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Description of key information

Two endpoints (skin irritation) are available.

1) In Vitro EpiDermTM

The test was performed according to the OECD Test Guideline No. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method (2015) and Protocol for: In Vitro EpiDermTMSkin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT. GLP study, Klimish score 1.

Value: The effect of the test substance was positive in EpiDermTMmodel (tissues were damaged). The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1).

As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.

2) QSAR Toolbox prediction: Model name: BfR skin irritation/corrosion

Predicted value: not irritating or corrosive to skin

 

Two endpoints (eye irritation) are available.

1) BCOP

The test was performed according to the OECD Test Guideline No. 437, Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage, Adopted 26thJuly 2013.

The basic values for the IVIS calculation (opacity values) could not be measured because of dark black colouring of corneas which were applied by the test substance. The In Vitro Irritancy Score (IVIS) for test substance, Acid Black 227, could not be calculated. For this reason, the classification was not performed. The results of permeability showed damage of corneas after exposure by the test substance.

2) In vitro EpiOcularTM

The test was performed according to theOECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcularTMEye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).GLP study, Klimish score 1.

Value: The effect of the test substance was positive in EpiOcularTMmodel (tissues were damaged). The test substance is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

3) QSAR Toolbox prediction: Model name: BfR eye irritation/corrosion

Predicted value: not irritating or corrosive to eye

Based on the available results of the test substance Acid Black 227 is classified as a skin irritant Category 2.

Based on the available results of the test substance Acid Black 227 is classified as an eye irritant Category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17.01. - 31.03.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
yes
Remarks:
See any other information...
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
other: normal human-derived epidermal keratinocytes
Cell source:
skin obtained from plastic surgery from multiple donors
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE- Model used: model EpiDermTM- Tissue: The reconstructed human epidermal model EpiDerm™ (EPI-200 ver. 2.0, MatTek, Bratislava, Slovakia); Lot No. 25802 kit C)- Date of initiation of testing: 30.6.2016The EpiDerm™ tissues (surface 0.63 cm²) are cultured on specially prepared cell culture inserts and shipped as kits, containing tissues on shipping agarose together with the necessary amount of culture media.TEMPERATURE USED FOR TEST SYSTEM- Temperature used during treatment / exposure: 37±1°C
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL- Amount(s) applied: 25 gNEGATIVE CONTROLPBS MatTek 101816ZSA, exp. 18/10/2017 - Amount(s) applied: 25 µLPOSITIVE CONTROL5 % SDS (sodium dodecyl sulphate), MatTek, Lot No. 031617MGKA, exp. 16/03/2018
Duration of treatment / exposure:
60 min.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
in the first experiment
Value:
36.9
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
in the second experiment, after correction
Value:
25.8
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
Direct MTT reduction - functional check in tubes The test was not performed due to blue colour of the test substance. The next step was performed directly.

Table 1: The first experiment: OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

 

Treatment

OD570

 

 

 

 

Avg

SD

%NC

PBS (NC)

2.022

1.940

2.050

NT

NT

2.004

0.047

 

viability (%NC)

100.9

96.8

102.3

-

-

100.0

2.3

100.0 

394/16 (C4)

0.455

1.727

0.730

0.364

0.416

0.738

0.510

 

viability (%NC)

22.7

86.2

36.4

18.2

20.8

36.9

25.5

36.9 

5% SDS (PC)

0.044

0.051

0.050

NT

NT

0.048

0.003

 

viability (%NC)

2.2

2.5

2.5

-

-

2.4

0.2

2.4

Table 2: The second experiment: OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities

Treatment

OD570

 

 

Avg

SD

%NC

PBS (NC)

1.961

1.946

1.941

1.949

0.008

 

viability (%NC)

100.6

99.8

99.6

100.0

0.4

100.0

394/16 (C3)

0.282*

0.264

1.098

0.548

0.389

 

viability (%NC)

14.5

13.5

56.3

28.1

20.0

28.1

394/16 (C3_CC)

0.049*

0.042+

NT

0.046

0.004

 

viability (%NC)

2.5

2.2

 -

2.3

0.2

2.3

5% SDS (PC)

0.059

0.053

0.056

0.056

0.002

 

viability (%NC)

