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Theme A Posters: Replacement And Reduction AlternativesA25: Biomimetic Extraction: An Effective In Vitro Tool for Determining the Aquatic Toxicity of Complex Petroleum Products Many effluents and chemical products consist of complex mixtures of non-polar components that vary in physical-chemical properties but cause toxicity in aquatic organisms by a common mode of action (non-polar narcosis). According to critical body residue theory, adverse effects are expected when the total molar sum of non-polar chemicals in an organism's tissue exceeds a critical threshold. Solid phase micro-extraction (SPME) fibers provide a simple analytical tool for estimating the tissue residue concentration and thereby predicting the effects of mixtures of non-polar chemicals. We used a complex petroleum product (#2 fuel oil) to develop a parallel data set of toxicity to a number of aquatic species and the corresponding SPME fiber molar concentrations of hydrocarbon. From the observed effect--SPME fiber residue relationship, a critical fiber residue (CFR) for each test species was deduced. Subsequent experiments showed that these CFR's could be used in conjunction with SPME concentration data for other chemicals to predict aquatic toxicity. SPME biomimetic extraction can provide a sound basis for estimating aquatic toxicity of non-polar chemicals and consequently reduce product-specific laboratory animal toxicity testing. A26: Toxicity of Phenethylisothiocyanate and Dithiothreitol on Fa32 Cells
and their Effect on Glutahione S-Transferase The toxicity of dithiothreitol (DTT), a potent disulfide reducing agent, and phenethylisothiocyanate (PEITC), an anticarcinogen, was determined after 24 hours of treatment in rat hepatoma derived Fa32 cells using the neutral red uptake inhibition assay. The influence on the toxicity of buthionine-sulfoximine (BS), a specific inhibitor of y-glutamycysteine-synthetase and vitamin E was investigated. One way anova analysis indicated that there was no significant difference in the toxicity expressed by the NI50 (the concentration of compound reducing neutral red uptake by 50%) of DTT for the different treatments (NI50 = 4.0 mM, + 50 µM Vit E : NI50 = 138 µM). An increase in reactive oxygen species production of 61% was observed for PEITC after 1 hour of treatment at a maximum concentration of 8.4 mM. For both compounds, an increased glutathione S-transferase activity was found: 29% for PEITC and 120% for DTT after 3 days treatment with the NI50 concentration. The use of DTT in buffers for glutathione-related research should thus be considered with care. A27: Gingival Tissue Model for Prediction of Irritancy of Oral Care Products In vivo-like, highly differentiated human tissues of the oral cavity would be used to assess the biocompatibility of dental materials, dentifrices, and oral care consumer products and to determine drug delivery rates through the oral mucosa. Recently, we have harvested normal human gingival cells from primary tissue and induced them to form a three-dimensional tissue using a serum-free medium. Histologically, the resultant tissue is 8-12 cells layer thick with cells becoming increasingly squamous as the apical surface is approached. The tissue is cultured in easily manipulated cell culture inserts, and the tissue extends from wall-to-wall, so that oral care products can be directly applied. To assess the reproducibility of the tissue, tissues were exposed to the common surfactant, 1% Triton X-100 (TRI), and MTT viability dose response curves were constructed. An Effective Time-50 (ET50), i.e. the time of exposure after which viability is reduced to 50%, was determined. The ET50 for 3 lots of tissue is more susceptible to damage compared with the EpiDerm™ skin model (ET50 = 6.74 ± 0.99 hrs) but more robust than the EpiOcular tissue model (ET50 = 24.9 ± 6.3 minutes). Exposure to "sensitive gums" and normal strength dentifrices showed that the tissue viability was compromised to a greater degree by the normal strength toothpastes. Based on the initial results, further investigation of the tissue model's ability to predict potential gingival irritation from oral care products is warranted. A28: Estimation Method of ET50 Scores for Three-dimensional Human Skin Models Recently developed three-dimensional human skin models (3D-skin model) are promising as reasonable alternatives to in vivo assays for skin irritation. Although the irritancy is evaluated by the time ET50 yields 50% damage on these models, the estimation method has not been established. In a pre-validation study conducted in Japan, we were forced to measure OD values for the damage at only three time points. To get reasonable estimates of ET50 scores based on these measurements, we devised a