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Theme E Posters: Test Development, Validation, And ImplementationE13: In Vitro Methods for Assessing the Safety and Toxicity of Australian Essential Oils Essential oils have been applied extensively in medicine, pharmacy, aromatherapy, perfumery, and flavoring. During the last decade, there has been an increase in the incidence of poisoning following oral ingestion or topical application of such oils. The aim of this research was to develop a range of procedures to be used in generating safety data of Australian essential oils, such as tea tree (Melaleuca alternifolia) and lemon myrtle (Backhousia citriodora). Procedures included: composition identification by gas chromatography-mass spectrometry, antimicrobial activity screening, in vitro cytotoxicity using human cell lines, and in vitro percutaneous absorption using freshly excised human abdominal skin. These procedures enabled the measurement of toxic effects from the cellular to the tissue level. A risk evaluation process was performed, which involved calculating human cell toxicity (measured as IC50--50% inhibitory concentration) and then extrapolation to give the NOAEL (no observable adverse effects level). NOAEL's for tea tree and lemon myrtle oils were 1 mg/kg and 0.5 mg/kg, respectively. The percutaneous absorption of essential oil components through skin was measured and compared to the NOAEL by assuming a one-compartment model of distribution within the human body. This information could potentially be used to set standards for the essential oil industry, and, most importantly, to offer product safety data to consumers, industry, and regulators without the need for animal experimentation. E14: In Vitro Oral Irritation Assessment of Dental Formulations Using the SkinethicTM Human Oral Epithelium Model This study investigated the SkinEthic human oral epithelium (HOE) model for assessing the oral irritation of dental formulations. SkinEthic HOE is a multilayered, non-keratinized tissue, grown using the TR146 oral keratinocyte cell line on filter inserts at the air/water interface. Test materials were toothpastes of known irritation potential: A (1.2% sodium lauryl sulphate (SLS)); B (SLS-free); C (5% SLS); D (1.8% cocoamidopropyl betaine, no SLS); E (1.8% SLS/3.11% pyrophosphate); F (1.8% SLS); G (similar to E). The time-course of cytotoxicity (10 minutes-24 hours) of 30 ml 30% diluted formulation (in ultra-pure water, topical application) was assessed by MTT reduction. The cytotoxicity order was compared with irritation potential; Cytotoxicity: C > G > E = F > A > B = D; Irritation: C > G > E > F > A > B > D. The cytotoxicity order was similar to that of known irritation potential (consumer trial/complaint data), except that E could not be distinguished from the less irritant F. These results suggest that this assay detected some differences in irritation potential between formulations, but not all. Cytotoxicity may relate to SLS content (known to be related to frequency); materials without SLS were non-toxic and the cytotoxicity order followed that of SLS content. This assay may be of use for comparison of novel formations with benchmark materials of known oral irritation. E15: Effects of Nicotine in Genetically Engineered PC12 Cells Expressing p53 and bcl-2 Several studies have suggested that nicotine has beneficial effects, both as a therapeutic and prophylactic agent, in some neurodegenerative brain diseases (i.e. Alzheimer's Disease, Parkinson's Disease). One of the pathophysiological features of neurodegenerative diseases is the apoptosis of neurons in specific brain regions. Two novel genetically engineered PC12 cell lines with different sensitivity towards apoptosis have been developed at ECVAM (Stingele et al. 1999). In these cell lines, the expression of human bcl-2 or human wt-p53 proteins is controlled by a tetracycline-regulated gene expression system. The aim of this study was to determine whether the effects of nicotine are at least in part due to inhibition of apoptosis in neuronal cells by this substance. The possible implications of bcl-2 and p53 protein regulation in the observed nicotine effects were studied. For this purpose, bcl-2 and wt-p53 genetically engineered PC12 cells were differentiated with nerve growth factor (7S NGF), pretreated with nicotine for 24 hours, and challenged to apoptosis using known toxic