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1	Adsorção multi-micotoxinas: revisão e estudo de carvões ativados / Multi-mycotoxin adsorption: review and study of activated carbons Cabral, Susana Maria 01 August 2019 (has links) Elevada incidência de contaminação de micotoxinas em grãos de cereais e alimentos processados tem sido relatada em todo o mundo, principalmente em produtos destinados à alimentação animal. O objetivo desse trabalho foi explorar as tecnologias para redução das concentrações de micotoxinas, que estão disponíveis ou em desenvolvimento. A tecnologia de adsorção, assim como das metodologias disponíveis para avaliação in vitro da capacidade de adsorção foram abordadas com mais ênfase. Na etapa experimental deste projeto, avaliaram-se cinco carvões ativados quanto a suas propriedades e capacidade de adsorção de micotoxinas presentes individualmente e combinadas (multi-micotoxinas) (Aflatoxina B1, Desoxinivalenol, Fumonisina B1 e Zearalenona). O carvão que apresentou maior capacidade de adsorção multi-micotoxinas foi selecionado para um estudo das interações entre adsorvente/adsorvato e possíveis fatores interferentes. Foram avaliados parâmetros físico-químicos do carvão, o efeito do tempo de contato, efeito dose, isotermas e efeito da composição do meio e da mudança do pH. A adsorção de Aflatoxina B1 e Zearalenona não sofreu efeito dos fatores estudados, diferente da adsorção de Fumonisina B1 e Desoxinivalenol. Os resultados auxiliam na análise da interação das moléculas de micotoxinas e do carvão ativado, bem como de quais fatores podem afetar o processo de adsorção de cada uma delas. Porém mais estudos são necessários para esclarecer melhor o papel de cada um dos fatores na adsorção das micotoxinas aqui estudadas. / The high incidence of mycotoxins contamination in cereal grains and processed foods have been reported worldwide, especially in products intended for animal feed. The objective of this work was to explore the available and upcoming technologies applied for the reduction of mycotoxins present in foods with a focus on adsorption, as well as in the in vitro methodology. Following, five activated carbon were evaluated about their properties and its individual and multi-mycotoxin adsorption capacity (Aflatoxin B1, Deoxynivalenol, Fuminisin B1 and Zearalenone). The activated carbon that presented the highest capacity of multi-mycotoxin adsorption was selected to continue the study in order to explore the interactions between adsorbent / adsorbate and possible interferents. Physicochemical parameters were evaluated, the effect of the contact time, concentration, isotherms and effect of the composition of the medium and the pH change. The adsorption of Aflatoxin B1 and Zearalenone had no effect of the factors studied, different from the adsorption of Fumonisin B1 and Deoxynivalenol. The results help in the analysis of the interaction of mycotoxins and activated carbon molecules, as well as which factors may affect the adsorption process of each of them. However, more studies are needed to clarify the role of each of the factors in the adsorption of these mycotoxins studied. In vitro test Activated carbon Adsorbent Adsorvente Carvão ativado Co-ocorrence Coocorrência Micotoxinas Mycotoxins Teste in vitro
2	Methodische Untersuchungen zu Eigenschaften, Nachweis, Reinigung und Antigenität des alpha-Toxins von <i>Clostridium septicum</i> / Methodical studies on properties, detection, purification and antigenicity of the alpha-toxin of <i>Clostridium septicum</i> Jansen, Katja 10 May 2000 (has links) No description available. 630 Landwirtschaft Veterinärmedizin Agricultural Sciences alpha-Toxin anaerob Antigenität Bioreaktor Chromatografie Clostridium <i>Clostridium septicum</i> Fermentation Filtration Gasödem Immunologie in-vitro-Test lokalspezifisch Reinigung Vakzine Zellkultur alpha-Toxin anaerobe antigenicity bioreactor chromatography cell culture Clostridium <i>Clostridium septicum</i> cytotoxicity fermentation filtration gas edema immunology in-vitro-test locality specific purification vaccine 35.70
