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EC number: 235-518-8 | CAS number: 12262-26-9 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53450.
The results of the optical density (OD) measured at 570 nm of each replicate and the calculated % viability of the cells is presented below:
Table 1: OD values and viability percentages of the controls:
Optical Density (OD)
Negative Control:1x PBS
standard deviation (SD)
Positive Control:SDS (5 % aq.)
Table 2: OD values and viability percentages of the test item (including corrected values):
Relative Viability (%)
Leuco Sulfur Blue 13
Table 3: OD values of additional controls for MTT-interacting test item:
Negative control killed tissues:1x PBS
Test item treated killed tissues:Leuco Sulfur Blue 13
Table 4: OD values and NSC % of additional control:
Additional colour control
Non Specific Colour %(NSC %)
Leuco Sulfur Blue 13(test item treated tissueswithout MTT incubation)
An in vitro skin irritation assay (EpiSkin SM) has been performed according to OECD 439 to predict the skin irritation potential of the test item.
Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in 5 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid.
The test item has an intrinsic colour (bluish black), therefore two additional test item treated tissues were used for the non-specific OD evaluation.
The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement.
The test item is a possible MTT-reducer and has an intrinsic colour (bluish black). To avoid a possible double correction [TODTT (MTT and NSC)] for colour interference, a third control for non-specific colour in killed tissues (NSCkilled) was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference.
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
In this in vitro skin irritation test using the EPISKIN model, the test item did not significantly reduce cell viability in comparison to the negative control (mean viability: 100 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin.
The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test is considered to be not irritating to the skin and is therefore not classified (UN GHS No Category).
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