substances (camptothecin, MPP+, 6-hydroxydopamine). E16: EpivaginalTM, a Human Tissue Model for Vaginal Irritation Studies Recently, a tissue culture-based model of the vaginal epithelium has developed. Normal, human ectocervico-vaginal (ECV) epithelial cells were induced to form a three-dimensional tissue, using specially formulated serum-free medium. The in vitro tissue reproduces many of the histological, ultra-structural, and protein expression properties of native tissue, including inter-digitation of cells, glycogen production, and cytokeratin expression. Initial experiments investigated the use of this tissue model and the MTT tissue viability assay for predicting ECV irritation. Vaginal anti-fungal products, contraceptives, and lubricants were exposed to the tissue model, and the exposure time, which causes a 50% reduction in tissue viability (ET-50), was determined. The ET-50s were compared to rabbit vaginal irritation scores for seven products that are currently marketed or in clinical trials. Using an ET-50 cutoff of 9.0 hours, the products could be successfully categorized into minimal or mild irritation classes. Based on these results and the problems associated with obtaining and the handling of human vaginal tissue, it is anticipated that the tissue model will be very useful in assisting product development scientists in developing safe, efficacious products. E17: Effect of Chemicals on Squamous Differentiation in the Beas-2b Cell Line Under pathological conditions, the bronchial epithelium becomes squamous. This is, in part, controlled by transglutaminase (TG) catalyzed formation of cross-linked envelopes. Manipulation of differentiation in the BEAS-2B bronchial cell line, in vitro, was investigated using TG modifying agents, nicotine, and cigarette smoke condensates(CSC)as inducers and cystamine and retinoic acid as inhibitors. Squamous differentiation was assessed at 6, 12, and 18 days, using TG catalysed fluorescein cadaverine (FC) incorporation into cornified envelopes. Cell viability and total protein were also recorded, employing resazurin reduction and kenacid blue staining, respectively. Squamous differentiation, expressed as ngFC, occurred over 18 days in normal BEAS 2B cells cultured in Green's medium. Cell cultures that demonstrated enhanced differentiation, (2.3 ngFC/mgprotein) at day 6 when exposed to 5 µg/ml CSC, subsequently showed a significant increase in FC incorporation (p < 0.05) at day 18 (24.25 ngFC/mgprotein). Conversely, FC incorporation was not significantly increased at day 18 (7.1 ngFC/µgprotein) in cultures that did not demonstrate enhanced differentiation at day 6 (1.3 ngFC/µgprotein). This indicates that the effect of the compounds tested is predetermined by the differentiation status of the culture. The differentiation status is not directly related to the passage number. This work was funded by a grant from the industrial sponsors of the FRAME research program. E18: Assessment of Chemically Induced Photogenotoxicity by Complementary In Vitro Approaches Today's lifestyle is often associated with frequent exposure to sunlight, but some xenobiotics used in drugs, cosmetics, or food chemicals can produce adverse biological effects when irradiated. In particular, they can increase the risk of photogenotoxicity already due to UV radiation itself. There is thus a need to design appropriate approaches in order to obtain relevant data at the molecular and cellular level in this field. For ethical and practical reasons, in vitro models can be very convenient, at least for first evaluation tests. Here, we propose a strategy based on complementary experiments to study the photogenotoxic potential of a compound. The fluoroquinolones BAYy3118 and lomefloxacin were used as standards to demonstrate the performance of each test: photo-induced interaction with supercoiled circular DNA, photomutagenicity in the yeast Saccharomyces cerevisae, induction of DNA photodamage in cultured human skin cells as revealed by comet assay, and finally, induction of specific phototoxic stress responses, such as p53 activation or melanogenesis stimulation. With this strategy, the yeast assay is particularly well adapted for a rapid and informative screening of ingredients, finished products, and alcohol-based formulas, and it is used to help to ensure the safety of products likely to undergo environmental sunlight exposure. E19: An In Vitro