3	Investigations of in vitro test systems for the detection of Glucocorticoid-induced skin atrophy as a tool in drug discovery Schoepe, Stefanie 12 August 2009 (has links) Topische Glukokortikoide (GCs) sind wirksam bei Therapie von entzündlichen Hauterkrankungen. Durch ihr Nebenwirkungspotential (z.B. Induktion von Hautatrophie) ist ihr Einsatz jedoch begrenzt. Für die Medikamentenentwicklung ist die Bestimmung des atrophogenen Potenzials neuer Verbindungen daher von großer Bedeutung. Derzeit stehen dafür keine prädiktiven in vitro Modelle zur Verfügung. Ziel dieser Arbeit war daher die Etablierung solcher Modelle. Es wurden kutane Zelltypen (3T3-Zellen, Rattenfibroblasten, HaCaT-Zellen, humane Keratinozyten [NHEK] und Fibroblasten) und Vollhautmodelle (CellSystems AST-2000 und Phenions FTSM) untersucht. Atrophie-Marker, die Proliferation, Kollagen-Metabolismus und Epidermisdicke betreffend, wurden auf mRNA-, Protein- bzw. zellulärer Ebene gemessen. Außerdem wurden mittels Genexpressionsanalysen von GC-behandelter Nagerhaut neue potenzielle Marker identifiziert, deren Regulation in vitro jedoch nicht bestätigt werden konnte. Nach Pilotexperimenten wurden 3 Modelle ausgewählt und für Evaluierungsexperimente mit Referenz-GCs behandelt: 1). MMP1, -2, -3 und -9 mRNA-Expression in NHEK, 2). COL1A1 und COL3A1 mRNA-Expression in 3T3-Zellen, 3.) Epidermisdicke, Kollagen- und MMP-Synthese in FTSM. Die Messparameter der 3 Modelle erwiesen sich als dosisabhängig reguliert und korrelierten mit dem atrophogenen Potenzial der GCs. Schließlich wurde die Prädiktabilität der 3 in vitro Modelle für die in vivo Situation im Nager analysiert. In allen 3 in vitro Systemen induzierte die Behandlung mit einem selektiven GC-Rezeptor-Agonisten weniger atrophogene Effekte als das Referenz-GC. Ähnliche Ergebnisse wurden auch in vivo im Rattenhautatrophie-Modell gefunden. Zusammenfassend wird eine Kaskade von 3 in vitro Modellen empfohlen, um das atrophogene Potential von GC-Rezeptor-Liganden zu bestimmen. Der tatsächliche prädiktive Wert für die klinische Situation sollte in weiteren Studien untersucht werden. / Topical glucocorticoids (GCs) are effective for the therapy of inflammatory skin diseases. However, their use is limited by their side effect potential, with skin atrophy being the most prominent one. Thus, determining the atrophogenic potential of novel compounds is of importance for drug development. Currently, there are no according predictive in vitro models available. The aim of this study was to establish such atrophy models. Rodent and human cutaneous cell types (3T3 cells, rat fibroblasts, HaCaT cells, human keratinocytes [NHEK] and fibroblasts) and human full-thickness skin equivalents (CellSystems AST-2000 and Phenions FTSM) were investigated. Atrophy markers related to proliferation, collagen metabolism and epidermal thickness were measured on mRNA, protein and cellular level, respectively. Additionally, by gene expression profiling of GC-treated rodent skin novel potential markers were identified, but subsequently not confirmed in vitro. After pilot studies 3 models were selected and treated with reference GCs for evaluation experiments: 1.) MMP1, -2, -3 and -9 mRNA expression in NHEK, 2.) COL1A1 and COL3A1 mRNA expression in 3T3 cells, 3.) epidermal thickness, collagen and MMP synthesis in FTSM. The read out parameters of all 3 test systems turned out to be regulated dose-dependently and correlated with the atrophogenic potential of the GCs. Finally, the predictability of the 3 recommended in vitro test system for the rodent in vivo situation was analyzed. In all 3 in vitro test systems, the treatment with a novel selective GC receptor agonist induced less atrophogenic effects than the reference GC clobetasol. Indeed, similar results were found in the hr/hr rat skin atrophy model. In summary, a cascade of 3 in vitro models is recommended to be applied for the characterization of the atrophogenicity of GC receptor ligands. Further experiments are necessary to eventually demonstrate the true predictability of these models for the clinical situation. Hautatrophie Glukokortikoid in vitro Testsystem humane Keratinozyten 3T3 Mausfibroblasten Vollhautmodell skin atrophy glucocorticoid in vitro test system human keratinocytes 3T3 mouse fibroblasts full-thickness skin model 570 Biowissenschaften, Biologie 32 Biologie WD 5800 ddc:570
