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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Remarks:
h-CLAT
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2019 to 21 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 442E: Skin Sensitisation: Human Cell Line Activiation Test (h-CLAT)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%

In vitro test system

Details on study design:
Experimental Design and Procedures of h-CLAT

The test item was tested in two independent runs. The tests were performed on different days. The test item was prepared separately for each run.

5.6.1 Treatment of the Cells
For the test item exposure the highest soluble test item concentration of the cytotoxicity test (without precipitations) was used instead of 1.2 × CV75, since no mean CV75 could be determined. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium.
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).

5.6.2 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).

5.6.3 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.

5.6.4 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 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.
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).

Results and discussion

Positive control results:
DMSO (Dimethyl sulfoxide, - Solvent Control for the Positive Control (h-CLAT)
DNCB (2,4-dinitrochlorobenzene - Positive Control (h-CLAT)

In vitro / in chemico

Results
Key result
Parameter:
other:
Remarks:
Dendritic cell activation potential
Run / experiment:
h-CLAT
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Acceptance Criteria of the first h-CLAT run for the Test Item Acid Orange 94 Refined

Cell viability of medium control and DMSO control should be more than 90%.
Medium = 94.84%
DMSO = 94.44%

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%.
3 μg/mL DNCB (CD 54): 208.5%
3 μg/mL DNCB (CD 86): 398.5%
4 μg/mL DNCB (CD 54): 289.8%
4 μg/mL DNCB (CD 86): 331.7%

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: 96.7%
CD86: 94.0%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%. Medium CD 54: 191.9%
Medium CD 86: 276.3%
DMSO CD 54: 183.8% DMSO CD 86: 256.2%

Any other information on results incl. tables

This 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 Acid Orange 94 Refined dissolved in 0.2% (v/v) DMSO 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 Acid Orange 94 Refined was previously determined by two cytotoxicity tests.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (334 μg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, the highest soluble test item concentration 334 μg/mL was used as highest test item concentration for the h-CLAT runs.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

93, 112, 134, 161, 193, 232, 278 and 334 μg/mL

The test item with a log Pow of -1.79 was tested in 2 independent runs. The RFI of CD54 was greater than 200% in all concentrations of both independent runs. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT.

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%. For details see Annex 1 and 2. All acceptance criteria met for the cytotoxicity test and the h-CLAT method.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test item Acid Orange 94 Refined with a log Pow of -1.79 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:

This 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 Acid Orange 94 Refined dissolved in 0.2% (v/v) DMSO 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 Acid Orange 94 Refined was previously determined by two cytotoxicity tests.

This study was performed in accordance to OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018, in accordance with GLP.

The test item Acid Orange 94 Refined with a log Pow of -1.79 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).