[x Sodium, y Potassium, z Lithium, x+y+z =2] 4-amino-3-[[[(2,4-diaminophenyl)diazenyl]phenyl]diazenyl]-5-hydroxy-6-(phenyldiazenyl)naphthalene-2,7-disulfonate

Endpoint summary

Description of key information

The Skin Sensitization endpoint has been assessed using a weight of evidence approach, as indicated in Annex VII, 8.3.1 of the REACH Regulation.

-An in silico assessment on skin sensitisation potential of the substance was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox. Weight of evidence of this in silico assessment suggested that consistency in the skin sensitization alerts triggered for the query compound by Derek and Toxtree, and for simulated autoxidation and skin metabolites by OECD QSAR Toolbox profiler might indicate the likelihood of skin sensitizing property for the substance.

-A positive result was obtained in a study using the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that teh substance is likely to be a skin sensitizer.

-An in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of DIRECT BLACK RBK dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of DIRECT BLACK RBK was previously calculated from two cytotoxicity tests. The test item activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

The substance EC number 824-263-3 , CAS number 2196165-14-5 is predicted to be sensitizing to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE : QSAR Toolbox and others

2. MODEL (incl. version number) : TOPKAT 4.5, CAESAR (VEGA 1.1.4), Derek 6.0.1, Toxtree 2.6.13.

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL : NC1=CC=C(\N=N\C2=CC=C(C=C2)\N=N\C2=C(C=C3C=C(C(\N=N\C4=CC=CC=C4)=C(O)C3=C2N)S([O-])(=O)=O)S([O-])(=O)=O)C(N)=C1.[Na+].[Na+].[K+].[K+].[Li+].[Li+]

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See report with explanations for each model

5. APPLICABILITY DOMAIN
See report with explanations for each model

6. ADEQUACY OF THE RESULT
Only with the results of the in silico assessement it is not possible to conclude on classification and labelling.

Principles of method if other than guideline:

QSAR has been applied

Key result

Parameter:

other: QSAR prediction

Remarks on result:

other: likelihood of skin sensitizing property for the substance

Interpretation of results:

study cannot be used for classification

Conclusions:

Taking into account the consistency in the skin sensitization alerts triggered for the query compound by Derek and Toxtree, and for simulated autoxidation and skin metabolites by the OECD QSAR Toolbox profiler, there is a likelihood that the substance could be a skin sensitizer.

Executive summary:

According to Annex VII (Regulation (EC) No 1907/2006), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step. Due to the complexity of the skin sensitisation endpoint, a combination of alternative test methods (e.g. in silico, in chemico and in vitro) in a weight of evidence approach needs to be considered to increase confidence in the final assessment of skin sensitisation (ECHA Guidance, Chapter R.7a, 2017).

The present in silico assessment was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox.

In the Appraisal of (Q)SAR Modelling, result of each computational tool was discussed if applicable including statistical performance, mode of action, eventual metabolism and reliability. The results were summarized in the appraisal section table. For each tool, the QSAR Prediction Reporting Format (QPRF) section, if any, and the respective software printout section were provided.

Weight of evidence of this in silico assessment suggested that consistency in the skin sensitization alerts triggered for the query compound by Derek and Toxtree, and for simulated autoxidation and skin metabolites by OECD QSAR Toolbox profiler might indicate the likelihood of skin sensitizing property for the substance.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)

Version / remarks:

October 2017

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Details of test system:

THP-1 cell line [442E]

Details on the study design:

