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Theme A Posters: Replacement And Reduction AlternativesA13: Detection of Endotoxins and Other Pyrogens by the In Vitro Pyrogen Test (IPT) as an Alternative to Rabbit Experiments A new pyrogen test (In Vitro Toxicology 9, 353-359, 1996) exploits the human fever reaction for the detection of pyrogenic contamination: In a simple set-up, human whole blood from healthy volunteers is used to carry out the in vitro pyrogen test. After incubation at 37°C, the release of interleukin is assessed by ELISA as a measure of pyrogenic contamination. This assay has been optimized and pre-validated in a collaborative effort with about 40 partners for about 100 pharmaceuticals. Currently, the test takes part in a European validation study. The new test has several advantages compared to the rabbit test: namely, it is less laborious, and it is quantitative, as well as more sensitive. Furthermore, it does reflect the reagibility of the target species (which can differ for a given pyrogen by a factor of 10,000 within mammals) and can replace the rabbit test. In contract to the LAL, the whole blood test is not restricted to endotoxins from Gram-negative bacteria, but detects all classes of pyrogens. Furthermore, in contrast to LAL, it reflects the potency of different endotoxins in mammals, and it is not disturbed by endotoxin-binding components. Pre-tested cryo-preserved blood is under development and non-endotoxin standards were developed. A14: Replacement of Animal Tests for Cyto- and Genotoxicity by Recombinant Mammalian Cells Alternatives for toxicity prediction based on bacteria, yeast, insect, or mammalian cells can reduce utilization of animal experiments and provide information concerning the mechanism of toxicity. In this work, two recombinant cell lines were produced using the reporter protein Enhanced Green Fluorescent Protein (EGFP) in order to contribute to the risk assessment of an increasing environmental toxic--UV radiation. The first recombinant cell line constitutively expresses EGFP on a high level. The fluorescence of the cell population correlates with the cell mass growth. Measurements in the microplate fluorimeter enable the fast detection of cytotoxic effects. The second stably transfected cell line, human embryonic kidney cells, shows an increased expression of EGFP in response to several kinds of stress. To this end, a synthetic promoter was used, which consists of four NF-kB binding sites and the minimal thymidine kinase promoter. Different UV-radiation qualities had, in equitoxic does, different effects on NF-kB-dependent gene activation: UVC and UVB had no effect; UVA induced a strong activation of NF-kB. This can be an important factor in skin cancer formation by natural sunlight, due to its anti-apoptotic effects in some cell types. A15: Evaluation of Cd86 and Mhc Class II Molecule Expression on THP-1 Human Monocyte Cells as Predictive Endpoint for Contact Sensitizers Several studies have shown that contact sensitizes induced CD86 expression and enhanced internalization of MHC class II molecules on Dendritic cells (DC's). We used THP-1, human monocyte cell line, as a replacement for DC's for evaluation of these phenotypical alterations on the cells as a predictive endpoint for contact sensitizers. Known sensitizers and irritants were evaluated. After 24-hour exposure to samples, the expression of CD86 on THP-1 cells was measured by flow cytometry. Of these chemicals, some of sensitizers, such as dinitrochlorobenzene, 2-mercaptobenzo- thiazole, p-phenylenediamine, and ammonium tetrachloroplatinate enhanced the CD86 expression on THP-1 cells, while nikel sulfate, cobalt sulfate, or all of the irritants such as methylsalicylate, sodium dodecyl sulfate, and dimethyl sulfoxide, could not augment CD86 expression. Synergistic effect was observed when DNCB and IFN-g were added to a culture of THP-1 cells simultaneously. Furthermore, internalization of MHC class II was observed when some sensitizers were treated for 2 hours. Effects of chemicals on the two phenotypical alterations were the same. These results suggest that these test systems are useful to predict contact sensitizers as an in vitro sensitization assay. A16: The Third Frame Toxicity Committee, A Strategy for Implementing Alternatives in Toxicity Testing The recently established FRAME Third Toxicity Committee is reviewing progress made in the application of the Three R's in the development and safety evaluation of medicines, biologicals, cosmetics, agrochemicals, and other products, as well as industrial chemicals, and to make recommendations as a basis for further sensible progress according to sound scientific and ethical criteria. The committee will: (a) co-ordinate the activities of several working parties on targeted risk-assessment versus hazard identification, data sharing, endocrine disruption, carcinogenicity testing, and workshops to discuss these and other issues; (b) monitor the application of genomics, proteomics, and metabonomics to toxicity testing; and (c) publish a status report on the use of alternatives in toxicity testing with a series of objective and achievable recommendations, to be discussed at a subsequent international conference, the proceedings of which will also be published. A17: Factors Effecting the Attachment and Penetration of Schistosome mansoni Larvae in Response to a Human Epidermal Model Whilst models for assessing attachment and penetration of the human parasite S. mansoni have included animals and human skin, the use of an in vitro epidermal model has clear potential. Keratinocyte, fibroblast, and endothelial cells were