Method for Visualizing the Time Course of Cytotoxic Effects on the Corneal Epithelium Steven Matsumoto, William Way, Kirk Tarlo, Brian Short, and Balbir Brar. Toxicology-Safety Evaluation, Allergan Inc., 2525 Dupont Drive, Irvine, California 92612, USA. Matsumoto_Steven@allergan.com. The ocular effect of potential irritants depends upon the time of exposure to the corneal surface. In vivo exposure time is decreased by the flow of tears. This process was investigated in vitro using full thickness rabbit cornea biopsies imaged by a fluorescence microscope using time-lapse images, captured with an intensified CCD camera. A continuous flow of isotonic buffer containing the fluorescent nuclear dye, ethidium bromide (EtBr), was perfused over the epithelial surface of the corneal biopsy. EtBr stains the nuclei of cells with damaged plasma membranes. When the perfusion was changed to a solution containing a cytotoxic chemical plus EtBr, the number of stained nuclei gradually increased with time. Treatment with higher concentrations of the cytotoxic agent produced a faster rate of staining. For example, treatment with 50, 100, or 200 mg/ml benzalkonium chloride gave rates of 0.4, 1.9, or 3.4 fluorescence units/minute, respectively. A pulse exposure to a cytotoxic agent gave a lower rate of staining providing a visualization of effects of simulated ocular tear washout. E20: A Tissue-Engineered Conjunctiva-Sclera for In Vitro Toxicology Testing Our objective was to develop a tissue engineered innervated and vascularised conjunctiva-sclera with very basic immune and inflammatory components that responds to chemotactic stimuli. Human umbilical vein endothelial cells (HUVEC's) and conjunctiva epithelial cells with extended life spans were obtained by transfection of the human papilloma virus 16 E6E7 region into low passage primary cells. HUVEC's were seeded within hydrated composite polymer matrices comprising fibrin-poly (N-isopropylacrylamide) PNIPAAM or fibrin-poly(N-isopropylacrylamide)-co-acrylic acid P(NIPAAM)-co-AAC. Dorsal root ganglia dissected from chick embryo served as the nerve source and were embedded within the matrices, while epithelial cells were seeded on top of the matrix. Results show that immortalized HUVEC's expressed appropriate markers, such as Factor VIII related antigen, and showed Dil-Ac-LDL uptake. Tissue engineered constructed showed nerve extension into the epithelium. These nerves were neurofilament positive. HUVEC's differentiated into vessel-like structures within the stroma and retained their endothelial markers. We also showed in separate experiments that the polymeric matrices supported migration of neutrophils and monocyte-macrophage cell lines, and production of MMP's, in response to chemotactic stimuli. We therefore conclude that it is possible to tissue engineer a conjunctiva-scleral tissue susbstitute. It is anticipated that with further fine-tuning, such a model may be useful as an alternative to animals in in vitro toxicology testing. E21: Three-step Method of Data Analysis, Incorporating Multiple Contrast Method to Detect Carcinogenic Substances in Cell Transformation Assay The BALB/c 3T3 cell transformation assay is used as an in vitro test to screen carcinogenic substances. It counts the number of foci in dishes to which one of several doses of a test substance was applied. The carcinogenicity is judged as positive if a monotonous dose-response relationship is observed. For this judgment, statistical tests for detecting positive trend have conventionally been used. In the course of the validation study, however, we found that such a procedure is not appropriate, due to high sensitivity; biologically irrelevant increases are likely to be judged as positive. We, therefore, devised a new statistical procedure with three steps, i.e., a Dunett-type test to detect high level of response in dose groups, a Margolin-type test to detect downturn in higher doses, and a multiple contrast method to detect monotonicity. In the application of this procedure, the choice of nominal significance level is very important. Actually, the adoption of the conventional 5% significance level for each procedure yields too big false positive rates from a toxicological viewpoint. We found that 0.1%, 50%, and 0.1% are reasonable to balance false positive and negative rates through our experience in the validation study. E22: Development of an Assay