4	Dental composite properties evaluation : from experimental approaches to the prerequisite of a chewing bench / Évaluation des propriétés des composites dentaires : prérequis pour l’élaboration d'un banc de mastication Abouelleil Sayed, Hazem 03 April 2017 (has links) La littérature scientifique révèle que les résultats in vitro sur les matériaux dentaires ont une faible corrélation avec le comportement clinique. Les tests standardisés aux normes fournissent des informations précieuses et pertinentes sur les propriétés des matériaux dentaires, et permettent aussi de comparer les résultats de différents instituts. Cependant, le développement de nouveaux matériaux à partir de nouvelles formulations chimiques nécessite une amélioration des méthodes d'évaluation. Ce travail de recherche est réalisé dans le but d'approfondir les connaissances sur les méthodes d'évaluation des matériaux dentaires avant insertion dans la cavité buccale. Une grande importance a été donnée au choix des matériaux à tester ; nous nous sommes basés sur les dernières tendances actuelles et les derniers développements de composition de matériaux dentaires. La même importance a été donnée à des méthodes et des techniques d'essai au laboratoire ; leur corrélation avec les résultats cliniques a été mise en évidence. Les modifications apportées à la méthodologie de ces tests ont exploré davantage les aspects cachés des différentes interactions de paramètres. La caractérisation et l'évaluation des matériaux dentaires nécessitaient une meilleure compréhension de l'interaction entre les différentes propriétés pour expliquer le vieillissement des matériaux. Notre travail a consisté à combiner de nombreuses études pour répondre à ce sujet. Les études ont porté sur les propriétés mécaniques et physiques, le composite fibré et Bulk, les matériaux CAM CAD, les adhésifs dentaires, le choc thermique et le cyclage thermique, le bisphénol A. L'objectif final était de développer un simulateur oral qui permettrait la reproduction de différents paramètres chimiques, physiques et mécaniques de l'environnement buccal, permettant ainsi de combler l'écart entre les tests in vitro et in vivo de matériaux dentaires / Scientific literature reveals that in vitro results are poorly correlated to materials clinical behavior. ISO standardized testing provides valuable information about the dental materials properties, and enables result comparison between different institutes. Conversely, new materials chemistry and formulations requires improved methodology and testing methods. Throughout our studies included in this work, the main objective was to reach a more global knowledge of the way dental materials are evaluated before being inserted into the oral cavity. A great deal of emphasis was given to the choice of materials to be tested, and that it would represent the current trends in dental practice and the latest developments in material composition. Equal highlight was given to the choice of testing methodology and laboratory testing techniques and their correlation to the clinical outcome. The modifications made to the methodology of these tests explored further the concealed aspects of different parameter interactions. Dental materials characterization and assessment required more understanding about the interaction between different properties to explain material aging; our work was to combine numerous studies to answer this topic. The studies included mechanical and physical properties, bulk and fiber composite, CAD CAM block materials, dental resin adhesive, thermal shock and thermal cycling, Bisphenol A. The final objective was to develop an oral simulator that would enable the reproduction of different chemical, physical and mechanical parameters of the oral environment, thus permitting to bridge the gap between in vitro and in vivo testing of dental materials Matériaux dentaires Résine composite Résine adhésive Essais normalisés Essai in vitro Simulateur de mastication Choc thermique CAD CAM Dental materials Resin composite Resin adhesive Standardized testing In vitro test Chewing simulator Thermal shock CAD CAM 617
5	Anwendung des Comet Assay (Einzelzell-Gelelektrophorese) an Zellen von Fischen zum Nachweis gentoxischer Wirkungen im aquatischen Biomonitoring Nehls, Sebastian 14 October 2013 (has links) Gewässer sind Lebensgrundlage, jedoch gleichzeitig Schadstoffsenken für eine Vielzahl von Kontaminanten. Biologische Wirkungstests und das Biomonitoring aquatischer Proben sind daher besonders wichtig, um Umwelt-Gefahrenpotenziale erkennen zu können. Der "Comet Assay" (Einzelzell-Gelelektrophorese) ist ein Indikator von DNA-Strangbrüchen und wurde hier als Test auf gentoxische Wirkungen erprobt und angewandt. Mit bekannten, gentoxischen Substanzen wurden Nachweisgrenzen und Dosis-Wirkungs-Beziehungen für die Zelllinien RTG-2 und RTL-W1 (aus der Regenbogenforelle, Oncorhynchus mykiss) in vitro ermittelt und methodische Parameter an die Zellen angepasst. Der Test reagierte sehr sensitiv auf 4-Nitrochinolin-1-oxid. Die Substanz war daher geeignet, um in weiteren Versuchen als Positivkontrolle zu dienen. Zur Bewertung der Messdaten wurde ein geeignetes statistisches Verfahren gefunden, das auch historische Kontrollen