method which, as the estimate, identifies the abscissa of the crossing point of the horizontal line for 50% and the regression line drawn on the plane with logarithm log(t) of the time period t as the x-axis and viability p as the y-axis. Most estimates by participating laboratories were based on simple graphical methods ignoring the convex property of the time-viability relationship and, therefore, the inter-laboratory variation was fairly significant. The variability was reduced to 2/3 by using the devised method. Comparing the recalculated ET50 scores with the scores in in vivo experiments, scores by two models turned out to be highly correlated with those in the Draize skin irritation test on the rabbit and in the human skin patch test, respectively. A29: Drug-Drug Interactions Between Antiarrhythmic Drugs in Chick Embryos In the present study, we examined the drug-drug interactions of antiarrhythmic drugs using chick embryonic electrocardiograms (ECG's). Anesthetic (urethane+a-chloralose) was injected into the air sac of fertilized eggs on the 16th day of incubation. After 20 minutes, procainamide, disopiramide, flecainide, or propranolol were each injected into the same site of eggs. Each drug was injected alone or in combination with propranolol. ECG's were recorded after injection of drugs and heart rate was calculated from R-R intervals. All drugs on alone treatment decreased the HR in a dose-and-time-dependent manner. When propranolol and other anti-arrhythmic drugs used were combined at the small dosage that did not change HR, there was a decrease in HR, including arrhythmics, i.e., A-V blocks. In conclusion, our ECG recording systems using chick embryos may be applicable for screening drug-drug interactions of anti-arrhythmic drugs. A30: Development of Rat Skin Transcutaneous Electrical Resistance Test for Skin Corrosion Testing of Different Substances Dermal corrosion is a production of irreversible tissue damage in skin. Several alternatives methods have been developed for skin irritation/corrosion assessment. The Rat Skin Transcutaneous Electrical/Resistance Test is an in vitro test based on the capacity of corrosive substances to destroy the skin's natural protective barrier, and corrosive action is measured by a fall in the transcutaneous electrical resistance (TER) of rat skin below a predetermined threshold. The study was developed based on the INVITTOX protocol No 115 in order to evaluate the reproducibility and reliability in our laboratory. Twenty-four substances were evaluated, and the results were compared with the existing in vivo data. The test was able to identify the substances with 86% accuracy, 70% sensibility, and 100% specificity. The associations between in vivo/in vitro were significant, taking into account the statistical analysis by the exact test of Fisher. These results affirmed a clear relationship among electric resistance and the potential of corrosion of a substance on the skin. As a conclusion, the test showed an acceptable reproducibility in our conditions, and it is a useful and reliable in vitro method for discriminating corrosive from non-corrosive substances. A31: CAM-TBS Assay to Predict the Eye Irritation of Arginine-based Dimeric Surfactants The CAM-TBS assay (INVITTOX, No. 108) was used as an alternative to the Draize test to assess the potential eye irritation of new arginine-based dimeric surfactants related to their structural characteristics. The amount of trypan blue absorbed onto the chorioallantoic membrane is an indicator of the injury to the membrane. We have introduced a modification on the method by expressing the results as a function of the membrane weight. The compounds can be classified as their potential eye irritation depending on the amount of trypan blue stain absorbed (Vinardell, Garcia, Toxicol. In Vitro, 14, 551-556, 2000). This method has advantages over some other alternatives tests, particularly cytotoxicity tests, because the technique is applicable to all types of chemicals, irrespective of their appearance and solubility, in addition to being a more objective method than the het-cam. Results obtained in the present work indicated that these compounds constituted an interesting novel surfactant class with lower potential eye irritation activity compared to commercial surfactants. A32: Growth of Human Epithelial and Stromal Fibroblast Cells in Serum-Free Media Human cell cultures are generated from un-transplantable corneo-scleral discs, the layers of which are separated by selective sectioning with a novel mini-microtome. Pure epithelial and fibroblast cells are grown in EMEM-20% FCS. As these cells will be used in corneal constructs for toxicity studies, the purpose of this study is to find effective serum-free (SF) media. Each of six SF media was tested 10 times