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Theme E Posters: Test Development, Validation, And ImplementationE25: Refined EPISKIN‚ Protocol for the Assessment of Acute Skin Irritation of Chemicals The human reconstituted epidermis, EPISKIN‚ took part in the prevalidation study on in vitro tests for acute skin irritation of chemicals, which was carried out during 1999 and 2000. This prevalidation study was coordinated and supported by the European Centre for the Validation of Alternative Methods (ECVAM). The protocol was based on cytotoxicity assessment (MTT test), after topical application of chemicals for 18 hours. The associated predictive model was as follows: if cell viability % < 50%, the chemical was classified as irritant. During Phase 1 and Phase 2 of the study, reproducibility and transferability of the method were verified. Unfortunately, during Phase 3, its performance in terms of predictive ability was insufficient, due to a low specificity. In order to improve the performance of the method, the protocol was refined, reducing the exposure time of epidermis to the chemicals, and the 20 chemicals of Phase 3 were evaluated once again. Sensitivity, Specificity, and Accuracy were 70, 80, and 75 percent, respectively, thus meeting the acceptance criteria, as defined by the MT. The EPISKIN method is now ready to enter a validation study of in vitro tests for acute skin irritation of chemicals. E26: Cell Tolerance Investigation of Four Commercial Multipurpose Contact Lens Solutions Purpose: To investigate cell tolerance of four commercial multi-purpose contact lens solutions. Methods: Cytotoxicity tests were done on an immortalized human conjonctival cell line using microplate cold light cytofluorimetry. IC 50 (neutral red test), DNA condensation (Hoechst 33342/Propidium iodide test), mitochondrial mass (orange nonyl-acridin test), and mitochondrial activity (Rhodamine123 test) were evaluated after 24 hours of treatment. In addition, CompleteTM, Opti FreeTM, RenuTM and Solo CareTM were tested at their commercial concentration and after dilutions. Results: The IC50 determination allows the classification of products into two different groups: Complete and Solo Care are weakly cytotoxic, because their IC50 borders are 80%, where Renu and Opti Free are more cytotoxic, having an IC 50 in the order of 46%. The chromatin condensation test and mitochondrial mass activity tests show similar results for the four solutions. Conclusion: In our model of conjonctival cells in vitro, commercial multi-purpose contact lens solutions induced cytotoxic damage, indicating characteristics of apoptosis. The preservatives included in the contact lens solutions are most likely responsible for the cytotoxic mechanism. In vitro, toxic effects of commercial multi-purpose contact lens solutions could, in part, explain some ocular surface disorders in patients treated long-term. E27: Integration of Alternative In Vitro Procedures for the Ecotoxicological Evaluation of Pentachlorophenol More than 100,000 chemicals are currently in use, and 2,000 new chemicals are introduced to the market every year. Accordingly, there is increasing concern about the effects that these compounds may exert in the environment and on human beings. Their study should be realized by means of representative, cost-effective and quantitative test batteries and alternative in vitro methods, as soon as there is a high public pressure to minimize the use of vertebrates in toxicity testing. The following alternative model systems and indicators have been selected to investigate the effects of the pesticide pentachlorophenol: immobilization of Daphnia magna; bioluminiscence inhibition in the bacterium Vibrio fischeri; growth inhibition of the alga Chlorella vulgaris; micronuclei rate, mitotic index, chromosome aberrations and root growth in Allium cepa, MTT reduction and cell proliferation in Vero monkey cells; neutral red uptake, MTS reduction and cell proliferation in SH-SY5Y human neuroblastoma cells; and finally, neutral red uptake, cell growth, MTT reduction, lactate dehydrogenase leakage, and activity in the fish cell line RTG-2, assay that replaces the classic fish test. The most sensitive systems to pentachlorophenol were micronuclei induction in Allium cepa, immobilization in Daphnia magna, bioluminiscence inhibition in Vibrio fisheri, cell proliferation in RTG-2, and neutral red uptake in SH-SY5Y. Inhibition of cell proliferation and MTT reduction on Vero monkey cells showed intermediate sensitivity. E28: An Alternative Strategy for the Assessment of Chemicals for Endocrine Disrupting Effects There is currently no consensus as to a satisfactory definition of an "endocrine disrupter" (ED), and no direct evidence for a causal link between exposure to such agents and the development of adverse health effects in humans. Also lacking is information regarding human exposure to suspected ED's. The myriad of possible mechanisms by which an ED could affect the human endocrine system, compounded by the absence of an adequate operational definition or evidence of a causal association and human exposure data, makes the development of a meaningful chemical-assessment strategy highly problematic. However, several