isolated from human skin and cultured in their respective media. Keratinocyte differentiation to form a multilayer was carried out at the air/liquid interface for 21 days. Monolayers of fibroblast and endothelial cells were produced 48 hours prior to infection. Infection of the models was carried out with a known density of larvae (cercariea), and counts were taken at 5 and 10 minutes. No significant attachment was observed with fibroblast and endothelial cells; however, the multilayer demonstrated an initial attachment of 78%, increasing to 80% at 10 minutes. The results demonstrate a selective parasite response to the presence of appropriate human cells. Microscopy showed that, as the cercariae interact with the cellular surfaces, initial attachment is followed by entry, tail detachment, and burrowing between the cell layers. In addition, once a point of ingress has been made, other cercariae may follow. Parasite attachment and penetration may follow. Parasite attachment and penetration are affected by cercarial age and viability. Work funded by a grant from the industrial sponsors of FRAME research program. A18: EpiAirway, Differentiated Airway Epithelial Cultures for Pre-clinical Respiratory Studies Normal human tracheal-bronchial epithelial cells (NHBE) are cultured using serum-free medium to form EpiAirway, a three-dimensional tissue that closely resembles the epithelial tissue of the respiratory tract. Histological cross-sections of both the in vitro tissue and a normal human bronchiole reveal a pseudo-stratified structure. Transmission electron microscopy revealed numerous cilia on the apical surface of the cultures and dot blot analysis was used to quantify mucin secretion from the cultures. The tissue forms tight junctions and has a transepithelial resistance of 584 ± 139 Ohm cm2 (n = 24 lots). Quality control testing is based on TER measurements; all tissue lots must exhibit a TER of at least 500Ohm cm2. Results utilizing the tissue for enhanced delivery of inhaled pharmaceuticals will be presented. In optimal formulations, TER values decreased by 99%, permeation rates of the active molecules (MW 20,000) increased 85 fold, and tissue viability remained at 97% of control tissues, as measured by the MTT assay. These results demonstrate that EpiAirway can serve as a reproducible, cost-effective, easily accessible in vitro alternative in place of much more expensive and more difficult to perform animal or human pre-clinical experiments. A19: Full Thickness EpidermTM: A Dermal-Epidermal Skin Model to Study Epithelial-Mesenchymal Interactions In contradistinction to previous dermal/epidermal models, Full Thickness EpiDerm is cultured in an easily manipulated cell culture insert, and the tissue extends from wall-to-wall. In terms of ease of use, these characteristics greatly facilitate testing of potential allergens or irritants in that direct topical application is possible. Topical exposure to the common surfactant, 1% Triton X-100, results in MTT tissue viability dose response curves that fall within the normal range of the keratinocyte-only tissue, EpiDerm. Currently, in order to produce a standardized, reproducible organotypic tissue, all lots of EpiDerm are compared to a reference database of Effective Time-50 (ET-50) values, i.e. the time of exposure, after which, viability is reduced to 50% following exposure to 100 L of Triton X-100. The database average (184 tissue lots) is 6.74 ± 0.99 hours (± 1 standard deviation); initial lots of the full thickness tissue, tested in an identical manner, average 7.14 ± 1.33 hours (n = 3). Histological cross-sections of the full thickness tissue show an epidermal layer that is very similar to EpiDerm and native epidermis atop a fibroblast-containing collagen matrix dermis-like layer. Based on these initial results, investigation of the tissue response to stimuli specifically affecting the dermis or epidermal/dermal "crosstalk" will likely prove rather informative. A20: Measurement of Antibody Responses Against E Coli F4 Antigens in Sows after Vaccination as an Approach to Replace Efficacy Challenge Procedures in Newborn Piglets Acute diarrhea is a common disease in newborn piglets, associated with a number of infectious agents including E coli. During their first weeks of life, piglets acquire passive immunity by maternal antibodies derived from colostrum and milk, which exert a protective effect against pathogens in the gut. In the present study, we conducted a field trial, vaccinating eight groups of ten pregnant sows with eight different colibacillosis vaccines, which contained F4 pili antigens. Each animal was immunized twice before parturition. Two groups of non-vaccinated animals served as controls. Blood samples were taken from all sows before the first vaccination and around farrowing. Colostrum samples were collected within a few hours after parturition. Specific antibodies to E. coli F4ab and F4ac adhesions were determined by means of an indirect sandwich ELISA. There were significantly higher levels of specific antibodies in serum and colostrums of vaccinated animals compared to controls even in the presence of pre-existing antibodies. This indicates the potential of the serological method to replace challenge experiments with E. coli F4 strains in piglets. A21: In Vitro Quantitative Determination of Ophthalmic Irritability by Chorioallantoic Membrane Test with Tryptan Blue Staining as Alternative to Eye Irritation Test The damage provoked by some substances on the chorioallantoic membrane (CAM) of the chicken egg is used as an alternative assay to determine ophthalmic irritation, with which, good results have been obtained