MatTek Corporation and MatTek In Vitro Life Science Laboratories (IVLSL), MatTek’s subsidiary located in Bratislava, Slovakia, are presenting posters and talks at the Eurotox 2016 meeting in Seville, Spain. Dr. Patrick Hayden is presenting a talk on in vitro inhalation toxicology using the EpiAirway tissue model. Dr. Silvia Letasiova is also presenting several posters on MatTek’s latest in vitro innovations including the new EpiIntestinal and EpiCorneal models for drug toxicity.
Visit us at booth #22, attend our talk or poster
Presentations
Development of an {in vitro} inhalation toxicity test using the EpiAirway model for improved protection of human health
P. J. Hayden1, G. R. Jackson, Jr.1, A. Hunter1, S. Coughlin1, A. Maione1, S. Letasiova2, H. Kandarova2 1MatTek Corporation, Ashland, MA, USA 2MatTek In Vitro Life Science Laboratories, Bratislava, Slovak Republic
Auditorium ITuesday, September 6 – 4pm-6pm
Oral Communication Session I – Presentation #OSC01-009
Posters
Assessment of the Phototoxicity of Three Different TiO2 Nano-forms using Reconstructed Human Tissue Model EpiDerm
Abstract Reference No: 1187
Alzbeta Liskova1, Tatiana Milasová2, Sona Jantová1, Vlasta Brezova3 and Helena Kandarova2
1MatTek IVLSL, Bratislava, Slovakia, 2Cyprotex US LLC, Kalamazoo, MI, United States, 3RIVM, Bilthoven, Netherlands, 4MatTek Corporation, Ashland, MA, United States, 5Division of Medical Devices – NIHS, Tokyo, Japan, 6Medtronic, Inc, Minneapolis, MN, United States.
Absorption of the solar light by photo-sensitive substances and consequent formation of reactive oxygen species (ROS) and other photo-products may lead to the cellular damage as well as to responses of the immune system. Taken that into the consideration, the determination of phototoxicity of substances absorbing UV and visible spectra of the solar light (VIS) belongs therefore to the basic toxicology tests.
One of the methods used for the determination of phototoxicity is a test based on the use of 3D in vitro reconstructed human skin tissue model-EpiDermTM, the EpiDerm H3D-PT. This test was developed and pre-validated by organization ZEBET already in 1997. The main objective of this work was to verify and determine the phototoxicity and phototoxic potential of the selected reference substances and three different types of TiO2 nanoparticles using the EpiDermTM H3D-PT.
We firstly evaluated and standardized the measurement conditions of the sunlight simulator SOL-500 and verified the sensitivity of the EpiDermTM tissues towards UV/VIS light. Next, we evaluated correct prediction of phototoxicity of the EpiDermTM H3D-PT using six reference substances, of which four were known phototoxins (chlorpromazine hydrochloride, two types of bergamot oil and anthracene) and two compounds were UV-absorbing, but without phototoxic potential (cinnamaldehyde, p-aminobenzoic acid). Finally, we have used this method to predict the phototoxicity of three different types of titanium dioxide (P25 AEROXID, Eusolex T-2000, TIG-115).
Based on the results obtained in this work, we conclude that the EpiDermTM H3D-PT is a reliable test for the detection of phototoxicity and prediction of the phototoxic potential of selected substances. This conclusion is supported by the fact that during the measurements we obtained the same or better results as published by Liebsch et al. (1997). Phototoxicity of TiO2 has not been demonstrated in any of the three samples tested. This is because TiO2 nanoparticles do not penetrate deep enough into the epidermis to cause cytotoxicity by irradiation with UVA/VIS.
