cosmetic ingredients are also destined for use in other industries. These include the LD50 test, the Up-and-Down Procedure and the Fixed Dose Procedure, which are regarded as having notable scientific deficiencies and low transferability to humans. By expanding on previous in vitro tests, such as the animal cell-based 3T3 Neutral Red Uptake (NRU) assay, this project aims to develop a truly animal-free predictive test for the acute toxicity of cosmetic ingredients in humans, by using human-derived cells and a prediction model that does not rely on animal data. The project, funded by Innovate UK, will incorporate the NRU assay with human dermal fibroblasts in animal product-free culture, to generate an in vitro protocol that can be validated as an accepted replacement for the currently available in vivo tests. To date, the project has successfully completed an assessment of the robustness and reproducibility of the method, by using sodium lauryl sulphate (SLS) as a positive control, and displaying analogous results to those of the original studies with mouse 3T3 cells. Currently, the testing of five known ingredients from key groups (a surfactant, a preservative, a fragrance, a colour and an emulsifier) is under way. The testing consists of initial range-finding runs followed by three valid runs of a main experiment with the appropriate concentration ranges, to generate IC50 values. Expanded blind trials of 20 ingredients will follow. Early results indicate that this human cell-based test holds the potential to replace aspects of in vivo animal acute toxicity testing, particularly with reference to cosmetic ingredients.
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