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EC number: 947-432-4
CAS number: -
Cell proliferation numbers and Stimulation index
Objective: To screen for possible sensitising potential of topically applied test items. The flow cytometry-modified LLNA protocol on which this screen is based is designed to be an alternative assay for the Buehler Guinea Pig Sensitisation Assay defined in the NIH report 'The Murine Local Lymph Node Assay: A test method for assessing the Allergenic Contact Dermatitis Potential of Chemicals/Compounds NIH No. 99-4494, 1999 and the LLNA as defined in EPPA OPPTS 870.2600, Final Guideline (March 2003) and OECD Guideline for the Testing of Chemicals No. 429, revised July 2010.
Method Synopsis: The test article was tested for solubility in Acetone:Olive Oil (4:1, AOO) and N,N-Dimethylformamide (DMF). The test article was not soluble at 25% (w/v) in AOO, but was soluble at 25% in DMF. One group of five healthy female CBA/J mice was treated with a 25% (w/v) concentration of test article by topical application to the dorsum of each ear once daily for three consecutive days. A Vehicle Control group of five mice was treated with DMF and another group was treated with the Positive Control (alpha-Hexylcinnamaldehyde, 85% DMF 25% HCA) in the exact same manner. The mice were given an intraperitoneal injection of the thymidine analog 5-bromo-2'-deoxy-uridine (BrdU) five days following intial dose and approximately five hours prior to sacrifice. At sacrifice, the auricular lymph nodes were isolated, single-cell suspensions of lymph node cells (LNC) were generated by flow cytometry for BrdU incorporation and the total number of LNC. The amount of proliferating (#BrdU+) LNC was determined as a measure of the proliferative response of the local lymph node. The Simulation Index (SI) was calculated by dividing the proliferative response (#BrdU + LNC) of each test article treated animal by the mean proliferative response of the Vehicle Control group. The mean SI ± SD was calculated for each group from the individual animal data. Test article groups that yielded ≥3 were determined as sensitising substances.
Summary: All animals survived the in-life phase of the study and were observed to be normal. Body weight changes were normal. Ear thickness measurements and individual animal obervations indicated that the test article treatment did not result in dermal irritation. The SI of the Positive Control, 25% HCA was 5.4. The SI for the test article at 25% (w/v) was 0.8.
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