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Development, Optimization and Validation of an In Vitro Skin Irritation Test for Medical Devices using the Reconstructed Human Tissue Model EpiDerm
Abstract Reference No: 1189
Kandarova, Helena1, 4; Willoughby, Jamin A.2; de Jong, Wim H.3; Bachelor, Michael A.4; Letasiova, Silvia1; Milasova, Tatiana1; Breyfogle, Bridget4; de la Fonteyne, Liset3; Haishima, Yuji 5; Coleman, Kelly P.6
1MatTek IVLSL, Bratislava, Slovakia, 2Cyprotex US LLC, Kalamazoo, MI, United States, 3RIVM, Bilthoven, Netherlands, 4MatTek Corporation, Ashland, MA, United States, 5Division of Medical Devices – NIHS, Tokyo, Japan, 6Medtronic, Inc, Minneapolis, MN, United States.
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 (OECD TG 439). However, medical device (MD) extracts are diluted solutions with low irritation potential, therefore the validated RhE-methods needed to be modified to reflect needs of ISO 10993.
A protocol employing RhE EpiDerm was optimized in 2013 using known irritants and spiked polymers (Casas et al., TIV, 2013).
In 2014, a second laboratory assessed the transferability of the assay. Two additional exposure times were tested along with other medical device materials. After the successful transfer and standardization of the protocol, nine EU and USA laboratories were trained in the use of the protocol in the preparation for the validation. All laboratories produced data with almost 100% agreement of predictions for the selected references.
Two of the laboratories performed additional tests with heat-pressed PVC sheets spiked with Genapol X-080 (Y-4 polymer), Vicryl suture, and polymers spiked with heptanoic acid and sodium dodecyl sulfate. All materials were extracted for 24 or 72 hours in both saline and sesame oil at either 37°C.
Significant irritation responses were detected for Y-4 under all conditions. These results were consistent with those reported by other research groups involved in the upcoming validation study. Vicryl suture was negative and spiked polymers were either positive or negative dependent on the extraction solvent.
We conclude that a modified RhE skin irritation test has the potential to address the skin irritation potential of the medical devices, however, standardization and focus on the technical issues is essential for accurate prediction. Round Robin validation of the Skin irritation test in vitro for assessment of Medical devices extracts has started in March 2016.
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CON4EI: EpiOcular Eye Irritation Test (EIT)
Abstract Reference No: 1188
Helena Kandarova1, An R. Van Rompay2, Els Adriaens3, Nathalie Alépée4, Agnieszka Drzewiecka5, Przemyslaw Fochtman5, Katarzyna Gruszka5, Robert Guest6, Gareth Maglennon6, Jane Schofield6, Jamin A. Willoughby7, Sandra Verstraelen2
1MatTek In Vitro Life Sciences Laboratories, Bratislava, Slovakia; 2Flemish Institute for Technological Research (VITO NV), Applied Bio & Molecular Systems, Mol, Belgium; 3Adriaens Consulting bvba, Aalter, Belgium; 4L’Oréal Research & Innovation, Aulnay-sous-Bois, France; 5Institute of Industrial Organic Chemistry Branch Pszczyna, Department of Toxicological Studies, Pszczyna, Poland; 6Envigo, Derbyshire, United Kingdom; 7Cyprotex US, LLC, Kalamazoo, MI 49008, USA
Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project is to develop tiered testing strategies for eye irritation assessment for all drivers of classification. For this, a set of 80 reference chemicals (38 liquids and 42 solids) was tested with eight different alternative methods. Here, the results obtained with reconstructed human cornea-like epithelium (RHCE) EpiOcular and the EpiOcular Eye Irritation Test (EIT) -adopted as OECD TG 492 – are shown.
The primary aim of this study was an evaluation of the performance of the test method to discriminate chemicals not requiring classification for serious eye damage/eye irritancy (No Category) from chemicals requiring classification and labeling (Category 1 and 2). In addition, the predictive capacity in terms of in vivo driver of classification was investigated. In a second step, it was investigated whether the EpiOcular EIT can be used as part of a tiered-testing strategy for eye irritation assessment. The chemicals were tested in two independent runs by MatTek IVLSL.
For the EpiOcular EIT, a sensitivity of 96.9% and specificity of 86.7% with an accuracy of 95% was obtained overall and for both runs separately (100% concordance). The results of this study seem promising with regard to the evaluation of inclusion of this test method in an integrated testing strategy for eye irritation assessment.
