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Theme E Posters: Test Development, Validation, And ImplementationE1: The Evaluation of an Alternative Mucosal Irritation Test Using Slugs The objective of this study was to evaluate an alternative mucosal irritation test using slugs as a test organism. The effect of 28 reference substances on the mucosal tissue of the slugs was determined by the mucus production, the reduction in body weight, and the release of proteins from the body wall. The results were compared with the available Draize scores for eye irritation (MMAS). The mucus production and the reduction in body weight caused by a 60-minute contact period (CP) were significantly correlated (r = 0.73) with the MMAS. However, the mucus production was the best endpoint to classify the chemicals into the three categories corresponding to the EU classification (NI, R36, and R41). Since irritating alcohols were underestimated (no mucus production), two prediction models were developed. Sixty-five percent of the non-alcohols were correctly classified, according to the mucus produced (60-minute CP). For alcohols, a second 60-minute CP was required, using the protein release as the endpoint (concordance: 91%). The sensitivity and specificity (considering NI and I category) were 83% and 100% for the non-alcohols, respectively, and 100% for the alcohols. We can conclude that the mucosal irritation test seems to be a reliable method for screening the irritating potency of chemicals on mucosal tissue. E2: Study of Alternatives to Testing for Endocrine Disrupters in Rat Whole Embryo Culturetes Bisphenol A (BPA), an endocrine disrupter (EDC), is a chemical produced in a large amount and used principally as a monomer in the manufacture of polycarbonate plastic and epoxy resins. Although the effects of BPA have been investigated in in vivo animal tests, there is little information on its toxicological effects. We have already reported that rat embryos treated with BPA at a low dose, (1 ppm) for 48 hours in a whole embryo culture, showed a decrease in embryonic heart rate, but no malformation was observed. In the present study, we examined the effect of BPA at a low dose on rat embryos for a prolonged time in a whole embryo culture. Rat embryos on day 11.5 of gestation (plug day = 0) were cultured for 72 hours in a medium containing 1 ppm BPA. After cultivation for 72 hours, morphological anomalies of hypolastic mandible, cleft lip, curly tail, and edema were found in all embryos treated with BPA. Furthermore, the total number of somites was less in treated embryos. As the result, BPA at a concentration of 1 ppm significantly inhibited some physiological functions and was effective in morphogenesis of cultured rat embryos. BPA at a low dose produced major malformations in rat embryos in culture for a prolonged time, as found with BPA at a high dose (100 ppm). It is suggested that more detailed investigations may be needed on the effects of BPA at low doses. E3: Percutaneous Absorption of Mexoryl SxTM in Human Volunteers: Comparison with In Vitro Data The potential human health risk of UV filters depends on their intrinsic toxicity and potential human systemic exposure. Using a mass-balance approach, a study was designed to investigate the systemically absorbed dose of [14C]-Mexoryl SX in humans after topical application of a typical sunscreen emulsion. To assess the correlation with in vitro experiments, the percutaneous absorption of this UVA filter through isolated human skin was measured in diffusion cells under similar exposure conditions. In vivo, when applied as a finite dose for a period of 4 hours, 89-94% of applied radioactivity was recovered from the wash-off samples. In the urine samples collected, radioactivity slightly exceeded background levels. No significant levels of radioactivity were measured in blood or feces sampled up to 120 hours after application. The systemically absorbed dose of [14C]-Mexoryl SX is less than 0.1%. In vitro, [14C]-Mexoryl SX remained primarily on the skin surface. 0.19% of applied radioactivity was found in the stratum corneum. The total average in vitro absorption over 24 hours was 0.16% of the applied dose. This value correlates well with corresponding in vitro data and demonstrates that, under realistic exposure conditions, human systemic exposure to this UVA filter is negligible and, therefore, poses no risk to human health. E4: The Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) Peer Review Panel Evaluation of the Revised Up-and-Down Procedure (UDP) for Acute Oral Toxicity In 1999, the U.S. Environmental Protection Agency (EPA) asked ICCVAM to evaluate the validation status of the Revised UDP as a substitute for the conventional acute oral toxicity test (e.g., OPPTS 870.1100 OECD Test Guideline 