mit einbezog. Der zeitliche Verlauf der DNA-Schädigung des Testsystems mit RTG-2-Zellen wurde ermittelt, und durch Inhibition der DNA-Reparatur mit Aphidicolin wurden Zusammenhänge zwischen der Entstehung von DNA-Strangbrüchen, der DNA-Reparaturkapazität sowie der Metabolisierungskapazität untersucht. In einer zweiten Phase wurden unbehandelte Wasserproben aus Rhein, Elbe sowie weitere Oberflächenwasserproben mit dem Comet Assay an RTG-2-Zellen getestet. Bei 15 von 49 Proben zeigten sich gentoxische Effekte. In einer dritten Phase wurden Erythrozyten von freilebenden Döbeln, Leuciscus cephalus, aus der Mosel mit dem Comet Assay untersucht. Die Fische von drei Messstellen zeigten erhöhte Werte von DNA-Schädigungen, gegenüber einer vierten, stromabwärts gelegenen Messstation. Korrelationen mit den Ergebnissen zusätzlicher Biomarker ergaben sich nur teilweise. Chemische Analysen von Wasser- oder Gewebeproben ließen keine Rückschlüsse auf verursachende Kontaminanten zu - gerade dies unterstreicht jedoch die Wichtigkeit biologischer Tests bei komplexen Proben. / Bodies of Water are both vital resources and pollutant sinks for a multitude of contaminants. Therefore, biological effect tests and biomonitoring of aquatic samples are of particular importance to detect potential environmental hazards. The "comet assay" (single cell gel electrophoresis) is an indicator for DNA strand breaks and was explored and applied as a genotoxicity test in the present study. Known genotoxic substances were used to determine the detection limits and dose-response relationships for the cell lines RTG-2 and RTL-W1 (from rainbow trout, Oncorhynchus mykiss) in vitro, and to adapt methodological parameters to the cells. The test was very sensitive to 4-Nitroquinoline-1-oxide. This substance was therefore well-suited to serve as positive control in further experiments. In order to evaluate the measurement data, an appropriate statistical procedure was developed, which also took "historical" controls into account. The time course of DNA damage in the test system using RTG-2 cells was determined, and relationships between the origin of DNA strand breaks, DNA repair capacity and the metabolizing capacity of the cells was investigated by means of inhibition of DNA repair with Aphidicoline. In the second stage, native water samples from the rivers Rhine and Elbe and further surface waters were tested with the comet assay, using RTG-2 cells. 15 out of 49 samples showed genotoxic effects. In a third stage, erythrocytes of feral chub, Leuciscus cephalus, from the Moselle river were examined with the comet assay. The fish from three measuring stations showed elevated values of DNA damage compared to fish sampled from a downstream station. There were only partly correlations with the results from additional biomarkers. Chemical analyses of water and tissue samples did not permit conclusions on effect-causing substances.However, this emphasizes the importance of biological tests in dealing with complex environmental samples. Comet Assay Flüsse Biomonitoring DNA-Reparatur DNA-Schäden Fische Gentoxizität Gentoxizitätstest In vitro-Test Leuciscus cephalus Mutagenität Ökotoxikologie Oncorhynchus mykiss RTG-2-Fischzelllinie RTL-W1-Fischzelllinie Umweltverschmutzung Wasserverschmutzung DNA repair Biomonitoring Comet assay DNA damage Ecotoxicology Environmental pollution Fish Genotoxicity Genotoxicity test In vitro test Leuciscus cephalus Mutagenicity Oncorhynchus mykiss Rivers RTG-2 fish cell line RTL-W1 fish cell line Water pollution 570 Biowissenschaften, Biologie 32 Biologie WC 3440 ddc:570
6	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology
7	Development of Sensitive In Vitro Assays to Assess the Ocular Toxicity Potential of Chemicals and Ophthalmic Products McCanna, David January 2009 (has links) The utilization of in vitro tests with a tiered testing strategy for detection of mild ocular irritants can reduce the use of animals for testing, provide mechanistic data on toxic effects, and reduce the uncertainty associated with dose selection for clinical trials. The first section of this thesis describes how in vitro methods can be used to improve the prediction of the toxicity of chemicals and ophthalmic products. The proper utilization of in vitro methods can accurately predict toxic threshold levels and reduce animal use in product development. Sections two, three and four describe the development of new sensitive in vitro methods for predicting ocular toxicity. Maintaining the barrier function of the cornea is critical for the prevention