This research is funded by CEFIC-LRI. We acknowledge Cosmetics Europe for their contribution in chemical selection.
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Determination of contact sensitization potential of chemicals using in vitro reconstructed normal human epidermal model EpiDerm: impact of the modality of application
Abstract Reference No: 1094
Letasiova S.1, Corsini E.2, Galbiati V.2, Kandarova H.1, Lehmeier D.3, Gehrke H.3
1MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia, 2Laboratory of Toxicology, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy, 3Eurofins BioPharma Product Testing Munich GmbH, Germany
Assessment of skin sensitization potential has traditionally been conducted in animal models, such as the Mouse Local Lymph Node Assay (LLNA) and the Guinea Pig Maximisation Test (GPMT). However, a growing focus and consensus for minimizing animal use have stimulated the development of in vitro methods to assess skin sensitization. Interleukin-18 (IL-18) release in reconstructed human epidermal models has been identified as a potentially useful endpoint for the identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability (1).
The purpose of this study was to investigate the impact of the modality of chemical exposure on the predictive capacity of the assay. EpiDerm tissue viability assessed by MTT assay and IL-18 release assessed by ELISA were evaluated after 24 h topical exposure to test chemicals either impregnated in 8 mm diameter paper filters or directly applied to the surface of EpiDerm. Acetone: olive oil (4:1) was used as the vehicle in all cases. A total of five chemicals from 3 different sources were tested. The testing set included 3 sensitizers, namely 2,4-dinitrochlorobenzene, cinnamaldehyde and isoeugenol/eugenol, and 2 non-sensitizers, lactic acid and salicylic acid. Four independent dose – response experiments were conducted in 3 laboratories, resulting in correct prediction of the sensitizing potency of test chemicals.
The assessment of IL-18 release using in vitro reconstructed normal human epidermal model EpiDerm appears to be a promising tool for in vitro determination of contact sensitization potential.
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Use of ECVAM validated EpiDerm skin corrosion test (EpiDerm SCT) for sub-categorization according to the UN GHS
Abstract Reference No: 1097
Letasiova Silvia1, Kandarova Helena1, Hayden Patrick2, Klausner Mitchell2
1 MatTek In Vitro Life Science Laboratories, Mlynské Nivy 73, 821 05 Bratislava, Slovak Republic, 2 MatTek Corporation, 200 Homer Avenue, 01721 Ashland, MA, USA
Skin corrosion refers to the production of irreversible damage to the skin. OECD adopted four reconstructed human skin model assays for predicting skin corrosion in vitro (OECD TG 431). The guideline, however, does not yet fully satisfy international labelling guidelines for transport of dangerous goods.
The UN-GHS utilizes 3 corrosion sub-categories (1A-very dangerous,1B-medium danger and 1C-minor danger). Labelling a chemical as 1A has important consequences for transport and animal tests are still utilized for assessing the packaging subclasses. An in vitro method that could discriminate at least between the 1A vs 1B/1C classes would therefore have a substantial impact on reducing animal tests for this purpose.
The current study evaluates prediction of the sub-classess using the EpiDerm SCT and 80 chemicals selected by the OECD expert group for skin irritation and corrosion. Using tiered classification strategy, sensitivity for class 1A was 86% using 3 min exposure time-point. None of 1A chemicals were under-predicted as NC. Specificity for NC chemicals was 74%. As demonstrated, the EpiDerm SCT allows a partial sub-classification of corrosives into sub-category 1A, 1B/1C,and NC. Adoption of the new prediction model based on a 3 min endpoint into the validated EpiDerm SCT and the OECD TG 431 will allow identification of severely corrosive substances without use of animals.