401). ICCVAM and NICEATM organized an independent scientific peer review evaluation of the Revised UDP by an international panel of expert scientists. The Panel met publicly to evaluate the extent to which the Revised UDP met ICCVAM validation and acceptance criteria and to develop conclusions regarding the usefulness and limitations of the Revised UDP. The Panel agreed that the UDP Primary and Limit Tests would perform as well as, or better than, the conventional LD50 test and would also reduce and refine animal use. Based on the Panel's conclusions, the EPA UDP Technical Task Force modified the UDP test guideline and added a computational procedure to calculate the LD50 confidence intervals (CI). The EPA also developed a user-friendly software program to use in performing the Revised UDP. At a second meeting, the UDP Panel endorsed the modifications to the Revised UDP, the CI calculation procedure, and the software program. Based on the Panel's evaluation, ICCVAM forwarded recommendations to Federal agencies, supporting the use of the Revised UDP as a substitute test for the conventional LD50 test. Supported by NIEHS Contract N01-ES-85424. E5: Development of an In Vitro Test Battery for the Estimation of Acute Human Systemic Toxicity: Presentation of the Edit Project The aim of the EDIT program (Evaluation-Guided Development of In Vitro Test Batteries), is to validate in vitro tests on toxicokinetics and organ-specific toxicity to be incorporated into optimal test batteries for estimation of human acute systemic toxicity. The scientific basis of EDIT is the good prediction of human acute toxicity by three human cell lines (R2 = 0.77), found in the MEIC (Multicenter Evaluation of In Vitro Cytotoxicity tests) study. The results from this study indicated that at least two types of in vitro tests ought to be added to the existing test battery to improve the prediction of human acute systemic toxicity. The tests should determine key kinetic events (e.g. passage over biological barriers and biotransformation), and crucial mechanisms not covered by the MEIC battery. The EDIT programme is a case-by-case project, but the validation of new tests will be carried through by a common procedure: The Scientific committee of EDIT defines the need for specific sets of toxicity and toxicokinetic data. Laboratories are invited to perform the defined tests to provide "missing" data for the 20 EDIT reference chemicals. The obtained results are evaluated against human acute lethal and toxicity data compiled in the MEMO (the MEIC Monograph program) database. E6: EpiskinTM as a Product Development Tool: Comparison of In Vitro (Reconstructed Epidermis) and In Vivo (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 3 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 Episkin. Cellular viability was measured by the MTT assay after 18 hours with a topical application. The same product batches were used in both test systems. The following results were obtained: in vivo, 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 with in vivo, at similar concentrations, C-22 was found to be well tolerated in vitro. In conclusion, the in vitro reconstructed 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 product development and risk assessment before clinical tests. E7: An Improved In Vitro Protocol to Assess Ocular Irritating Potential of Chemicals Using a human corneal epithelial cell line (CEPI17), we have improved the previously described protocol (Bologna; 1999) to predict ocular irritancy. Cells were grown at about 80% confluency in a modified medium and treated every 15 minutes with dilutions of chemicals. After rincing, cells were incubated for 24 hours. Interleukin-8 (IL-8) release was quantified by ELISA on cell supernatants. Cell viability was determined using Alamar Blue® at 24 hours. After rincing, cells were re-incubated with a modified medium, and viability was determined again at 48 and 72 hours to assess the cells' ability to undergo growth. We have tested 17 chemicals, and we have used in vivo data from the ECETOC data bank. This new protocol has helped us to improve the classification of chemicals. E8: 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 of performing 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. We will present 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). 