countries, in association with the Organization for Economic Cooperation and Development (OECD), are pushing forward with a large-scale chemical-assessment strategy based almost exclusively on animal tests. This poster (1) discusses the animal welfare implications of current and proposed programs, (2) presents an alternative strategy for screening and testing chemicals for ED effects, and (3) recommends priority targets for research funding to accelerate the development and validation of promising non-animal tests and testing strategies. E29: A Reproducible Tissue Model for Ocular Irritancy Testing The utility of any toxicological test system or method critically depends on the reproducibility of the target tissue. Over the past six years, quantitative quality control (QC) test methods utilizing the MTT assay have established a high level of intra-lot and inter-lot reproducibility for MatTek's organotypic skin model, EpiOcularTM. QC testing on each lot of tissue consists of exposing the tissue to 0.3% Triton X-100 (TX). Using the MTT assay, an exposure time which reduced the tissue viability to 50% (ET-50) is determined. For OCL-200 produced in 1996 (47 lots) versus 2001 (77 lots), the ET-50 for TX averaged 24.9 ± 6.3 and 23.8 ± 5.7 minutes, respectively. Interestingly, the MTT assay can distinguish between standard EpiOcular (OCL-200) and hydrocortisone-free EpiOcular (OCL-200-HCF); during 2000-2001, the ET-50 for OCL-200-HCF was 15.3 ± 5.5 minutes (17 lots). Direct comparison between laboratories shows that the shipping effects on the tissue are negligible. During 2000 and 2001, 50 lots of OCL-200 were tested by the Institute of In Vitro Sciences (IIVS). ET-50 values at IIVS for TX averaged 26.7±5.3 minutes; these same lots tested at MatTek gave 23.4 ± 6.2 minutes, which corresponds well, despite small changes in testing methodology. Two prediction models have been developed that allow companies to interpret their in vitro EpiOcular results in terms of in vivo irritancy. Both methods have been shown to be useful and are used in industry. Thus, EpiOcular provides a reproducible, non-animal means to test materials and/or products for ocular irritancy. E30: Validation Study Design to Evaluate In Vitro Cytotoxicity Assays for Predicting Rodent and Human Acute Systemic Toxicity The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and NICEATM convened an international workshop in October 2000, to evaluate the validation status of in vitro methods for predicting acute systemic toxicity. Workshop participants recommended that two in vitro cytotoxicity methods should be further evaluated to determine their usefulness for predicting rodent and human acute toxicity. The NICEATM and ECVAM subsequently designed a multi-laboratory validation study to evaluate the relevance and reproducibility of two neutral red uptake assays, using one rodent cell line and one human cell type. Seventy-two coded chemicals representing twelve chemicals from each of six hazard classification categories will be tested in each of three laboratories. The study will proceed in three phases. Twelve chemicals will be tested in Phases I and II, followed by sixty chemicals in Phase III. The Registry of Cytotoxicity prediction model will be used to evaluate the prediction of rodent oral LD50 tests. Prediction of human toxicity will be evaluated using a prediction model based on human poisoning data. This study will further characterize the usefulness and limitations of these basal cytotoxicity tests for predicting acute systemic toxicity and for reducing and replacing animal use. Supported by NIEHS contract N01-ES-85424. E31: Selection of Reference Chemicals for the Validation of In Vitro Cytotoxicity Assays for Predicting In Vivo Acute Systemic Toxicity The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and NICEATM convened an international workshop in October 2000, to evaluate the validation status of in vitro methods for predicting acute systemic toxicity. Workshop participants recommended that in vitro basal cytotoxicity methods should be further evaluated. NICEATM and ECVAM subsequently designed a multi-laboratory validation study to evaluate the utility of two in vitro cytotoxicity tests for predicting acute oral toxicity in rodents and humans. A critical aspect of the study design was the selection of appropriate reference chemicals. Selection criteria included: (1) representation of chemicals across the full range of acute toxicity; (2) availability of high quality rodent acute toxicity test data; (3) availability of human toxicity data and/or exposure potential, and (4) representation of the types of regulated chemicals. A list of 117 candidates was compiled by mining several publicly available databases, including chemicals from the Multicentre Evaluation of In Vitro Cytotoxicity and the Registry of Cytotoxicity. Seventy-two chemicals were selected for testing that included twelve chemicals for each of six hazard classification categories. These reference chemicals and data will now be used to evaluate the performance of the proposed in vitro test methods. Supported by NIEHS contract N01-ES-85424. E32: Using the Bovine Corneal Opacity and Permeability (Bcop) Assay to Evaluate Ingredient Interactions