Abstracts / Toxicology Letters 196S (2010) S37–S351 S151 suspension aggregates termed as embryoid bodies in which cells start to differentiate and in monolayer culture, where the dif- ferentiation also takes place. We have studied the expression of several genes in D3 mouse embryonic stem cells in presence of a wide range of concentrations of 5-fluorouracil (from 0.00001 nM to 200 nM). This well-established embryotoxic compound com- pletely inhibited cell viability after exposure of monolayer cultures to 200 nM during 20 days. The exposure of embryonic bodies dur- ing 21 days to 0.00001 nM 5-fluorouracil caused a reduction of 40% in the expression of the alpha-fetoprotein gene, a marker of the visceral endoderm. However, the expression of several mesoder- mal gene markers was not significantly affected at this 5-fluoracil concentration. These results suggest a high sensitivity of the vis- ceral endoderm differentiation to 5-fluorouracil. The expression of other visceral endoderm gene markers might be studied, although the quantification of the alpha-fetoprotein gene after exposure to potential embryotoxic compounds might be considered as an addi- tional end-point in in vitro embryotoxicity assays with embryonic stem cells. Acknowledgments: The authors thank Encarna Fuster for her technical assistance. Our research has been supported by: the Fun- dación Médica Mutua Madrile ˜ na to JAR and ER, the Generalitat Valenciana (AP-089/08 and AP-100/09) and the Instituto de Salud Carlos III (PS09/01093) to ER and Ministerio de Medio Ambiente y Medio Rural 051/2007/3-14.4) to MAS. doi:10.1016/j.toxlet.2010.03.520 P201-066 Neuropathy target esterase (NTE) as potential biomarker of embryotoxicity during early stages of development A.C. Romero, D. Pamies, C. Estevan, E. Vilanova, M.A. Sogorb Instituto de Bioingenieria. Universidad Miguel Hernandez de Elche, Spain Neuropathy Target Esterase is the target for a neurodegenerative syndrome caused by exposure to certain organophosphorus com- pounds, but it is believed that also plays a critical role in the embryonic development and therefore in cell differentiation. The exposure of monolayer cultures of D3 embryonic stem cells to 50 ng 5-fluoracil/ml (a well-established teratogenic compound) caused a modest (although statistically significant for p < 0.05) increment in the expression of the gene codifying for NTE. It is expectable that this exposure to 5-fluoracil caused no significant alterations in cell viability since the IC50 reported in the literature for ten days of exposure is higher than this. The expression of other genes con- sidered as biomarkers of neuroectoderm differentiation were also increased by exposure to 5-fluoracil. In this way, the expression of acetylcholinesterase and Nestin were increased by 5.70- and 4.7- fold, respectively (both statistically different from the control for p < 0.005). The expression of intermediate neurofilament was also increased, although in minor extension (only 1.6-fold). In conclu- sion, although further studies are needed, our data suggest that the expression of NTE might be a good biomarker for exposure to teratogenic and or embryotoxic compounds during early stages of development. Acknowledgments: Ministry of the Environment of the Govern- ment of Spain (Grant A051/2007/3-14.4). doi:10.1016/j.toxlet.2010.03.521 P201-067 Meeting requirements of the new OECD TG for in vitro skin irritation testing: Reproducibility of the EpiDerm Skin Irritation Test (EpiDerm-SIT) following the ECVAM validation and acceptance as a full replacement method M. Klausner, W. Deng, J. Kubilus, H. Kandarova, P. Hayden, J. Sheasgreen MatTek Corporation, United States In autumn 2007, an international validation study was performed to evaluate the reproducibility and confirm the predictive abil- ity of the EpiDerm Skin Irritation Test (SIT) method. In November 2008, ECVAM endorsed the EpiDerm SIT as a full replacement method for the in vivo rabbit skin irritation test. As reflected draft OECD guidelines for skin irritation testing, US and EU reg- ulators have appropriately maintained that a test method must demonstrate reproducibility on an ongoing basis so that regula- tors and commercial end users are assured that the assay method is stable and continues to give valid test results over time. The purpose of the present study was to investigate the reproducibil- ity of the EpiDerm SIT post-validation. Over a 4-month period in 2009, 12 independent lots of EpiDerm tissue were exposed to 3 irritants (alpha terpineol, heptanal, and butyl methacrylate), 3 non- irritants (benzyl benzoate, benzyl salicylate, and isopropanol) and the positive control (sodium dodecyl sulphate) and negative con- trol (Dulbecco’s phosphate buffered saline) using the validated SIT protocol. As per the SIT method, tissue viability was determined using the robust and cost effective MTT assay following a single, 60-min exposure and 42-h post-exposure incubation. In all cases, the EpiDerm SIT method correctly identified the irritants and non- irritants. Coefficients of variation (CV) between the tests (n = 12) for the tissue viability for all test articles were <8%, except for iso- propanol (IPA) which had a CV of 10.3%. Further study of the IPA results, revealed the importance of thoroughly rinsing the tissue following the 60-min exposure (as outlined in the SIT method). These results together with quality control results for EpiDerm over the past 13 years confirm that the EpiDerm SIT method is REACH ready and compliant with the EU 7th Amendment to the Cosmetic Directive. doi:10.1016/j.toxlet.2010.03.522 P201-068 Reproducibility of the MATTEK ocular irritation test (EpiOcular TM -OIT) in response to the COLIPA/ECVAM REACH initiative Y. Kaluzhny, H. Kandarova, S. Letasiova, P. Hayden, J. Sheasgreen MatTek Corporation, United States Purpose: In vitro tests for assessing ocular irritancy of con- sumer/household products and a broad range of organic chemicals are urgently needed to comply with EU legislation such as the 7th amendment of the Cosmetics directive and the REACH directive. Eye care cosmetics (ECC) also need to be tested for ultra-mildness in order to fully service the marketplace. This poster summa- rizes extensive testing of the ocular irritation test protocols for use with the in vitro EpiOcular tissue model in order to accom- modate both purposes. Methods: For REACH irritation testing, a single exposure period is used: 30 min with a 2-h post-exposure incubation (liquids) or 90 min with 18-h post-exposure incubation (solids). A single cut-off in relative survival is used for classifica-