on primary cultures. The criteria of culture success were initiation speed and growth to confluency, as compared with the control. Four-day quantitative growth experiments were performed on P=1 cultures and estimation of cell growth was by measurement of total protein content. The fibroblasts thrived in all the SF media, whether as primary or passaged cells. All six SF media sustained the initiation of epithelial culture, but with much variation in confluency time. The P=1 in four of the SF media consistently showed a faster rate of growth, and of these, two displayed double that of EMEM+FCS. Human epithelial and fibroblast cells can be successfully grown in some defined SF media. A33: In Vitro Cytotoxicity of Benzalkonium Chloride on Sv40 Transformed Human Corneal Epithelial Cells (Hce-T) Purpose: To determine the cytotoxicity potential of benzalkonium chloride (BAK) with several colorimetric in vitro assays using an immortalized human corneal epithelial cell line. Methods: Solutions of BAK/media were tested at 20, 10, 5, 2.5 and 1.25 ppm, by serial dilution on 96 well plates. The tests used to determine the cytotoxicity of BAK were the cell viability assay using MTS/PES, cell membrane integrity assay using neutral red uptake/release, and the proliferation assay using BrdU incorporation and cell. Results: Based on these studies, the following solutions of BAK were significantly different in comparison to the control at 24 hours exposure: 20, 10, and 5 ppm in all three test methods. The BAK solution at 2.5 ppm was not significantly different with a mean MTS/PES value of 80% and NR release values of 98% while BrdU incorporation was considered significant with a value of 68. Conclusions: The use of these three in vitro assays has allowed the evaluation of BAK cytotoxicity in HCE-T cells through the analysis that only living cells are able to metabolize MTS/PES, incorporate BrdU, or uptake NR. Cytotoxicity due to BAK increased in a linear fashion from 4 hours to 24 hours exposure. The IC 5024 value for BAK was found to be 6.25 ppm. A34: In Vitro Pig Heart Perfusion--A Powerful Tool in Research and Development In order to study cardiovascular diseases and drug impact on heart physiology in vitro, complex experimental models are necessary to mimic the in vitro situation as far as possible. The isolated hemoperfusion of porcine hearts allows a broad spectrum of biochemical, physiological, morphological, and pharmacological parameters to be addressed. The data presented describe a standardized in vitro model to study arrhythmia, proarrhythmia, or ischemia, respectively. A comprehensive database provides detailed information on the effects of common reference substances on heart function. In comparison to the common Langendorff-perfusions of small animal hearts, the model presented here enables researchers to (1) reduce animal testing by using hearts of regular slaughter pigs; (2) extend perfusion time up to 6 hours; (3) view heart anatomy and physiology that is comparable to the human heart; and (4) simulate a compound's plasma protein binding by the use of blood-based perfusate. In conclusion, the isolated hemoperfused porcine heart is a highly reproducible standardized preparation for research and development in the field of cardiac arrhythmia, proarrhythmia, and ischemia. The high predictive value for the feasibility in humans is an adequate alternative for the assessment of drug potentials and safety. A35: In Vitro Approach to Determine the Bioavailability of Topically Applied Cationic Hair Dyes In Europe, the principles of cosmetic ingredients safety are regulated within the European Council Directive [76/768/EEC]. Additional technical guidelines exist to define criteria necessary for specific aspects relevant for the safety assessment of individual ingredients. In this respect, the bioavailability of topically applied chemicals is of crucial interest. Convinced of the Three R's approach for animal testing, hair-dyeing compounds are routinely tested in our laboratories with appropriate in vitro methods. The current presentation summarizes the principles of the used in vitro technique to analyze percutaneous penetration/dermal absorption of cosmetic ingredients topically applied to excised pig skin. Examples are given for the routine application, demonstrating the bioavailability of cationic azo hair dyes out of an aqueous-alcoholic solution and out of a realistic standard hair dyeing formulation. This in vitro technique is recommended by the European SCCNFP (The Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers) for the safety evaluation of particular ingredients. In 2001, this in vitro method became officially accepted by the OECD and will be published as OECD guideline no. 478.
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