TEST ITEM PREPARATION
On the day of the experiment (prior to start) DIRECT BLACK RBK was dissolved in culture
medium.
As tested by a solubility test, 5000 µg/mL in culture medium (the OECD 442E guideline
recommended maximal to be tested test item concentration) was used as highest test item
concentration in the cytotoxicity tests. Due to a technical error in the preparation of the test
item stock solution, 4910 µg/mL in culture medium was used as highest test item
concentration in the first cytotoxicity test.
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were
analysed. For this, dilutions were prepared by 1:2 serial dilutions.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.
THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1
cells also show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS
GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 106 cells/mL) allowing the
repeated use of the same cell culture batch in experiments. Therefore, the parameters of the
experiments remain similar, because of the reproducible characteristics of the cells. Thawed
stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice
weekly. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is
incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to
two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 11, 19 and 24 in the cytotoxicity tests and
25, 26, 28, 21, 26 and 22 in the h-CLAT for runs 1 to 6, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented
with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium
pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin)
is used to culture the cells during the assay. Medium with supplements has to be stored at
2 - 8 °C and used within one month. The culture medium has to be warmed to room
temperature just before use.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity or main experiment (h-CLAT) directly before the treatment of
the cells, a volume of 500 µL with a cell density of 1.8 - 2 × 106 THP-1 cells/mL was seeded
in each corresponding well of a 24-well flat bottom plate.

Experimental Design and Procedures of the Cytotoxicity Test
Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined
with three cytotoxicity tests, but only the results of the first and the third test were used for
the calculation of the mean CV75. The tests were performed on different days. The test item
was prepared separately for each run.
-Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each volume (500 µL) of the dilutions of the test item and culture medium was added to the
cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested
in one replicate for each cytotoxicity test. At the end of the incubation period, the cell
cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, culture medium and solvent control were prepared for the
7-AAD staining.
- Staining of the Cells
Each test item-treated and not test item treated cells were collected in sample tubes
centrifuged (approx. 250 × g, 5 min), washed twice (2 - 8 °C) with 2 mL FACS buffer (PBS
with 0.1% (w/v) BSA) and re-suspended in a final volume of 2 mL/tube FACS buffer. At
least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added
in each sample tube.
-Flow Cytometry Acquisition (Cytotoxicity Test)
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was
calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The
7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD
fluorescence signal.
Preparation of the acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of the FL-3 channel
The voltage of FSC and SSC was set to appropriate levels. FSC and SSC are not needed for
the analysis, but the FSC/SSC plot should be checked to make sure that a single population
appears without contamination or excessive debris. The FL-3 voltage was set and compensate
to appropriate position (FACSCalibur, Becton Dickinson GmbH, software FACSComp 6.0).
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of
10,000 living cells were analysed.
The maintenance of the flow cytometer was in accordance with the manufacturer’s
instructions. The process of washing was conducted very carefully since insoluble chemicals
could flow in the flow line.
-Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) or is calculated
according to the following equation:
Cell Viability [%] = ( Number of living cells /Number of acquired cells )× 100

The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is
calculated by log-linear interpolation using the following equation:
Log CV75 = (75 −𝑐) × 𝐿og (𝑏) − (75 −𝑎) × 𝐿og (𝑑) / (𝑎 −𝑐)

Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively

-Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
• The cell viability of the medium control should be more than 90%.

-Calculation of the Test Doses for the Main Experiment (h-CLAT)
The mean of two CV75 values (first and third cytotoxicity experiment) was used to determine
the dose-range for the main experiment (h-CLAT).
Eight final concentrations (µg/mL) were used for the test item in the main experiment
(h-CLAT). The highest concentration used was 1.2 × mean CV75 and seven further
concentrations of the test item were prepared by serial 1:1.2 dilution.
Due to strong cytotoxicity (cell viability < 50%) observed in all test item treated cells of the
first h-CLAT run, the test item concentrations were adjusted for the following h-CLAT runs.

Experimental Design and Procedures of h-CLAT
The test item was tested in six independent runs. The tests were performed on different days.
The test item was prepared separately for each run.
- Treatment of the Cells
Each volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO
control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At
the end of the incubation period, the cell cultures were microscopically evaluated for
morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was
prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54
antibody or mouse IgG1).
- Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and
equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5
min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA).
Thereafter, the cells were centrifuged, re-suspended and blocked with 600 µL of blocking
solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and
the cell pellets were re-suspended in 100 µL FACS buffer. The cells were stained with FITC-
labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis
procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected
for 30 ± 5 min. at 2 - 8 °C (on ice).
-Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS
buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes
before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

-Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was
calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using
the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal
detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set
for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC
are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single
population appears without contamination or excessive debris. The FL-1 and FL-3 voltage
were set and compensate to appropriate position. The FL-1 voltage was set using the FITC
labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set
in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo
Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson
GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining
with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between
the peak of the negative fraction and the positive fraction in the FL-3 histogram using
DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the
subsequent analyses. For each control and all test item concentrations, the cell viability was
recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the
CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability
evaluation.
Since the peak in the FL-3 histogram showed a shift to the right side (due to possible
interference of the test item) and no clear cytotoxicity could be observed in the 2D plot
consisting of FSC (Forward Scatter) versus SSC (Side Scatter), the R1-gate was shifted to the
right side to obtain a reliable result for the cell viability of the test item treated cells. The
R1-gate in the h-CLAT runs was set comparable to the R1-gate set in the dose range finder
tests (cytotoxicity tests).
The maintenance of the flow cytometer was in accordance with the manufacturer’s
instructions. The process of washing was conducted very carefully since insoluble chemicals
could flow into the flow line.
Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC
(side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence
intensity (MFI) of viable cells and viability for each sample were used for analysis. The other
tubes were acquired without changing the settings of the cytometer. The MFI was recorded
for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded
from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of
cytoplasmic structures that could be generated due to cell membrane destruction). for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded
from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of
cytoplasmic structures that could be generated due to cell membrane destruction).

Vehicle / solvent control:

cell culture medium

Negative control:

other: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD86 RFI value of the positive control (2.0 µg/mL DNCB) in the third h-CLAT run did not exceed the positive
criterion (CD86 ≥ 150%) and the CD54 RFI value of the positive control (2.0 µg/mL DNCB)
in the first, third, fourth and sixth h-CLAT run did not exceed the positive criterion (CD54 ≥
200%). However, this is considered to be acceptable since the CD86 and CD54 RFI value of
the positive control (3.0 µg/mL DNCB) in the first, third, fourth and sixth h-CLAT run
exceeded the positive criteria.

Group:

test chemical

Run / experiment:

other: run/experiment:4

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

>83%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

>72%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

<50%

Remarks on result:

other: not valid

Key result

Group:

test chemical

Run / experiment:

other: run/experiment: 6

Parameter:

RFI CD54>150 [442E]

Value:

215.2 %

Cell viability:

73.89%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: run /expeiment: 5

Parameter:

RFI CD54>150 [442E]

Value:

263.4 %

Cell viability:

88.56%

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

RFI CD54>150 [442E]

Value:

245.8 %

Cell viability:

68.

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium
control of both CD86 and CD54 should not exceed the positive criteria (CD86
≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86
and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the
positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should
be > 50% in at least one concentration of the two tested positive control
concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested
concentratins in each run.

ACCEPTANCE CRITERIA OF THE h-CLAT
Acceptance Criteria of the first h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 94.00%
DMSO = 93.69%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 154.0%#
2 µg/mL DNCB (CD 86): 579.8%
3 µg/mL DNCB (CD 54): 261.5%
3 µg/mL DNCB (CD 86): 538.0%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 114.1%
CD86: 93.7%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 244.5%
Medium CD 86: 331.4%
DMSO CD 54: 265.0%
DMSO CD 86: 316.8%
# CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Acceptance Criteria of the second h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 96.51%
DMSO = 96.39%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 360.7%
2 µg/mL DNCB (CD 86): 384.8%
3 µg/mL DNCB (CD 54): 519.7%
3 µg/mL DNCB (CD 86): 408.9%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 66.3%
CD86: 83.1%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 138.8%
Medium CD 86: 237.1%
DMSO CD 54: 125.7%
DMSO CD 86: 213.9%

