Human precision-cut lung slices (hPCLS) are a highly relevant 3-dimensional model of the lung. They offer native architecture and cells of the lung tissue including respiratory parenchyma, small airways, and immune competent cells involved in inflammatory and sensitization processes. The scarcity of human lung tissue available for research and the inability to conduct larger scale testing has limited the use of hPCLS as a test system for routine, high-throughput testing. To overcome this barrier, IIVS has refined the methodology behind the cryopreservation, storage, thaw and post-thaw maintenance of human lung slices. As primary tissues have varied quality and responsiveness, a standardized performance characterization (PC) is conducted on all donor batches. (more…)
The Organization for Economic Cooperation and Development (OECD) guidance document on Good In Vitro Method Practices (GIVIMP) details a set of quality standards to improve both the quality of and confidence in newly developed, and routinely executed in vitro methods. An important part of (and a source of potential high variability in) any method is the test system. Microphysiological Systems (MPS), including organ-on-a-chip platforms have been used in novel methods for many years. Despite their potential benefits, there are several quality concerns associated with MPS. Two of these concerns are reproducibility and well defined and understood biological relevance, both of which are covered in the GIVIMP guidance. (more…)
The demand for toxicology data from human-relevant, New Approach Methodologies (NAMs) continues to increase. Fortunately, the scientific community has responded with new tools based on human tissues and cells. The creators of these systems and test methods utilizing them have invested in their development and also in studies designed to demonstrate their relevance, reproducibility, and transferability. Despite these efforts, scientific confidence in these methods at the regulatory level remains comparatively low. (more…)
IIVS presented several posters at SOT 2023. This post includes links to the full PDF versions of each poster presented. (more…)
The Institute for In Vitro Sciences (IIVS) is sponsoring a series of workshops to identify, discuss and develop recommendations for optimal scientific/technical approaches for utilizing in vitro assay data within and across tobacco and nicotine product categories. Workshops provide a unique opportunity for invited expert stakeholders to share experiences and to develop recommendations that may serve as a resource for developing optimal approaches and data to evaluate the toxicity of tobacco and nicotine products. It is envisioned that some of these recommendations would form the basis for the generation of guidance documents and/or serve as authoritative reference publications for optimal methodologies and data interpretation and to support regulatory submissions. Invited experts for the IIVS workshops include scientists from tobacco companies, contract research organizations, US regulatory agencies, and other in vitro assay experts with tobacco product and/or genetic toxicology experience. The format for this workshop series is primarily discussion among participants which provides an environment to tackle issues in detail. Participants are expected to actively participate by collecting relevant published and unpublished non-proprietary research information, to offer experiences and expert opinions, and to actively share with other workgroup members. While the focus will be on the widely used regulatory in vitro genetic toxicology assays, it is important to note that much of the discussion will be applicable to all in vitro assays. As a part of the workshop discussion, data gaps will be identified. Thus, in addition to recommendations based on current information, this workshop series will provide key research questions that need to be addressed by the scientific community. This will provide a useful roadmap for research that can have direct impact on the regulation of tobacco products and on protecting human health related to consumer use of tobacco products. The product of these workshops will be a series of scientific publications and meeting presentations that can be utilized by all stakeholders. Prior to the first workshop (November 27-28, 2018) important issues for using in vitro genotoxicity assays for evaluating tobacco and nicotine products were identified. During the first workshop issues were triaged into three priority categories based on the amount of available information.
Respiratory Toxicology | Tobacco | NGTP | P. Desai | R. CurrenThe Family Smoking Prevention and Tobacco Control Act gave the FDA regulatory authority over next generation tobacco products (NGTP) such as E-vapor products. E-vapor product liquids contain a variety of ingredient combinations that should be assessed for human risk. One human lung-relevant testing platform with reasonable throughput, is human precision-cut lung slices (HuPCLS). HuPCLS are arguably the most complex non-animal model of the lung, retaining native architecture and immune-competent cells over multi-week culture periods. HuPCLS were exposed to three concentrations (0.1%, 0.5%, and 1.2%) of propylene glycol (PG ; an E-vapor product constituent) continuously for 16 days. Exposure-effects were evaluated biochemically (WST-8 assay) and histologically viability assessment of H&E stained slides). Positive control treatments consisted of 10μM Phortress and 13μM bleomycin. HuPCLS were fed every day with fresh medium ± treatment and harvested at days 4, 8, and 16. Untreated control UC) HuPCLS viability was confirmed using protein and a denylate kinase assays. Over 16 days in culture, UC lost 30% viability while WST-8 results indicated no loss over 16 days in culture. Phortress caused severe damage by day 4 and bleomycin by day 8 (histologically & WST-8 viability). Prolonged 1.2% PG exposure diminished WST-8 viability by ~30% at day 16 which agreed with histological results. High osmolality is the suspected mechanism of toxicity. There was no effect histologically or via WST-8 viability for prolonged exposure to 0.1% and 0.5% PG. In summary, PG, a common E-vapor product ingredient, at 1.2% had adverse effects in a human pulmonary model in an exaggerated exposure regimen(prolonged exposures with changes in osmolarity). The HuPCLS platform has huge potential to serve as a screening tool for e-liquid(and other materials of concern) by elucidating potentially relevant, long-term events following NGTP ingredient exposure.
