Abstracts / Toxicology Letters 189S (2009) S57–S273 S83

V58International validation of an in vitro skin irritation test pro-tocol (EpiDerm-SIT) to replace the in vivo rabbit test for hazardidentification of chemicals

Manfred Liebsch 1,∗, Armin Gamer 2, Rodger Curren 3, JürgenFrank 4, Elke Genschow 1, Julian Tharmann 1, Marina Remmele 2,Britta Bauer 2, Hans Raabe 3, Nicole Barnes 3, Allison Hilberer 3,Nathan Wilt 3, Mohammad Reza Lornejad-Schäfer 4, ChristineSchäfer 4, Patrick Hayden 5, Helena Kandarova 5

1 Bundesinstitut für Risikobewertung, Unit 37: Centre for AlternativeMethods to Animal Experiments - ZEBET, Berlin, Germany, 2 BASF SE,Experimentelle Toxikologie und Oekologie, Ludwigshafen, Germany,3 Institute for In Vitro Sciences, Inc., Gaithersburg, United States,4 zet–LSL; Centre for Alternative and Complementary Methods toAnimal Testing, Linz, Austria, 5 MatTek Corporation, Ashland, UnitedStates

In April 2007, ECVAM endorsed 2 alternative test methods (EPISKINand EpiDerm Skin Irritation Tests (SIT)) as replacements of thein vivo rabbit skin irritation test. While EPISKIN was recognizedas a stand alone method, EpiDerm-SIT was endorsed for use in atiered testing strategy (OECD TG 404), where irritating results areaccepted and non-irritating results may require further testing byanother method.

Based on published data and analysis of results of the ECVAMvalidation study, there was evidence that differences in the barrierproperties between the 2 models were responsible for the lowersensitivity of EpiDerm-SIT when using an identical protocol as usedfor EPISKIN. Therefore, modifications of the exposure conditionswere introduced to the EpiDerm-SIT protocol: (a) exposure timewas increased from 15 min to 60 min; (b) the temperature duringthe exposure was increased to 37 ◦C. With these modifications, sig-nificant increase in sensitivity was obtained, while maintaining anacceptable specificity of the method.

In autumn 2007, an international validation study was per-formed to evaluate reproducibility and confirm the predictiveability of the modified EpiDerm-SIT method. Results of the studyare presented here. Overall, sensitivity and specificity of 80% wereobtained, which is comparable to results for the EPISKIN SIT forthe same set of chemicals (sensitivity of 70%, specificity 80%). Theinter-laboratory reproducibility of the modified EpiDerm-SIT andits concordance with the in vivo rabbit data was also very good.The method was endorsed by ECVAM in November 2008 as fullreplacement method.

doi:10.1016/j.toxlet.2009.06.249

V59MucilAir: A novel human 3D airway epithelium model for longterm toxicity testing

Song Huang ∗, Jean-Paul Derouette, Samuel Constant, MireilleCaulfuty, Ludovic Wiszniewski

Epithelix, In vitro Toxicology, Geneva, Switzerland

Most of the in vitro cell models for long term testing of chemicalssuffer of at least two shortcomings: (1) The failure of reproducingthe in vivo physiological characteristics of the corresponding tis-sues, such is the case for the immortalized cell lines. (2) A limitedshelf-life, for example, the freshly established primary cell cultures.Our company, Epithelix, has developed and is commercializing a

novel in vitro cell model of the human airway epithelium (MucilAir)which is free of these limitations.

MucilAir is not only morphologically and functionally differenti-ated; but it can also remain at a homeostatic state for more than oneyear. Under the electronic microscope, the typical ultra-structuresof the human airway epithelium, such as the tight junctions, thecilia, the basal cells, the mucous cells, could be observed. Theepithelium is electrically tight with a TEER about 450 W cm2. Theion channels like the sodium channel and chloride channel are fullyfunctional and respond normally to their specific inhibitors andactivators when measured in modified Ussing chambers. More-over, the epithelial cells react to pro-inflammatory mediators suchas TNF-� in a physiological manner. Remarkably, the epitheliumhas a strong capacity of regeneration after mechanical or chemicalinjuries.

Due to its unique long shelf-life of one year, this model can beused for studying the human respiratory diseases, and for testingthe long-term/chronic effects of drugs/chemicals on human respi-ratory tracts. Epithelix offers ready-to-use products and servicesfor toxicity tests of chemicals.

doi:10.1016/j.toxlet.2009.06.250

V60Chlorpyrifos induces apoptosis in murine thymocytes

Atul Prakash ∗, Saleem Khan, Manoj Aggarwal, Avinash G. Telang,Jitendra Kumar Malik

Indian Veterinary Research Institute, Pharmacology and Toxicology,Bareilly, India

Chlorpyrifos is an organophosphate (OP) pesticide used widelythroughout the world in agriculture and for controlling residen-tial insect pests. Human exposures to OP pesticides constitute amajor public health hazard and their widespread use brings highrisks of severe health problems including oxidative stress, hep-atic dysfunction, immunological abnormalities, embryotoxicity,genotoxicity, teratogenicity, neurochemical, and neurobehavioralchanges. To investigate the mechanism of immunotoxicity, weexamined whether chlorpyrifos induces apoptosis in murine thy-mocytes and studied underlying mechanism. Murine thymocyteswere isolated and cultured in RPMI-1640 medium as per the stan-dard protocol. The cells were exposed to different concentrationsof chlorpyrifos ranging from 20 to 80 �M for 4–8 h at 37 ◦C in a 5%CO2 incubator. After the incubation period, we determined apop-totic DNA and reactive oxygen species by using propidium iodideand 2′,7′-dichlorofluorescein diacetate (DCFH-DA), respectively byflowcytometry. Chlorpyrifos treatment resulted in concentration(0–80 �M)- and time-dependent increase in apoptotic DNA. Chlor-pyrifos at the highest concentration (80 �M) induced apoptosisto the extent of 44.10 ± 2.68% at 8 h. Chlorpyrifos treatment alsocaused a concentration-dependent increase (6–7-fold) in reactiveoxygen species generation at 4 and 8 h. Our results indicate thatchlorpyrifos can induce apoptosis in murine thymocytes and thiseffect may be mediated through generation of reactive oxygenspecies.

doi:10.1016/j.toxlet.2009.06.251