Acceptance Criteria of the third h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 96.11%
DMSO = 95.96%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 163.6%#
2 µg/mL DNCB (CD 86): 132.4%#
3 µg/mL DNCB (CD 54): 329.1%
3 µg/mL DNCB (CD 86): 175.9%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 114.6%
CD86: 112.8%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 140.9%
Medium CD 86: 263.0%
DMSO CD 54: 145.6%
DMSO CD 86: 279.3%
# The CD86 and CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Acceptance Criteria of the fourth h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 96.69%
DMSO = 97.35%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 89.2%#
2 µg/mL DNCB (CD 86): 255.2%
3 µg/mL DNCB (CD 54): 305.4%
3 µg/mL DNCB (CD 86): 332.4%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 94.9%
CD86: 84.8%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 153.2%
Medium CD 86: 264.1%
DMSO CD 54: 147.6%
DMSO CD 86: 231.3%
# CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Acceptance Criteria of the fifth h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 93.87%
DMSO = 93.15%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2 µg/mL DNCB (CD 54): 350.0%
2 µg/mL DNCB (CD 86): 565.0%
3 µg/mL DNCB (CD 54): 245.5%
3 µg/mL DNCB (CD 86): 500.0%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 130.7%
CD86: 85.4%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 147.9%
Medium CD 86: 291.9%
DMSO CD 54: 172.5%
DMSO CD 86: 290.1%

Acceptance Criteria of the sixth h-CLAT run for the Test Item DIRECT BLACK RBK
Cell viability of medium control and DMSO control should be more than 90%.
Medium = 93.24%
DMSO = 92.91%
In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the
positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
2.7 µg/mL DNCB (CD 54): 181.6%#
2.7 µg/mL DNCB (CD 86): 292.9%
4 µg/mL DNCB (CD 54): 287.7%
4 µg/mL DNCB (CD 86): 340.6%
In the DMSO control, RFI values compared to the medium control of both CD54 and
CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 107.6%
CD86: 99.5%
For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control
should be > 105%.
Medium CD 54: 260.2%
Medium CD 86: 422.0%
DMSO CD 54: 282.8%
DMSO CD 86: 439.7%
# CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

Interpretation of results:

other: the study is part of a WoE assessment on skin sensitisation

Conclusions:

The test item DIRECT BLACK RBK with a log Pow of -1.84 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

An in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of DIRECT BLACK RBK dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of DIRECT BLACK RBK was previously calculated from two cytotoxicity tests.
Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2455 µg/mL up to the highest tested concentration (4910 µg/mL) in the first cytotoxicity test, starting with the concentration of 625 µg/mL up to the highest tested concentration (5000 µg/mL) in the second cytotoxicity test and starting with the concentration of 1250 µg/mL) up to the highest tested concentration (5000 µg/mL) in the third cytotoxicity test (threshold of cytotoxicity: < 75%). Due to a calculation error in the preparation of the test item stock solution for the first cytotoxicity test, observed at the end of the study, the calculated CV75 value was 1835.66 µg/mL instead of 1802.61 µg/mL.
Subsequently, the mean CV75 calculated with the CV75 of the first and third cytotoxicity test was 1411.01 µg/mL instead of 1394.49 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):
472, 567, 680, 816, 980, 1176, 1411 and 1693 µg/mL in the first main experiment, 132, 158, 190, 228, 273, 328, 393 and 472 µg/mL in the second to sixth main experiments.

The test item with a log Pow of -1.84 was tested in 6 independent runs. Since the peak in the FL-3 histogram showed a shift to the right side (due to possible interference of the test item) and no clear cytotoxicity could be observed in the 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter), the R1 gate of all six h-CLAT runs was shifted to the right side to obtain a reliable result for the cell viability of the test item treated cells.

The first h-CLAT run was not valid, since all test item treated concentrations showed a cell viability < 50%. Therefore, the test item concentrations were adjusted for the following h-CLAT runs. In the second h-CLAT run the RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively, at any test item concentration. In the third h-CLAT run the RFI of CD54 was equal or greater than 200% at the highest tested test item concentration.

The first to third h-CLAT run were conducted with THP1 cells which were slightly older than two month, therefore additional h-CLAT runs were performed and used for the evaluation.

