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EC number: 947-876-9 | CAS number: -
In an in vitro skin irritation assay (recontructed human epidermis) according to OECD 439, the test item was non-irritant to skin.
In an ex vivo assay using bovine cornea epithelium(BCOP) according to OECD guideline 437, the test item did not induce eye damage, but no final conclusion could be made on the eye irritation potetnial.
In a follow-up in vitro eye irritation test (RhCE) according to OECD guideline 492, a relative cell viability of 104% was determined, indicating no eye irritation potential. Thus, the test item is considered as non-irritant to the eye (UN GHS No Category).
OD values and viability percentages of the controls:
Optical Density (OD)
Negative Control:1x PBS
standard deviation (SD)
Positive Control:SDS (5 % aq.)
OD values and viability percentages of the test item:
Relative Viability (%)
OD values of additional controls for MTT-interacting test item:
Negative control killed tissues:1x PBS
Test item treated killed tissues:Black DB
OD values and NSC % of additional control:
Additional colour control
Non Specific Colour %(NSC %)
Black DB(test item treated tissueswithout MTT incubation)
An EpiSkinTMSM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. 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±1°C for 42 hours in an incubator with 5±1% CO2. The viability of each disk was assessed by incubating the tissues for 3 (±5min) hours with MTT solution at 37±1°C in 5±1% 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. The test item has an intrinsic colour (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 (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 show significantly reduced cell viability in comparison to the negative control (mean relative viability: 71 %). 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. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. 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 item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Results after 240 Minutes Treatment Time
Opacity value = Difference (t240-t0) of Opacity
Permeability at 490 nm (OD490)
Proposed in vitro Irritancy Score
Standard Deviation of in vitro Score
No prediction can be made
This in vitro study was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspension in saline of the test item Black DB, the positive, and the negative controls were applied to the different corneas and incubated for 240 minutes at 32± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas and opacity was measured again (t240). After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
For the negative control (saline) an increase of neither opacity nor permeability of the corneas could be observed (mean IVIS = 0.15). The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control. Therefore the acceptability criteria were fulfilled. The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 164.26) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The positive control did not met the acceptance criteria of the assay since the mean IVIS is higher than twofold standard deviation of the upper limit of historical established boundaries. However, the raw data show that the IVIS of one cornea out of three of the positive control revealed a higher value compared to the other two corneas which values are in an acceptable range. This single cornea can be declared as outlier. Therefore this deviation has no impact on the validity or the outcome of the study. The Test Item was tested as suspension. Relative to the negative control, the test item caused an increase of the corneal opacity. The calculated mean IVIS was 8.60 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not requiring classification for eye damaging potential (UN GHS Category 1). No prediction can be made if the test item is requiring classification for eye irritation (UN GHS Category 2) or if the test item is not requiring classification (UN GHS no Category).
Table 2: OD values and viability percentages of the controls
Negative Control:Sterile deionized water
Positive Control:Methyl acetate
Table 3: OD values and viability percentages of the test item (including corrected values)
Table 4: OD values of additional controls for MTT-interacting test item
Negative control killed tissues:Sterile deionized water
Test item treated killed tissues:
Table 5: OD values and NSC % of additional control
Non Specific Colour %(NSCliving %)
Remark: Δ% is the difference of viability between the two relating tissues
In an in vitro eye irritation assay (RhCE) according to OECD guideline 492, the eye irritating potential of the test item on three-dimensional RhCE tissue in the EpiOcular™ model was determined. Before treatment the tissues were pre-wetted with ca. 20μL of Ca++Mg++Free-DPBS and were incubated at standard culture conditions for 30 ± 2 minutes. Disks of EpiOcular™ (two units) were treated with 50 mg test item and incubated for 6 hours (± 15 min) at standard culture conditions (37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere). Exposure of test material was terminated by rinsing with Ca++Mg++Free-DPBS solution. After rinsing, the tissues were incubated for a 25 ± 2 minutes immersion incubation (Post-Soak) at room temperature. At the end of the Post-Soak immersion, test item treated tissues were incubated for 18 hours ± 15 minutes at standard culture conditions (Post-treatment Incubation). The viability of each disk was assessed via MTT assay and quantified spectrophotometrically. Sterile deionized water or methyl acetate treated tissues were used as negative and positive controls, respectively. 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). Therefore, a third control for non-specific colour in killed tissues (NSCkilled) was performed with two killed treated tissues to avoid a possible double correction [TODTT (MTT and NSC)] for colour interference.
Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative viability: 104 %). Therefore the test item was considered to be non-irritant to eye (UN GHS No Category).
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008 The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the test item is not classified for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.
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