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EC number: 274-550-7 | CAS number: 70304-38-0
Direct MTT reduction: functional check in tubes
25 mg ofthe test substance was addedto 1.0 mL of MTT medium. Suspension was incubated for 1 hour at culture conditions. After incubation, the medium was coloured red-brown. The test substance does not reduce MTT directly.
The test substance is soluble in water for injection. OD570 of solution in water for injection was > 3 what is > 0.08.
The test substance is not soluble in isopropyl alcohol. Average OD570 value from 2 wells was 0.005 what is < 0.08.
It means that the test substance will go well washed from the tissue and any residue is not dissolved in isopropyl alcohol. On the basis of results obtained, it was decided do not use concurrent colorant control in the MTT test.
OD570values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities
The test item, Direct Brown 103, was assayed for the in vitro skin irritation in human epidermal model EpiDermTM. The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method test (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT.
In the preliminary experiments neither colour interference with the endpoint nor direct MTT reduction were found.
After pre-incubation of tissues, 25 mg of the test substance was placed directly atop to the previously moistened tissue and it was spread on the entire tissue surface. The length of exposure was 60 minutes. Three tissues were used for the test substance and for positive and negative controls.
After removal of the test substance, tissues were post-incubated for approximately 42 hours. Three hours incubation with MTT and two hours extraction period with shaking followed then. Optical density (OD570) of isopropyl alcohol extracts was measured on a spectrophotometer. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.
Under the above-decribed experimental design, average viability of tissues treated by the test substance was 60.9 %, i.e. viability was > 50 %.
The effect of the test substance was negative in EpiDermTMmodel (tissues were not damaged).
According to the classification criteria, the test substance, Direct Brown 103, is considered to have no category in regard to skin irritation.
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