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. E9: Application of a Genetically Engineered PC12 Cell Line as Part of an In Vitro Integrated Testing Strategy for Detecting Neurotoxic Effects The application of the genetically-modified PC12 cell line, displaying different sensitivities toward human wt p53-mediated cell death, has been evaluated as a part of an in vitro integrated testing strategy. This test system allows human wt p53 gene mechanistic studies to be conducted at Tier III of a tiered testing strategy, as proposed by Atterwill et al. (ATLA 22:350-362, 1994). The sensitivity and robustness of this system was tested using compounds with known neurotoxic inducers and inhibitors, such as camptothecin, cyclosporin A, L-Glutamate, and staurosporine. For example, camptothecin over a concentration range of 0-150mM, produced an IC50 value 20-fold higher in human wt p53-non-expressing cells in comparison with human wt p53-expressing cells. Increased human wt p53-expression clearly renders the cells more sensitive to camptothecin-induced toxicity, indicating that this genetically engineered cell line appears to be a sensitive model for detecting human wt p53-mediated neurotoxicity. E10: Comparison Between Permanent Female and Male Egc-Lines of Balb/cJ Mice for Reproductive Toxicity Testing In order to offer a sensitive and predictive in vitro method to assess the genotoxic potential of test chemical agents on male and female reproduction, we established primordial germ (PG) cell-derived permanent embryonic germ (EG) of the mouse (strain Balb/cJ). The sister chromatid exchange (SCE) test served as a test for gene mutation and the MTT assay for cytotoxic effects. To evaluate differences of the genotoxic potential between male and female EG cells, both cell lines were used. We started testing a series of in vivo genotoxic test chemicals (ENU, MNU, MMS, Hydroxyurea and Mitomycin C) that were recommended by an expert committee for sex-specific mutagenic and cytotoxic effects. In order to develop a preliminary prediction model using linear discriminant analysis, negative chemicals were tested as well (ascorbic acid, penicillin G, saccharin). Both cell lines obtained a correct classification of 100%. Although the female EG cell turned out to be more sensitive, the probability for correct classification for the male EG cell was even higher. E11: Inflammatory Response to Pyrogens Absorbed to Biomaterials Determined by a Novel Whole Blood Assay Without Animal Use The human immune system reacts vigorously to pyrogenic contamination, i.e. fever-inducing bacterial debris harboring endotoxins and non-endotoxin immune stimuli. This inflammatory reaction is very sensitive, making it necessary to control pyrogen contamination of medical devices. The impact of pyrogenic contamination of biomaterials has been discussed as a reason for impaired biocompatibility and inflammatory responses induced by implants. However, to date, the assessment of such pyrogenic contamination is limited. Both established pyrogen tests, i.e. the rabbit test and the Limulus assay, can only be performed with solutes (extracts or wash solutions). However, since most pyrogens are highly hydrophobic, they only partially detach from surfaces and often escape detection. The new in vitro pyrogen test (IPT), which is based on the human fever reaction, was adapted to materials: human whole blood is brought into direct contact with the device, monocytes react to a broad spectrum of pyrogens by the formation of inflammatory mediators, such as interleukin-1, which are detected by ELISA. The assay proved its suitability in evaluations for various types of biomaterials and is currently being discussed for inclusion in ISO guidelines. E12: Whole Blood Cytokine Response as a Measure of Airborne Microbial Contamination The pathogenic effects of inhaled airborne pyrogens (especially endotoxins) for humans is increasingly recognized. Various syndromes are described after contact with airborne microbial contaminations via the respiratory tract: sick-building syndrome, ODTS (organic dust toxic syndrome), chronic obstructive lung disease (COLD), etc. Air-conditioning systems intensify this problem, but storage of organic garbage in households also represents a considerable source of airborne pollutants. The in vitro pyrogen test (IPT) utilizes the natural reaction of the immune system in order to detect a broad spectrum of pyrogens (including Gram-positives and fungal spores) very sensitively in the relevant species. We adapted this whole blood system for different filter materials that are used in air-control pump systems. We show that it is possible to measure the cytokine response after incubating the contaminated filters directly with whole blood. The measurement was performed with samples from different sites, such as stables of pigs, sheep, and cows, as well as in flats. Microbiological burden with fungal and bacterial colony forming units were roughly correlated with the whole blood response. With this integral attempt, it might be possible to correlate contaminated air and the resulting reaction of the immune system, indicating, therefore, a potential risk for the person exposed.
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