The RFI of CD86 and CD54 in the fourth h-CLAT run was not equal or greater than 150% and 200%, respectively, at any test item concentration. In addition, the RFI of CD54 was equal or greater than 200% in at least one concentration of the fifth and sixth h-CLAT run. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT. However, a color interference by the intrinsic color of the test item cannot be fully excluded.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD86 RFI value of the positive control (2.0 µg/mL DNCB) in the third h-CLAT run did not exceed the positive criterion (CD86 ≥ 150%) and the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first, third, fourth and sixth h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD86 and CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first, third, fourth and sixth h-CLAT run exceeded the positive criteria.

In conclusion, the test item DIRECT BLACK RBK with a log Pow of -1.84 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX) Protocol: KeratinoSens™, 2009/vers.6.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

1. Cell culture: The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan. The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10270) and 5.5 mL Geneticin (Gibco 10131).
1.1. Cell Culture from Frozen Stocks: Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in a total of 10 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.
1.2. Cell Passage: Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks may have been pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages.
1.3. Preparation of Test Cell Cultures: The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except well H12 of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. Well H12 of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.
2. Positive control: Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using a formula where the purity of the chemical in % is used. The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.
Results from the positive control were shared with other studies performed in the same assay.
3. Test Item Solubility: Prior to commencing testing, the solubility of the test item, Direct Black RBK, in DMSO was assessed.
The test item, Direct Black RBK, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.
4. Test Item Solubility: Prior to commencing testing, the solubility of the test item, Direct Black RBK, in DMSO was assessed.
The test item, Direct Black RBK, was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.
5. Preparation of the Test Item: A stock solution of the test item, Direct Black RBK, was prepared by weighing between 20 - 40 mg into a tared glass container and diluting to 200 mM in DMSO using the same formula as for the positive control. As the test item had a range for molecular weight, the mid-point (705 g/mol) was used for calculating the volume of DMSO required to dilute the test item to 200 mM.
6. Test procedure:
6.1. Preparation of the 100x Solvent Plate: A 100x solvent plate was set up by adding 100 µL of DMSO to all wells of a 96 well plate except wells in column 12 and well H11 of the plate. 200 µL of the stock solution of the test item, Direct Black RBK, was added to one well in column 12. The test item was serially diluted across the plate by transferring 100 µL from column 12 to column 11 and then mixed by repeat pipetting (at least 3 times) and then 100 µL was transferred from column 11 to
column 10 and so forth across the plate.
200 µL of the 6.4 mM stock solution of cinnamic aldehyde was added to well H11 and serially diluted from column 11 to column 7.
6.2. Preparation of the Dilution Plate: The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.
6.3. Treatment of Cultured Plates: Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring
cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.
6.4 Cell viability measurement: A kit (Molecular Probes Vybrant MTT kit V13154) was used to determine cell viability. 1 vial from the kit was reconstituted by adding 1 mL of sterile PBS (Gibco 10010) and vortexed mixed until dissolved to give 5 mg/mL MTT in DPBS. After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution was added to each well of the 96-well plate. The plate was incubated at 37 ± 2C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes.
The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.
6.5. Luciferase measurement: Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for test 1. Frozen reconstituted reagent was used for both tests
and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Positive control results:

Positive Control (cinnamic aldehyde)
Results- test 1: Imax 3.64; EC1.5 16.09;
Results - test 2: Imax 4.92; EC1.5 8.88

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

2.99

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

3.79

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Value:

2.11

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Value:

0.98

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
Test1: As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 50% and approximately 30% at concentrations 125 – 500 µM. The cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.