3.0

2.7

2.9

2.9

0.1

2.9

NC, PC

negative, positive control

C3, C4, 394/16

test substance 

avg

arithmetic average

SD

standard deviation calculated from individual % tissue viabilities

viability (%)

viability of single tissues compared with negative control

NT

not tested

*

damaged tissue (with lost of part of tissue)

+

damaged tissue (without lost of part of tissue)

Interpretation of results:
other: The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Acid Black 227 was 36.9 % and 25.8% of negative control average value i.e. viability was < 50 % in both cases. The effect of the test substance was positive in EpiDermTM model. The test substance, Acid Black 227, is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.
Executive summary:

The test substance, Acid Black 227, was assayed for in vitro skin irritation in the 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 (2015) and Protocol for: In Vitro EpiDermTM Skin Irritation Test For use with MatTek Corporation’s Reconstructed Human Epidermal Model EPI-200-SIT (see par. 1.4).

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 on previously moistened tissue and 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-described experimental design average viability of treated tissues was 36.9 % in the first and 25.8 % (after correction) in the second experiment, i.e. viability was < 50 %.

The effect of the test substance was positive in EpiDermTM model (tissues were damaged).

The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1). As the test substance was not tested for skin corrosion, it is not possible to decide between these two categories.

Endpoint:
skin irritation / corrosion, other
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Justification for type of information:
1. SOFTWAREQSAR Toolbox 3.4.0.172. MODELBfR skin irritation/corrosion3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL[Na+].O=N(=O)c8ccc(/N=N/c6c7ccccc7ccc6O[Cr]3Oc4ccc5ccccc5c4N=Nc1c(cc(c2ccccc12)S([O-])(=O)=O)O3)c(O)c8
Qualifier:
no guideline required
Principles of method if other than guideline:
BfR skin irritation/corrosion
Specific details on test material used for the study:
SMILE: [Na+].O=N(=O)c8ccc(/N=N/c6c7ccccc7ccc6O[Cr]3Oc4ccc5ccccc5c4N=Nc1c(cc(c2ccccc12)S([O-])(=O)=O)O3)c(O)c8
Interpretation of results:
study cannot be used for classification
Conclusions:
Toxicity of the target chemical (Not Irritating or Corrosive to skin) is predicted by QSAR "BfR skin irritation/corrosion".
Executive summary:

Predicted value: not irritating or corrosive to skin

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20.06. - 22.06.2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
Adopted: 28 July 2015
Deviations:
yes
Remarks:
The difference of viability between the positive control tissues of was > 18%. Average viability was 28.9% what is < 50%. The highest (outlying) viability is 45.2% what is < 50% as well.
GLP compliance:
yes (incl. certificate)
Details on test animals or tissues and environmental conditions:
The reconstructed human cornea-like epithelial model EpiOcular™ (OCL-200 ver. 2.0) comes from MatTek, Bratislava, SK. The RhCE tissues are reconstructed from primary human cells, which have been cultured for several days to form a stratified, highly differentiated squamous epithelium, morphologically similar to that found in the human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.
Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test substance (50 mg of substance/surface ratio 39.7 mg/cm2) is placed directly atop to the tissue moistened with 20 µL of PBS.
Duration of treatment / exposure:
6h
Duration of post- treatment incubation (in vitro):
after removal:post-soaked-25 min.post-incubation-18 hours
Number of animals or in vitro replicates:
two replicate tissues
Details on study design:
Temperature: 37±1°C3 tissues for the test substance3 tissues for every control2 tissues as colorant controlTest ProcedureOn the day of receipt, EpiOcularTM tissues are conditioned to release transport stress related compounds and debris by incubation in assay medium delivered by MatTek for test performance for 1 hour at standard culture conditions and, after media replacement, overnight (following 16-24 hours) also standard at culture conditions. After pre-incubations, tissues are wetted with 20 μl of PBS spread across entire tissue surface. After 30 minutes incubation tissues are topically exposed to the test chemical (50 mg per tissue) for 6 ± 0.25 hours. Four tissues (2 for MTT test 2 for colorant control) are used per test substance (TS) and two for the positive control (PC) and negative control (NC). At the end of the 6±0.25 hours treatment time, the test articles should be removed by extensively rinsing the tissues with PBS brought to room temperature. After rinsing, tissues are immediately transferred to and immersed in 5 ml of previously warmed assay medium (room temperature) in a pre-labelled 12-well plate for a 25 ± 2 minute immersion incubation (post-soak) at room temperature. This incubation in assay medium is intended to remove any test article absorbed into the tissue. At the end of the post-soak immersion, each insert is removed from the assay medium, the medium is decanted off the tissue, and the insert is blotted on absorbent material and transferred to the appropriate well of the pre-labelled 6-well plate containing 1 ml of warm assay medium. The tissues should be incubated for 18±0.25 hours at standard culture conditions (post-treatment incubation). At the end of the post-treatment incubation, each insert is removed from the 6-well plate and gently blotted on absorbent material. Two tissues are placed into the 24-well plate containing 0.3 ml of MTT solution and incubated for 180±10 minutes at standard culture conditions. Two tissues undergo the same procedure with medium instead MTT solution (colorant control).In the end of staining (incubation with medium) the bottom of all inserts is blotted on absorbent material, and inserts are then transferred to a pre-labelled 6-well plate containing 2 ml of isopropyl alcohol in each well so that no isopropyl alcohol is flowing into the insert. The plates are sealed with parafilm, and are either stored overnight at 2-8°C in the dark or immediately extracted. To extract the MTT, the plates are placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. After extraction extracts are collected, mixed and two 200 μl aliquots are transferred to the appropriate wells of a pre-labeled 96-well plate for OD570 reading.
Irritation parameter:
other: viability of treated tissues
Run / experiment:
1
Value:
-2.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Table No. 1: OD570 values obtained at the MTT test, their averages, standard deviations (%) and relative viabilities.

