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Theme A Posters: Replacement And Reduction AlternativesA1: Involvement of Heat Shock Proteins and P-Glycoprotein in Cdcl2-induced Toxicity and Intestinal Barrier Function Caco-2 cells, human colorectal adenocarcinoma cells, represent one of the most widely used models for studying human cellular barrier functions. CdCl2 is a potent nephrotoxin that also causes damage to the intestinal epithelium. We aimed to establish the relationship between the effects induced by CdCl2 on Caco-2 cells at the barrier level and at the cellular and molecular levels. After 24 hours exposure to 0-50 mM CdCl2, trans-epithelial resistance (TER) and paracellular permeability (PCP) were measured. Damage at the molecular level was evaluated by measuring heat shock protein (HSP's) and P-glycoprotein (P-gp) expression, and reactive oxygen species (ROS) production. A concentration-dependent decrease of TER and a concentration-dependent increase of PCP were observed. In addition, a concentration-dependent increase in the expression of HSP70, starting at 15 mM, and a slight increase in HSP27 were found. The expression of P-gp increased only at 5 and 10 mM CdCl2. Production of ROS was not detected. Our results indicate that TER and HSP70 appear to be potential early markers of CdCl2 toxicity, while it seems that ROS are not involved in CdCl2-induced intestinal barrier dysfunction. A2: Expanding the Application of the Embryonic Stem Cell Test (Est) by using Tissue-Specific Molecular Markers Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse (cell line D3) are able to differentiate in culture into a variety of cell types of ecto-, meso-, and endodermal lineage. The embryotoxic potential of drugs can be assessed by the embryonic stem cell test (EST). This in vitro assay is based in ES cell differentiation into contracting myocardial cells to determine inhibitory effects of drugs. In a joint project with three drug companies in Germany, we are attempting to expand the EST by establishing molecular endpoints of differentiation in cultured ES cells. Therefore, we have tested several culture conditions that allow ES cells to develop into cell types of varying tissues. Tissue-specific proteins of cardiomyocytes, neuronal cells, chondrocytes, and osteoblasts were analyzed by immunofluorescent antibody techniques, e.g. microscopy and FACS-analysis. The inhibition of differentiation of ES cells into specific tissues may provide an objective endpoint for predicting the embryotoxic potential of chemicals. A3: Comparative Assessment of the Acute Skin Irritation Potential of Detergent Formulations using a Novel Human Four-hour Patch Test Method P. Casterton, F.H. Kruszewski, M. Robinson, N.K. Snyder, M.E. Blazka, F. Heitfeld, J.E. Swanson, D. Mallon, and R. Gingell. The Soap and Detergent Association, Washington, DC 20005, USA. fkruszewski@sdahg.org. Predictive skin irritation test methods that do not require use of animals are needed for the pre-market assessment of soap and detergent formulations. The utility of a novel and ethical human acute skin irritation patch test method, recently developed for the assessment of chemical skin irritation potential, was evaluated. The method uses a graduated exposure occluded patch test format (up to 4 hours in total duration of exposure) and compares the total incidence of positive skin reactions (generally limited to mild erythema) for test materials versus a concurrently tested positive control (20% aqueous sodium dodecyl sulfate). All studies were conducted in accordance with the Helsinki Declaration of 1975 and were independently reviewed by an institutional review board. Using this approach, 24 soap and detergent formulations of various types were tested in seven individual studies. Based on time-response criteria, the skin irritation profiles were generally consistent within product types, which were categorized as follows: mold/mildew removers>disinfectants/ sanitizers>aluminum wash=fabric softener concentrate>20% SDS>liquid fabric softeners>hard surface cleaners>powder laundry detergents=powder automatic dish detergents. In addition to formulation effects, some seasonal effects were noted. These results confirm the utility of this patch test method for the comparative skin irritation assessment of these product types. A4: Co-culture of Sensory Neural Cells with Human Corneal Epithelium Whilst the corneal epithelium is well innervated in vivo, many of the corneal in vitro models do not contain neural components. The in vitro media requirements for corneal epithelia are stringent, in particular, the calcium ion concentration required to achieve a multi-layered model. Corneal epithelial cells have been shown to release nerve growth factor and respond to substance P, whilst the sensory neural cells, ND7/23, respond to nerve growth factor, as well as producing substance P and calcitonin gene-related peptide. To identify the ND7/23 cells in co-culture, the transient uptake of fluorescein cadaverine was employed, as it can be monitored repeatedly. The ND7/23 cells will differentiate in the corneal medium. They reduce their growth rate, develop neurite-like projections, but maintain viability as determined by resazurin reduction. Corneocytes in co-culture grow in between the neural cells and can be maintained for more than 16 days, which is well within the 8 days required to produce the fully differentiated HCE-T corneal model. This work was sponsored by a grant from the industrial sponsors of the FRAME research program. A5: EpiskinTM as a Product Development Tool: Comparison of In Vitro (Reconstructed Epidermis) and In Vitro (Human) Skin Responses to Irritation Potential of Cationic Surfactants There is a need to investigate the irritation potential of new products or ingredients prior to human testing and launch. Keratinocyte monolayer cultures