by comparing the in vitro and the in vivo method. Nevertheless, this assay has some limitations as a subjective factor. Hagino et al developed an objective evaluation technique using the amount of trypan blue adsorbed at the treated site as an indicator of injury to the CAM. The present work was aimed at the determination of ophthalmic irritation of 22 substances (chemicals and cosmetics). For that we used the spectrophotometric quantification of the damage produced on CAM by trypan blue staining, using fertile chicken eggs (INVITOX protocol No108, 1996), comparing the results with the values obtained by the traditional Draize assay. On the other hand, the in vitro and in vivo assay costs were evaluated by the minimization costs method. As a result, we obtained a good correlation between the amounts of pigment adsorbed by the CAM and the scores obtained in the Draize eye irritation test (r = 0.90 p < 0.0001). For the cosmetics and chemical substances, it was 0.94 p < 0.0001 and 0.86 p < 0.01, respectively. There were three chemical substances as false positive and three cosmetics as false negative. Furthermore the trypan blue assay is an inexpensive method, compared with the Draize test. The CAM-TBS assay is a simple and inexpensive alternative method, used expressly to predict the damage that can be caused by chemical substances or cosmetics on the ocular structures. A22: Comparative Study of Red Blood Cell Assay in Rat and Calf Blood as Alternatives of Draize's Eye Irritation Test A. Lagarto, R. Vega, I. Guerra, and Y. Vega. Drug Research and Development Center, 17 No. 6208 entre 62 y 64, Playa, Ciudad Habana, Cuba. cinfa@infomed.sld.cu. Red blood cell assay (RBC) is used to estimate irritation potential of tensides and detergents. The assay is based on the differentiation between cell membrane lysis and cell protein denaturation. Both effects are measured photometrically. Data of media hemolysis concentration, denaturation index, and the ratio of both parameters are compared with in vivo data of eye irritancies (OECD Guideline No405, 1987 and ISO 10993-10, 2000). We evaluated 21 substances (20 cosmetics and 1 chemical) using rat and calf blood (INVITOX protocol No 37, 1992). A good correlation between in vitro and in vivo assay was found with both kinds of blood. For rat blood, it was 0.84 p < 0.0001, and it was 0.83 p < 0.0001 for calf blood. According to the exact probability of Fisher, taking as the approach the acceptance or rejection of the product, there are not significant differences between the in vitro assay with calf blood and in vivo results. For the RBC assay with rat blood, significant differences were found between the in vitro test and the in vivo test. The best results were obtained with calf blood. In rat blood, there were several false negatives, because the red blood cell membrane is most resistant to hemolysis by detergents. For that reason, this kind of blood (should not) be used for this in vitro assay. A23: Prediction of Acute Toxicity by In Vitro Assays in Drug Discovery The wide application of new technologies, such as combinatorial chemistry, give rise to a larger number of new chemical entities, which are available in limited quantities. In vitro methodology is, thus, the only way to overcome such limitations. This study presents the application of in vitro methods to determine acute cytotoxicity of a new family of antimicrobials, and the correlation with in vivo results. Cytotoxicity studies were carried out with four different cell lines derived from different target organs. Cuture cells were exposed during 24 hours to the corresponding drug, and protein synthesis was used as a cytotoxicity marker. The in vivo toxicological potential of these compounds (n = 12) was estimated in mice following the Up and Down methodology. In vitro data were able to rank projects according to their toxic potential within the same family, and to exclude those that showed highly toxic properties. In summary, it appears from the good correlation observed that in vitro methods have the potential to reduce and replace, if properly developed, the use of laboratory animals in early phases of drug discovery. A24: Toxic Effects of Ketamine Hydrochloride (VetalarTMV) on Cultures of Marmoset Bladder Epithelial Cells and Whole Rat Bladders In Vitro During pre-clinical safety studies, lesions resembling varying degrees of cystitis occurred in the bladders of the common marmoset in control and treated animals. An adverse reaction to ketamine, routinely used to sedate marmosets prior to electrocardiogram measurements, was considered. In vitro investigations on primary marmoset bladder epithelial cells were carried out as an alternative to an in vivo study using concentrations of ketamine likely to occur in marmoset urine following administration for sedation. Cultures were treated for 24 hours, and cytotoxicity was assessed by morphology, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction and lactate dehydrogenase (LDH) leakage. Also, whole rat bladders were treated for 6 and 24 hours in vitro to establish if the toxic effect of ketamine was species-specific. Toxicity was assessed microscopically by the degree of urothelial damage. In both in vitro models, significant compound-related toxicity was apparent at concentrations of ketamine that can occur in the bladder in vivo. These in vitro investigations concluded that high concentrations of ketamine hydrochloride in marmoset urine were probably responsible for the lesions seen in marmoset bladders. Lower concentrations have since been administered with no adverse effects.
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