Treatment

OD570 (tissue 1,2,3)

Avg

SD

%NC

water (NC)

1.906

1.919

1.967

1.931

0.026

 

viability (%NC)

98.7

99.4

101.9

100.0

1.4

100.0

394/16 (C5)

0.509

0.498

0.644

0.550

0.066

 

viability (%NC)

26.4

25.8

33.4

28.5

12.0

28.5

394/16 (C5_CC)

0.654

0.551

-

0.603

0.052

 

viability (%NC)

33.9

28.5

31.2

8.6

31.2

99% MA (PC)

0.872

0.442

0.359

0.558

0.225

 

viability (%NC)

45.2

22.9

18.6

28.9

40.3

28.9

Interpretation of results:
Category 2 (irritating to eyes) based on GHS criteria
Conclusions:
Under the above-described experimental design, average viability of tissues treated by the test substance Acid Black 227 was -2.7 % (after correction) of negative control average value. The effect of the test substance was positive in EpiOcularTM model (tissues were damaged). According to the classification criteria given in chapter 4.5., the test substance, Acid Black 227, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).
Executive summary:

The test substance, Acid Black 227, was assayed for the in vitro eye irritation in human epidermal model EpiOcularTM. The test was performed according to the OECD Test Guideline No. 492: Reconstructed human Cornea-like Epithelium (RhCE) test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Details of the procedure are given in Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals (MatTek 06/29/2015).

After pre-incubation and wetting of tissues, 50 mg of the test substance was placed directly atop to the tissue and it was spread on the entire tissue surface. Length of exposition was 6 hours at 37±1°C in humidified CO2 incubator (5±1% CO2). Three tissues were used for the test substance and every control. Two tissues more were used as colorant control to correction of possible colour interference, which was undergo the entire testing procedure excepting of incubation with MTT medium.

After removal of the test substance, tissues were post-soaked in medium for approximately 25 minutes and post-incubated for about 18 hours at culture conditions. 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 plate reader. Relative cell viability was calculated for each tissue as % of the mean viability of the negative control tissues.

Test for colour interference revealed direct reduction caused with the test substance. “Viability” of tissues treated by the test substance was corrected.

Test for direct reduction was performed as a part of another study. Direct reduction was not discernible. With regard to the result of MTT, test assay in frozen tissues was not performed.

Under the above-described experimental design average viability of treated tissues was -2.7 % (after correction) i.e. viability was ≤ 60 %.

The effect of the test substance was positive in EpiOcularTM model (tissues were damaged).

According to the classification criteria given in chapter 4.5., the test substance, Acid Black 227, is identified as substance potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1). Further testing with other test methods will be required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification