Global ETD Search

1	Development of a new screening assay to identify proteratogenic compounds using Zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT) Busquet, François 30 December 2008 (has links) (PDF) The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays e.g., in rats or rabbits. Following the 3R principles, the development of alternative methods is encouraged to reduce the number of animal tests. From this perspective, we have developed an in vitro assay (mDarT) using the zebrafish Danio rerio embryo teratogenicity assay (DarT) combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide, ethanol, benzo[a]pyrene and thalidomide were used as test materials to assess the efficiency of this assay. Briefly, the zebrafish embryos were co-cultured at 2 hpf (hours post fertilization) with the test material at varying concentrations, mammalian liver microsomes from different species and NADPH for 60 min at 32°C under moderate agitation in Tris buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterwards fish embryos were transferred individually into 24-well plates filled with fish medium for 48 hours at 26°C with a 12 hour-light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. The test was considered to be valid if a minimum of 90% of fish eggs developed normally for the two controls (test material alone and MAS alone). For each test material, the experiment was repeated three times with the controls satisfying the validation criteria (≤ 10% impaired embryos). Indeed, no significant teratogenic effects were observed compared to controls in fish embryos exposed to the proteratogens alone (i.e., without metabolic activation) or the MAS alone. In contrast, the four test materials induced significant abnormalities in fish embryos when co-incubated with animal liver microsomes. For cyclophosphamide, ethanol and thalidomide a concentration-response relationship was shown and the qualitative nature of the malformations was similar between fish embryos and humans. Benzo[a]pyrene was demonstrated to be significantly teratogenic in fish embryos in spite of no concentration-response and unspecific teratogenic fingerprints. We conclude that the application of animal liver microsomes will improve and refine the DarT as a predictive and valuable alternative method to screen teratogenic substances. zebrafish cyclophosphamide ethanol alternatives to animal testing teratogenicity metabolic activation zebrafish cyclophosphamide ethanol Alternative zum Tierversuch Teratogenität Aktivierung System ddc:520 rvk:WI 4400
2	Development of a new screening assay to identify proteratogenic compounds using Zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT) Busquet, François 18 September 2008 (has links) The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays e.g., in rats or rabbits. Following the 3R principles, the development of alternative methods is encouraged to reduce the number of animal tests. From this perspective, we have developed an in vitro assay (mDarT) using the zebrafish Danio rerio embryo teratogenicity assay (DarT) combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide, ethanol, benzo[a]pyrene and thalidomide were used as test materials to assess the efficiency of this assay. Briefly, the zebrafish embryos were co-cultured at 2 hpf (hours post fertilization) with the test material at varying concentrations, mammalian liver microsomes from different species and NADPH for 60 min at 32°C under moderate agitation in Tris buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterwards fish embryos were transferred individually into 24-well plates filled with fish medium for 48 hours at 26°C with a 12 hour-light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. The test was considered to be valid if a minimum of 90% of fish eggs developed normally for the two controls (test material alone and MAS alone). For each test material, the experiment was repeated three times with the controls satisfying the validation criteria (≤ 10% impaired embryos). Indeed, no significant teratogenic effects were observed compared to controls in fish embryos exposed to the proteratogens alone (i.e., without metabolic activation) or the MAS alone. In contrast, the four test materials induced significant abnormalities in fish embryos when co-incubated with animal liver microsomes. For cyclophosphamide, ethanol and thalidomide a concentration-response relationship was shown and the qualitative nature of the malformations was similar between fish embryos and humans. Benzo[a]pyrene was demonstrated to be significantly teratogenic in fish embryos in spite of no concentration-response and unspecific teratogenic fingerprints. We conclude that the application of animal liver microsomes will improve and refine the DarT as a predictive and valuable alternative method to screen teratogenic substances. info:eu-repo/classification/ddc/520 ddc:520
3	Development of a computational consensus model for the in silico prediction of the skin sensitising potential of organic chemicals in the context of REACH Hillebrand, Marcus 27 September 2018 (has links) Die Hautsensibilisierung (Typ 4 Hautallergie) nimmt in der Toxizitätsbewertung einen wichtigen Stellenwert ein, was u.a. daran zu sehen ist, dass sie unter dem europäischen Chemikaliengesetz REACH schon sehr früh, d.h. ab einer Jahrestonne, abgeprüft werden soll. Die Dissertation untersucht, ob die derzeit im Tierversuch stattfindende Toxizitätsprüfung durch computerchemische Methoden ersetzt werden kann. Dazu wurde eine Datenbank aus über 2000 Stoffen erstellt, aus der wichtige Unterschiede zwischen den eingesetzten Tiermodellen herausgelesen werden konnten. In den Untersuchungen trat auch zu Tage, dass – entgegen vorheriger Annahmen – die Bioverfügbarkeit, d.h. die Aufnahme von Substanzen über die Haut, im Tierversuch nur eine untergeordnete Rolle spielt. Zudem ist eine Abschätzung des hautsensibilisierenden Effekts von Stoffen anhand eines Read-across (Interpolation aus strukturähnlichen Verbindungen) und von Strukturalarmen (Substrukturelemente als Indikatoren für einen bestimmten Effekt) möglich. Wenn beide Ansätze im Rahmen einer Konsensmodellierung miteinander verschränkt werden, ergibt sich sogar eine gute Vorhersagestatistik. / Skin sensitisation (type 4 skin allergy) is an important parameter in the toxicity assessment of chemicals, which is underlined by the fact that it is evaluated even at the lowest tonnage (1 t/a), which can be registered under the european chemicals regulation (REACH). In this thesis it was investigated if the currently used animal models can be replace or refined with computational (in silico toxicological) methods. In this regard a data base consisting of about 2000 substances was build. With its data important differences between the currently applied animal tests could be derived. Furthermore, the investigation found that – in contrast to previous assumptions – the bioavailability of a chemical compound, i.e. the uptake via the skin, has only a minor impact on the test result of the evaluated animal models. Moreover, it was demonstated that the skin sensitising potential of chemicals can be predicted by read-across (interpolation with structurally similar substances) and with structural alerts (substructural elements which indicate a particular effect). Combining both prediction methods with consensus modelling lead to a good prediction regarding the question whether a particular chemical compound is a sensitiser or not. info:eu-repo/classification/ddc/540 ddc:540 Haut Allergodermatose Toxizität REACH-Verordnung Chemische Verbindung Bioverfügbarkeit
4	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
5	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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