Precision Cut Lung Slices (PCLS) | Respiratory Toxicology | Tobacco | Hans Raabe | Holger Behrsing | HuPCLS | New Generation Tobacco Products | NGTPThe Transient Receptor Potential Vanilloid type 1 (TRPV1) receptor is one of the most well characterized pain-inducing receptors and has been recently identified as a valuable tool to predict eye stinging potential of surfactant based formulations. In this study we sought to predict eye stinging of nonsurfactant based cosmetic formulations by studying TRPV1 activity using the NociOcular assay. In the NociOcular assay, TRPV1 expressing neuroblastoma cells are exposed to test substance and TRPV1 activity is measured by acute increases in intracellular calcium. Three of the formulations induced stinging in the human test and were also positive in the NociOcular assay. The other four formulations evaluated were classified as stinging in the human test, but a conclusive determination could not be made in the NociOcular assay as the formulations were not fully soluble in assay buffers. The formulations were also evaluated in the EpiOcularTM assay, an established in vitro model for eye irritation utilized by the cosmetics industry. The Epiocular™ assay results did not correlate with the human sting data. Our data support that the NociOcular assay may be a valuable in vitro tool to predict human eye stinging sensation for cosmetic formulations. Future efforts seeks to further expand the applicability of the assay to product types other than surfactant based formulations.
NociOcular Eye Sting | Cosmetic & Personal CareIn order to further promote the implementation of Directive 2010/63/EU, the European Commission issued calls for a number of related projects last year. One of these projects is aimed at facilitating the uptake of non-animal alternatives by developing two e-learning modules. The contract for this project was awarded to a consortium consisting of SYRCLE, the Swiss 3R Competence Centre, lnstitute for ln Vitro Sciences, Pharma Launcher and Ecorys UK. This consortium will develop two modules, i.e., one eLearning module focused on searching for existing non-animal alternatives (including systematic reviews) and one module targeted at researchers who want to develop reliable and relevant non-animal alternatives for regulatory use taking into account Good In Vitro Method Practices (GIVIMP). The quality of the developed modules will be assessed by external review groups. The learning outcomes will be presented to the commission along with the design of the assignments through which these outcomes will be realised.
alternatives to animal testing | Amanda. K. Ulrey | Erin Hill | GIVIMPWhile the skin sensitization hazard of substances can readily be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA (OECD TG 442C) wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide is evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times at 25°C. After the respective incubation time, the reaction is stopped by addition of the fluorescent dye monobromobimane (mBBr). The highly reactive and non-fluorescent mBBr rapidly reacts with unbound cysteine moieties of the model peptide to form a fluorescent complex. The remaining non-depleted peptide concentration is determined thereafter by fluorescence measurement at precisely defined time points. Kinetic rates of peptide depletion are then used to distinguish between two levels of skin sensitization potency, i.e. to discriminate between CLP/UN GHS sub-categories 1A and 1B. During an in house validation (Wareing et al., 2017) 35 of 38 substances with LLNA-based sensitizing potency were correctly assigned to the potency sub-categories, and the predictivity for 14 human data was similarly high. These results warranted the kDPRA for further validation. Here we present the results of a ring trial testing 24 blind-coded chemicals in seven labs. In parallel we present the extension of the kDPRA database to further assess the predictive capacity of the assay. Eventually the kDPRA should be used as a part of defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment.
kDPRA | Agro/Petrochemical | Cleaning Products | Fragrance & Flavors | Household & Consumer Products | Andreas Natsch | Barbara Birk | Britta Wareing | Cindy Ryan | Erin Hill