Contributing to the Eye Irritation Potential of Products The efficacy of many household products depends on materials (active ingredients) that may be eye irritants. Some authors suggest that a formula's irritation potential can be "estimated" on the basis of the known irritancy of its ingredients. However, we have recently shown, with the BCOP assay, that specific formulations can affect the irritancy potential. Here, we report on two types of products; an insect repellent (10% active ingredient) in an ethanol base, and the other is an aqueous household cleaner containing sodium hypochlorite (1%). The BCOP assay was employed because of its dynamic range and multiple end points (opacity, permeability, and histology). Treatment and scoring followed the standard protocol (Sina et al, 1995). In the insect repellent, the concentration of ethanol varied from 0 to 85% but the irritancy potential (mild to severe) was largely independent of the ethanol concentration. Combinations of the cleaner components showed relatively limited irritation potential. However, the complete formulation was highly irritating. These data suggest that the concentrations of individual components may be poor predictors of toxicity and highlight the importance of testing complete formulations. To this end, the BCOP assay can be an important tool for optimizing formulations during product development. E33: Use of Daphnia magna as an Alternative Bio-object in Pharmacology and Toxicology In pharmacological and toxicological investigations the development of various methods involves an active search for alternative test objects. Daphnia magna from a class of paddle-handed crabs holds a most unique position. It is well known that Daphnia is the most sensitive test object to the large amount of toxic compounds and drugs among all known bio-objects, including experimental animals. A comparative pharmacological analysis of Daphnia magna and rat's cholinergic and gamma-aminobutyric acid (GABA)-ergic system with the use of agonists and antagonists of muscarinic cholinergic receptors (M-ChR) and GABA-ergic receptors was carried out. For the first time in the experiments on the Daphnia magna, it was shown that the M-ChR and GABA-receptors of daphnids are similar to such receptors on rats and mice. The principal similarity in action of muscarinic and GABA-ergic agonists and antagonists to Daphnia magna and mammals allow recommending the Daphnia magna as an alternative bio-object for screening of such types of drugs. On the basis of the data obtained, new methods of bio-identification of anticholinesterase compounds (organophosphates and carbamates) and some organochlorines (lindane, DDT, aldrine, etc.) in water with usage of Daphnia magna have been developed. E34: Development of a New Opacitometer for the Bovine Corneal Opacity and Permeability (Bcop) Assay The BCOP assay is now widely used as an alternative to the in vivo rabbit eye irritation test (Draize test). In our laboratory, the assay is used to evaluate the irritating potential of drug candidates and their formulations. The test relies on the measurement of induced opacity and enhanced permeability evaluated in isolated bovine corneas. Opacity is determined by an OP-KIT opacitometer (Electro-Design, Riom, France), which measures changes in voltage when the transmission of white light through the cornea is altered. When performing routine tests, it was observed that when chemicals induced spots along the sides of the excised cornea, OD readings were smaller compared to spots induced in the corneal middle. Consequently, these findings question the reliability of the data obtained when opacity is manifested by spots. Further, non-linear readings were collected when increasing neutral density filters were analyzed. For these reasons, we developed a new opacitometer, which uses an adjustable laser beam in combination with a calibrated photocell. Results showed an improved method of opacity assessment. A refined in vitro method for evaluating the ocular irritating potential of test compounds in development was obtained with this new opacitometer. E35: Characterization of Genetically Engineered PC12 Cell Lines for Detecting p53-Mediated Cell Death A novel genetically engineered PC12 cell line was developed at ECVAM, which has a controlled expression of the human wt p53 gene according to the media used. In order to induce differentiation of the genetically modified PC12 cells into neuron-like cells, nerve growth factor (NGF) was introduced. The present study was undertaken to characterize the genetically modified PC12 cells after NGF treatment and to evaluate their applicability for detecting in vitro p53-mediated cell death in acute and chronic toxicity testing. Expression of the p53 protein was verified after addition of increasing concentrations of NGF and the use of low-serum culture conditions. In a second step, the growth rates and cell cycle distribution of the NGF-treated p53 genetically engineered PC12 cells were studied. NGF treatment of the genetically engineered PC12 cells strongly induces the expression rate of the p53 protein. This induction of the p53 protein did not lead to cell cycle arrest.
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