Test 2: As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 60% and approximately 50% at concentrations 125 – 250 µM. The confluency of the cells treated with a concentration of 500 µM of Direct Black RBK was approximately 30% and the cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
yes, The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 14.8% and 11.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.
- Acceptance criteria met for positive control:
yes. Luciferase activity induction with the positive control is statistically significant above the threshold of 1.5 in at least one of the test concentrations. Average induction in the three replicates of positive control at 64 µM is between 2 and 8. EC1.5 of positive control is within two standard deviations of the historical mean of the testing facility (4.94 – 50.00). The aveerage coefficient of variatton of the luminescence reading for the negative solvent control (DMSO) was 14.8% and 11.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Direct Black RBK results test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.16	1.42	2.47	2.99	2.45	2.31	1.02	0.89	0.55	0.24	0.00	0.00
Statistically significant	No	No	Yes	Yes	Yes	Yes	No	No	No	No	No	No
Viability (%)	90.49	87.22	78.01	81.53	73.58	77.68	71.82*	75.25*	82.11*	70.23*	52.57*	76.84*
Imax	2.99
EC1.5	2.11
IC30	506.48
IC50	N/A

* As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 50% and approximately 30% at concentrations 125 – 500 µM. The cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with  concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.

 

Direct Black results test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.81	1.90	2.55	3.17	3.68	3.79	3.09	1.64	1.42	0.61	0.30	0.07
Statistically significant	yes	yes	Yes	Yes	Yes	Yes	yes	yes	No	No	No	No
Viability (%)	116.03	107.74	102.03	98.91	96.32	89.09	120.59*	115.65*	112.15*	112.15*	112.15*	123.64*
Imax	3.79
EC1.5	<0.98
IC30	N/A
IC50	N/A

* As Direct Black RBK gave a coloured solution the cells were visually assessed for cytotoxicity prior to the addition of MTT as the colour of the test item interfered with MTT. The confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 60% and approximately 50% at concentrations 125 – 250 µM. The confluency of the cells treated with a concentration of 500 µM of Direct Black RBK was approximately 30% and the cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. The DMSO control produced approximately 70% confluency.

 

Foorr both Direct Black RBK tests (1 and 2):

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	Yes
Is the EC1.5value <1000µM	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

Interpretation of results:

other: the study is part of a WoE assessment on skin sensitisation

Conclusions:

It was concluded that Direct Black RBK gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that Direct Black RBK is a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Direct Black RBK, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imax for Direct Black RBK was 2.99 in test 1 and 3.79 in test 2. The Imax for both tests was >1.5 fold and statistically significant as compared to the DMSO control.  

The cellular viability was 52.57% at 1000 µM in test 1 and the IC30 was 506.48 µM. In test 2 the cellular viability did not fall below 89.09% and therefore the IC30 and IC50 could not be calculated. However, the cellular viability results were unreliable as Direct Black RBK gave a coloured solution which interfered with MTT and therefore the cells were visually assessed for cytotoxicity prior to the addition of MTT. In test 1, the confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 50% and approximately 30% at concentrations 125 – 500 µM. The cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent. In test 2 the confluency of the cells treated with a concentration of 62.5 µM of Direct Black RBK was approximately 60% and approximately 50% at concentrations 125 – 250 µM. The confluency of the cells treated with a concentration of 500 µM of Direct Black RBK was approximately 30% and the cells treated with the concentrations 1000 µM and 2000 µM of Direct Black RBK were dead. The cells treated with concentrations of the Direct Black RBK below 62.5 µM were approximately 70% confluent.  In both tests the DMSO control produced approximately 70% confluency.

The EC1.5 for Direct Black RBK was 2.11 µM and <0.98 µM for tests 1 and 2, respectively. Graphs for Direct Black RBK showed an overall dose-response for luciferase induction.

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.

The EC1.5 values of the positive control, cinnamic aldehyde were 16.09 μM and 8.88 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory.  

The average induction in the three replicates for cinnamic aldehyde at 64 µM were 3.64 and 4.92 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 14.8% and 11.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

It was concluded that Direct Black RBK gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

No conclusion on skin sensitisation category can be made, the substance is classified as Skin Sens. 1.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The predicted behavior of the substance as skin sensitizer was assessed in an weight-of-evidence approach by in vitro / in silico methods, taking into account the key events of skin sensitisation. The substance is predicted to be a skin sensitizer. As the classificacion in category 1A or 1B can not be derived, the substance is classified in category 1.