have been used as model test systems for investigating in vitro irritation potential, but reconstructed skin models with their in vivo-like structure (presence of a barrier function and stratum corneum) appear to be better suited to the task. Soluble cationic surfactants, widely used in hair conditioning products, come into contact with the stratum corneum and may penetrate into cell membranes, causing irritation. The aim of the present study was to directly compare in vivo and in vitro dose-response characteristics of two cationic surfactants formulated into identical hair conditioning products. The surfactants differed only by their carbon chain length: C-16 and C-22. In the in vivo studies, 22 healthy volunteers were patch tested using three applications of 24-hour occlusive Webril patches. The test sites were graded visually for erythema according to a 0-4 scale. In vitro tests were carried out using the reconstructed human epidermis model EpiskinTM. Cellular viability was measured by the MTT assay after an 18-hour topical application. The same product batches were used in both test systems. The following results were obtained: in vitro, a clear difference was observed in the irritation potential of the two surfactants despite their similar chemical structures. The C-16 was significantly more irritating with an unequivocal dose-dependent response. No such dose-response was obtained with the C-22 surfactant, which was also less irritating. In vitro, a clear dose-response effect of the C-16 was measured on epidermis viability with a significant vitro-vivo correlation of r = 0.98. Interestingly, as in vitro, at similar concentrations, C-22 was found to be well-tolerated in vitro. In conclusion, the in vitro reconstituted epidermis model is a sensitive tool for detection of the irritation potential of cationic surfactants in cosmetic formulations. As such, it is a valuable support in the product development and risk assessment processes before clinical tests. A6: Characterization of Genetically Engineered Hcyp2d6*1 Cell Lines (and Mock Transfected Control) in Serum-Free Medium Evaluation of metabolism-mediated effects is a vital part of the preclinical toxicity assessment. One of the recent and most promising innovations in the in vitro metabolism field is represented by genetically engineered cell lines expressing selected cytochrome P450 (CYP) enzymes. A major cause of variation of the metabolism of drugs by CYP enzymes is polymorphism of CYP 2D6, which is known to affect the metabolism of more than 40 drugs. Therefore, easy screening methods are needed to identify any CYP 2D6-mediated toxicity. Based on the increasing demand and application of these novel in vitro systems, and the need for official recognition of the system in a regulatory environment, the project is focused on the development of a serum-free validated in vitro test. Detection of metabolism-related effects will be done by using Chinese hamster lung cell lines (V79) expressing CYP 2D6 enzymes, in order to improve metabolism-related hazard assessment procedures. V79 CYP2D6*1 cells appear well adapted to serum-free medium; however, study on the metabolic activity of the adopted cell lines and on specificity, selectivity, linearity, precision, repeatability, and reproducibility of the test are ongoing (prevalidation phase). A7: In Vitro Corneal Recovery Assay Using a Reconstituted Human Corneal Epithelial Model Development of in vitro tests on reconstituted corneal tissues allows improving our knowledge on ocular sensitivity to chemicals, since they provide more accurate and objective information using an experimental design that is faster, cheaper, and more convenient than the Draize test. A human corneal epithelial model (HCE), closely mimicking the in vivo corneal epithelial structure [1], was used to measure in vitro corneal tissue toxicity and recovery. HCE tissues were exposed to 19 ECETOC reference chemicals. After washing, the cultures were cultivated 48 or 96 hours. Histology and tissue viability was performed before and after incubation. Non-irritants like Tween-20 exhibited results similar to that of untreated controls. Chemicals such as n-butyl acetate and ethyl acetate induced necrosis of the upper cell layers, but tissue regeneration was observed after incubation. On the opposite, no recovery was observed with Triton X-100 (10%) or benzalkonium chloride treatment. This test approach allows differentiating in vitro the irritation potential of chemicals, as well as their tissue recovery capacity. [1]: Nguyen D., Journal of Applied Toxicology, 2002. A8: Tissue-Specific Toxicity of Forty Chemopreventive Agents in Epithelial Cell Cultures from Normal Human Tissues The epithelial cell lines or primary epithelial cell cultures from up to ten normal human tissues were used to determine the comparative toxicity for forty potential chemopreventive agents in the Human Epithelial Cell Cytotoxicity (HECC) Assay. The endpoints assessed were three- and five-day growth inhibition, mitochondrial function inhibition, and inhibition of proliferating cell nuclear antigen (PCNA) or albumin expression. Examples of agents that produced little or no toxicity in any of the cell types include: Aspirin, anethole trithione, DHEA analogue 8543, curcumin, calcium glucarate, folic acid, limonene, lycopene, and vitamin E acetate. Examples of agents that produced log differences (or greater) in sensitivity in one or more cell types were: N-acetyl-L-cysteine, budesonide, DHEA, genistein, glycyrrhetinic acid, and Polyphenon E. Unique tissue-specific toxicity was observed for each toxic agent, suggesting that tissue-specific effects are the rule rather than the exception. For agents for which clinical plasma levels are known, the in vitro concentration response data will be used to determine relevance to clinically adverse events. The HECC Assay is useful