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Pre-Validation of human small intestinal tissue model system to screen drug-induced gastrointestinal toxicity and wound healing
Abstract Reference No: 1129
Seyoum Ayehunie1, Matthew Wagoner2, Herbert Barthlow2, Clay Scott2, Zachary Stevens1, Timothy Landry1, Alex Armento1. Mitchell Klausner1, and Patrick Hayden1
1MatTek Corporation, Ashland, MA and 2AstraZeneca, Waltham, MA – USA
Purpose: The mechanisms of off-target dose-limiting gastrointestinal toxicities for therapeutic compounds are often poorly understood, in large part due to the lack of physiologically-relevant in vitro models. The inherent differences in intestinal physiology between patients and preclinical species also add another layer of complexity in gastrointestinal (GI) toxicity studies. The purpose of this work is to evaluate the utility of an in vitro primary human cell based small intestinal 3D tissue (SMI) model as an investigational tool for drug induced GI toxicity and wound healing studies.
Methods: To achieve this goal, a blinded study was performed using N=5 therapeutic compounds for which dog and rat GLP toxicity studies were not predictive of human toxicities. These compounds were selected from stopped AstraZeneca clinical development programs representing a diversity of target classes and chemistries and tested at five concentrations. As negative controls, N=3 drugs that are well tolerated in humans were included. To model GI toxicity, we examined cytotoxicity by MTT viability and LDH release assays and tissue barrier integrity using transepithelial electrical resistance (TEER) measurements, following two repeat exposures over 96 hours. We also examine the utility of the tissue model for wound healing studies by inducing a 2 mm wound area using 2 mm biopsy punch. The wounded tissues were cultured for up to 10 days in medium with or without human serum. To examine the rate of wound healing, confocal imaging of tissues was performed at days 2, 4, 7, and 10.using anti-CK19 and anti-vimentin antibodies for epithelial cell migration and fibroblast spreading, respectively.
Results: The results showed that the SMI system detected drug induced disruption of intestinal barrier function (TEER) in 5/5 problematic drugs with human GI toxicity at concentrations within or below 30x clinical exposure levels. Importantly, the SMI system showed no effect within 1,000x clinical exposure levels for the three negative controls. In terms of wound healing, tissues cultured in human serum completed the wound healing process at day 6 of the culture period compared to > 10 days for control tissues cultured in the absence of serum.
Conclusion: Overall, the use of TEER as an endpoint makes the SMI tissue a sensitive predictive tool to assess clinically relevant exposures of drugs that induce intestinal toxicity in patients but incorrectly predicted in preclinical GLP toxicology studies, suggesting that the in vitro system may serve as a promising model for both investigational and traditional GI drug safety studies. Furthermore, the small intestinal tissue model can be used for evaluating therapeutics for their wound healing potential.
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Evaluation of inflammatory and genotoxic effects of smokeless tobacco using an organotypic model of human oral epithelium
Abstract Reference No: 1147
Bridget Breyfogle, Jonathan Oldach, Patrick Hayden, Mitch Klausner, Michael Bachelor
MatTek Corporation, Ashland, MA
In addition to the well-known effects of tobacco use on the causation of lung and cardiovascular disease, tobacco use is also implicated as a major cause of oral cavity disease that leads to thousands of deaths per year. Snus, a smokeless tobacco applied to the oral cavity, has been proposed as a less harmful alternative to smoking although its safety has not been adequately evaluated. The objective of this study was to evaluate the cytotoxic, genotoxic and inflammatory effects of snus using an in vitro model of human oral mucosa (EpiOralTM). EpiOralä tissues were treated topically with 5 or 25 milligrams of snus for 24-48 hours and evaluated for cytotoxicity by MTT. Tissues treated with 5 mg of snus had comparable viability to vehicle treated controls while those treated with 25 mg displayed approximately a 20% decrease in viability after 24 and 48 hours of exposure. Histological analysis revealed hyperchromic staining in tissues treated with 5 mg of snus at 24 hours post-treatment whereas tissues treated with 25 mg of snus displayed a significant amount of sloughing of the apical layers. Following treatment, an inflammation-specific cytokine panel was used to analyze markers of inflammation at 24 and 48 hours post treatment. Of the cytokines analyzed, significant increases (1.5-2 fold) in IP-10, GM-CSF and RANTES were observed at both 24 and 48 hours post treatment in tissues treated with 25 mg of snus. As a measure of genotoxicity, the presence of γ-H2AX foci (specifically, phosphorylation at Serine 139) was evaluated in treated tissues. γ-H2AX is a phosphorylated derivative of the H2AX histone and is tightly bound to double strand DNA break sites, therefore serving as a biomarker of genotoxic insult. γ-H2AX foci were readily detected in the apical layer of tissues treated with 25 mg of snus at 24 and 48 hours post treatment. These results demonstrate the utility of this organotypic oral tissue model to evaluate the safety of smokeless tobacco products.
