[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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2019

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

[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

EC Number:

824-263-3

Cas Number:

2196165-14-5

Molecular formula:

C28H21N9O7S2.xNa.yK.zLi

IUPAC Name:

[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

In vitro test system

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]

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI CD54 and RFI CD86

Cell viability:

<50%

Remarks on result:

other: not valid

Results 4

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

Results 5

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

Results 6

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%).

Applicant's summary and conclusion

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).