Method for Predicting Tumor Promoters Using V-Ha-Ras-Transfected Balb/C 3T3 Cells (Bhas 42 Cells) It is becoming urgent to develop a simple in vitro method for detecting non-genotoxic carcinogens which must include tumor promoters. Bhas 42 cells are v-Ha-ras-transfected BALB/c 3T3 cells and are regarded as initiated cells in the two-stage transformation process. We designed a method for predicting tumor promoters by the use of Bhas 42 cells. In our method, the cells are cultured in wells of 6-well plates for 17 days, during which, test chemicals are added in the medium for 1.5 weeks. When TPA was used, transformed foci appeared dose-dependently on the monolayer. In order to confirm the applicability of this method, various chemicals, including carcinogens and tumor promoters, were tested. Chemicals that showed significant increase of transformed foci were PDD, mezerein, anthralin, okadaic acid, lithocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, ursodeoxycholic acid, arsenic trioxide, cadmium chloride, zinc oxide, 1-nitropyrene, and so on. In addition, we confirmed the focus formation with extracts from tobacco smoke or diesel exhaust particles. Validation study on our transformation assay using Bhas 42 cells is going on with the participation of 12 laboratories in Japan. We propose that the method is reliable, reproducible, relevant, and useful for detecting tumor promoters. Supported by Grant-in-Aid from Japan Chemical Industry Association. E23: Establishment of LD50 Reference Values for Chemicals Used in Validation Studies of In Vitro Acute Toxicity Assays 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 (http://iccvam.niehs.nih.gov). Workshop participants recommended further evaluation of the usefulness of in vitro methods for predicting rodent and human acute toxicity. NICEATM and ECVAM subsequently designed a multi-laboratory validation study to evaluate the utility of two in vitro cytotoxicity tests using 72 chemicals. A major aspect of the study design was the selection of the rodent LD50 reference value for each chemical. LD50 studies were located through literature searches and secondary references. Studies were reviewed to identify the most appropriate LD50 reference value for each chemical. Criteria used to select references LD50 values included: (1) similarity of age, gender, and species to that recommended in current acute lethality testing guidelines, and (2) quality of the data, including conduct in accordance with standardized test guidelines and good laboratory practices. Chemical-specific examples of the selection decisions for reference LD50 values will be provided. These reference data will be used to evaluate the extent to which in vitro test methods can predict rodent LD50 values. Supported by NIEHS contract N01-ES-85424. E24: Assessment of Weak Phototoxic Potential of Chemicals Using Human Reconstituted Epidermis EPISKIN Skin photo-irritation is a non-immunological inflammatory reaction, occurring after topical or systemic administration of chemicals and concurrent exposure to UV. This reaction can now be partially reproduced in vitro, using human skin models. In this study, we investigated the ability of human reconstituted epidermis Episkin‚ to assess phototoxicity of weak phototoxic compounds, such as 6-Methylcoumarin and Ofloxacin, compared to a strong one, Chlorpromazine. Two negative controls were also studied: Sodium Dodecyl Sulfate (non-phototoxic, irritant) and Sulisobenzone (non-phototoxic, UVA absorber). After topical application of each chemical, epidermis modes were subsequently exposed or not to a non-cytotoxic dose of UVA (50J/cm). Eighteen hours later, cellular damage was evaluated measuring cytotoxicity by MTT conversion test; in addition, the release of pro-inflammatory mediator IL-1a was also investigated. After UVA exposure, 6-Methylcoumarin and Ofloxacin induced a dose-dependent decrease in cell viability, in concordance with an increasing IL-1a release. Moreover, the IL-1a release observed with 6-Methylcoumarin and Ofloxacin was lower than that induced by Chlorpromazine for an equivalent cell viability. This result could be related to their weak phototoxic potential. This study showed that Episkin‚ could be useful to investigate in vitro the onset of cutaneous phototoxic reactions, and particularly, to identify weak phototoxic chemicals.
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