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An In Vitro Reconstructed Normal Human Corneal Tissue Model: Applications to Dry Eye, Wound Healing, and Ophthalmic Drug Delivery
Abstract Reference No: 1148
Kaluzhny Y 1, Kinuthia MWK 1, Gremilogianni AM 2, Plotkin A 1, Megkoulas M 2, Hayden P 1, and Klausner M 1
1 MatTek Corporation – Ashland, MA – USA
2 Univ. of Athens, Lab. of Analytical Chemistry, QualiMetrix SA , Athens, Greece
Current methods to investigate corneal wound healing (CWH), pathogenesis of dry eye disease (DED)1, and permeation of topically applied ophthalmics utilize cell cultures or animals2. This study evaluated the utility of EpiCorneal human tissue model for CWH, DED, and drug permeation. EpiCorneal tissue model comprised of normal human corneal epithelial cells that are cultured at the air-liquid interface. Corneal wounds were introduced by abrasion or chemicals (1N NaOH). Wounded tissues were cultured in the presence or absence of human corneal keratocytes (HCK) or EGFR inhibitor (erlotinib, 10 mM). A DED model was generated by placing EpiCorneal tissues under desiccating stress conditions (DSC, 40% RH, 40°C, and 5% CO2) that stimulate morphological, cellular, and molecular changes relevant to dry eye. Corneal permeability was evaluated using compounds with a wide range of properties: hydrophilic and hydrophobic, low and high molecular weight dyes, ophthalmic related drugs at seven different formulations3.
CWH was analyzed by transepithelial electrical resistance (TEER), histology, confocal microscopy, and gene expression. TEER recovered to 933.7/502.4 Ω*cm2 in the presence/or absence of HCK in 4 days post-wounded cultures. mRNA expression was analyzed using a 96-gene wound healing microarray. 13 genes (including collagen, integrin, chemokine, and protein kinase families) were up-regulated in the EpiCorneal tissues 24h post-abrasion in the absence of HCK and 16 genes (including WNT, FGF, small GTPases, chemokine, and integrin families) were up-regulated in the presence of HCK, but not in control cultures. DED was analyzed by TEER, histology, tissue viability, mucins and tight junction (TJ) protein expression. Dramatic reduction in tissue thickness was observed after 48h in DSC that coincided with decreased expression of mucins, increased TEER and atypical expression of TJ proteins. Topical application (25 µl/tissue) of lubricant gel drops (GenTeal, Alcon) improved tissue morphology and barrier function.
Out of seven formulations of Latanaprost eye drops tested, Xalatan (containing 0.02% BAC) had the highest permeation (Papp=8.81cm×s-1) and Monoprost (preservative-free) had the lowest (Papp=1.15 cm×s-1). Formulations containing Poloxamer 407 had higher Papp (6.05 and 6.27) when compared to formulations without surfactants (1.69 to 2.57). Tissue integrity and viability were maintained in all experiments as evidenced by Lucifer Yellow and MTT results.
The reconstructed in vitro organotypic human corneal tissue structurally and functionally reproduces CWH and DED, and its permeability resembles that of the in vivo human cornea. This model is anticipated to be a useful tool to study molecular mechanisms of ocular surface damage, DED, and to evaluate new corneal drug formulations.
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