of the penetration of infections microorganisms and irritating chemicals into the eye. Chapter 2 describes the development of a method for assessing the effects of chemicals on tight junctions using a human corneal epithelial and canine kidney epithelial cell line. In Chapter 3 a method that uses a primary organ culture for assessing single instillation and multiple instillation toxic effects is described. The ScanTox system was shown to be an ideal system to monitor the toxic effects over time as multiple readings can be taken of treated bovine lenses using the nondestructive method of assessing for the lens optical quality. Confirmations of toxic effects were made with the utilization of the viability dye alamarBlue. Chapter 4 describes the development of sensitive in vitro assays for detecting ocular toxicity by measuring the effects of chemicals on the mitochondrial integrity of bovine cornea, bovine lens epithelium and corneal epithelial cells, using fluorescent dyes. The goal of this research was to develop an in vitro test battery that can be used to accurately predict the ocular toxicity of new chemicals and ophthalmic formulations. By comparing the toxicity seen in vivo animals and humans with the toxicity response in these new in vitro methods, it was demonstrated that these in vitro methods can be utilized in a tiered testing strategy in the development of new chemicals and ophthalmic formulations. in vitro Alternative Methods alamarBlue rhodamine tight junctions bovine lens viability toxicity mitochondria mitochondria integrity benzalkonium chloride sodium dodecyl sulphate sodium lauryl sulfate scanning electron microscopy Draize Draize maximal average scores zonula occludens cosmetic directive PETA humane society animal league ICCVAM ECVAM JaCVAM BCOP bovine cornea ocular irritants ocular irritation ocular toxicity human corneal epithelial cells vitro confocal confocal microscopy metabolic activity optical quality reactive oxygen species contact lens contact lens care solutions barrier function microbial keratitis mulitipurpose solutions tight junction cell damage claudin ZO-1 occludin MDCK Madin Madin-Darby canine kidney cells cornea human cornea human corneal epithelium epithelial cell line human corneal epithelial cell line sodium fluorescein sodium fluorescein permeability fluorescein permeability ocular surface cell monolayer Araki-Sasaki cell physiology risk assessment safety assessment ScanTox scanning laser lens epithelium corneal cells in vitro model ophthalmic formulations multiple instillation back vertex animal testing rabbit testing alternatives to animal testing cultured bovine lens toxicity of chemicals irritation irritants laser scanner organ culture delayed toxicity toxins hydrogen peroxide cornea toxicity cornea toxicity models prediction of human toxicity no observable adverse effect level lowest observable adverse effect level NOAEL LOAEL toxicity thresholds safety factors cornea uncertainty factors preservatives disinfectants ophthalmic products preclinical preclinical testing epithelial barrier drug penetration clinical confocal microscopy animal rights rabbit cornea human cornea human clinical effects toxic animal rights activist sensitive measures toxic effect toxicity threshold agar overlay agar diffusion agar overlay method agar diffusion method cytochrome cytochrome c apoptosis necrotic apoptotic necrosis caspase rhodamine 123 resazuran resorufin cell death cell viability metabolic dye microsomal microsomal enzymes cytotoxicity cytotoxic cytotoxic effect MTT XTT WST-1 plasma membrane mitochondrial mitochondrial morphology ocular toxicity potential ocular toxicity human corneal epithelial cell line confocal analysis corneal epithelium cell fluorescence alamarBlue assay rhodamine dye animal welfare toxic injury degraded mitochondria epithelial monolayer disinfectants membrane integrity eye toxicity eye viability dye toxicity in humans human toxicity effects on the mitochondria mitochondrial toxicity in vitro battery in vitro test battery ophthalmic eye drop direct contact product safety cytotoxicity potential molecular molecular biology refine reduce replace sensitivity and relevance sensitivity relevance rabbit ocular irritation test product development cytotoxicity models cytotoxicity alternative methods replacements for animal testing three r's beagle tiered testing tiered testing strategy replacements animal testing mechanistic toxicity cornea mitochondria dose response threshold Vision Science and Biology

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