for identifying tissue-specific toxicity for chemopreventive agents and should also be useful for other types of agents. A9: Correlation of Difluoromethylornithine Efficacy and Biomarker Response in the Human Epidermal Cell Assay with Clinical Trial Biomarker Modulation The Human Epidermal Cell (HEC) Assay was used to test for chemoprevention efficacy of difluoromethylornithine (DFMO) with inhibition of propane sultone induced growth and induction of involucrin expression as endpoints. In addition, we have determined the effects of DFMO on polyamine content in the HEC Assay. Clinical trials for colon cancer use suppression of polyamine content as a biomarker for the chemopreventive efficacy of DFMO. The data show 1) the effective concentrations in vitro overlap the plasma concentrations in the clinical trial and 2) the percent change in spermidine to spermine ratio and the depletion of putricine show excellent correlation with in vitro chemopreventive efficacy. These data provide further validation of the utility of the HEC assay as a screen for chemopreventive efficacy. Supported by the Division of Cancer Prevention, National Cancer Institute under Contract No. N01-CN-65115 (EE and JLR) and Grant NO. CA-72008 (E.W.G.). A10: Evaluation of a Human Red Blood Cell Lysis Test for Predicting the Eye Irritancy of Industrial Enzyme Preparations The irritant potential of new industrial enzyme preparations is currently established in vivo, using the rabbit as an experimental animal. In vitro models offer the possibility to perform toxicity tests on human tissues, thereby contributing to a more relevant safety evaluation. Enzyme preparations of various classes and even proteases are, in general, low irritancy products, giving predominantly conjunctival reactions. These effects, plus the haemorrhagic discharge that can be seen in vivo after exposure to certain protease preparations, are most likely related to membrane disruption. The red blood cell lysis test was an obvious model to use to predict the eye irritancy of this class of materials. The results of a human red blood cell model and its ability to predict in vivo eye irritancy of industrial enzyme preparations (proteases, amylases and lipases) is presented. In comparison with existing in vivo data, the human red blood cell test had a high ability to predict the irritancy of this class of materials. The conclusion is that the red blood cell lysis test using human erythrocytes is a predictive test that is a powerful addition to an in vitro testing battery for the assessment of ocular irritancy of new industrial enzyme preparations. A11: Hypochlorite-Anion as a Protector in Methylmethacrilate Cytotoxicity Arthroplasty, in certain cases, is possible only with the application of methylmathacrylate bone cement (MMA). Nevertheless, the risk of complications in the form of local and generalized toxic effects is statistically higher than in surgery without MMA-fixed prothesis. It was shown that in that the main aggressive factor is unbound MMA. We have demonstrated earlier hypochlorite-anion can de applied successfully for prevention of lipid globules formation in the bloodstream. An assumption that hypochlorite can also be used for oxidative degradation of unbound MMA was tested on cultured fibroblasts with the endpoints of oxidative stress detected by TBA-reactive products and cellular hypoxia estimated with MTT. It was shown that monomeric MMA and sodium hypochlorite, when they were previously mixed in equimolar ratio, significantly lost their toxicity and did not provoke oxidative stress and cell hypoxia. When the ratio of components was altered and the hypochlorite concentration was increased several times, cytotoxicity of the mixture exceeded the toxicity of each of the components applied separately. A12: A Preliminary Study for the Determination of Photohemolitic Potential of Five National Production Perfumes, using the 81's INVITTOX Protocol Phototoxicity is an acute reaction that appears in the skin with a single chemical treatment (topic or systemic) and subsequent exhibition to visible or Ultraviolet radiation. The objective of toxicological studies is determining the possible toxic effect of substances and, in this case, the phototoxic effect. Until recently, the studies that were carried out to determine toxicity and phototoxicity used different animal species. Our toxicologists didn't have a guide that provided a reliable and validated method for this purpose. Some alternative toxicological techniques have been developed; among those we can mention is the Neutral Red absorption for the 3T3 cells from the mouse's fibroblast (3T3 NRU phototoxicity), which is approved by the European Union for toxicologists' use. However, other alternative methods exist for the screening of the phototoxic potential of the chemicals or finished products by their ability to disturb the erythrocyte membrane and/or to oxidize haemoglobin under the effects of the ultraviolet and visible light. Our studies were carried out with rat erythrocytes. We used the 81's INVITTOX protocol with this modification to determine the possible photohemolitic effect of five National Production perfumes. In the assay, we also included the Chloropromacin and the Sodium Dodecil Sulphate, respectively, as positive and negative controls. None of the perfumes used with the Sodium Dodecil Sulfate showed photohemolic effects, since the P Factor (Phototohemolic Factor, relation among the half hemolytic concentration not irradiated with regard to the one irradiated) was smaller than 3, while for the Chloropromacin, it was observed that this exceeded the mentioned value. These results suggest that the five National perfumes are not potentially phototoxic for humans; the results also validate the utility of the mentioned protocol for this purpose.
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