This peer-reviewed, PubMed-indexed VIDEO technical article produced by scientists at MatTek Corp. and published in JoVE (Journal of Visualized Experiments) provides detailed instructions on how to perform the ECVAM-Validated EpiDerm Skin Irritation Test (SIT) Method, a stand-alone full replacement for traditional animal-based skin irritation tests.
NOTE: This 20 MINUTE video protocol is ONLY to be used to perform the EpiDerm Skin Irritation Test (SIT). You can view the FIRST 4 MINUTES for an overview of EpiDerm and the EpiDerm SIT protocol.
www.jove.com/index/details.stp <— Click here to view the video protocol.
The EpiDerm Skin Irritation test (EpiDerm SIT) was developed and validated for in vitro skin irritation testing of chemicals, including cosmetic and pharmaceutical ingredients. This procedure can be used as full replacement of the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in line with EU regulations.
The test is performed over the course of a 4 day time period, consisting of pre-incubation, 60 minute exposure, 42 hour post-incubation and MTT viability assay.
After tissue receipt and overnight pre-incubation (Day 0), tissues are topically exposed to the test chemicals (Day 1), which can be liquid, semi-solids, solid or wax. Three tissues are used for each test chemical, as well as for the positive control (5% aq. SDS solution), and a negative control (DPBS). Chemical exposure lasts for 60 minutes, 35 min of which the tissues are kept in an incubator at 37°C. The test substances are then removed from the tissue surface by an extensive washing procedure. The tissue inserts are blotted and transferred to fresh medium.
After a 24 hr incubation period (Day 2), the medium is exchanged. The medium can be saved for further analysis of cytokines or other endpoints of interest. After the medium exchange, tissues are incubated for an additional 18 hours.
At the end of the entire 42h post-incubation (Day 3), the tissues are transferred into yellow MTT solution and incubated for 3 hours. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, is extracted for 2 hours using isopropanol. The optical density of the extracted formazan is determined using a spectrophotometer.
A chemical is classified as an irritant if the tissue viability relative to the negative control treated tissues is reduced below 50%.
