Disodium 2,5-dichloro-4-[4-[[5-[[(dodecyloxy)carbonyl]amino]-2-sulphonatophenyl]azo]-4,5-dihydro-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonate

Endpoint summary

Description of key information

Skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 03rd to September 18th, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 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:

9 October 2017

Deviations:

yes

Remarks:

not impacting the reliability of test results

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

CELL USED
- Cells: human leukemia cell line THP-1 .
- Source: purchased from ATCC, #TIB-202.
- Characteristics: THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.
- Storage: stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of testing facility allowing the repeated use of the same cell culture batch in experiments.
- Sub-culturing: thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly.
- Cell density: the cell density should not exceed 1 × 10^6 cells/ml.
- Incubation: the THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere.
- 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.

TEST FORMULATION
- Vehicle: DMSO
- Preparation of substance: on the day of the experiment (prior to start) test item was dissolved in DMSO (0.2 %).

CONTROLS: concurrent controls were used.
- Medium control: culture medium
- Solvent control for the Test Item: DMSO (final concentration for XTT cytotoxicity 0.2 - 0.5 % and for h-CLAT 0.2 %).
- Positive Control (h-CLAT): DNCB (2,4-dinitrochlorobenzene) final concentration: 2 and 3 µg/ml), in DMSO.

TEST CONCENTRATIONS
14, 17, 20, 24, 29, 35, 42 and 50 µg/ml - first experiment
24, 29, 35, 41, 50, 60, 72 and 86 µg/ml - second experiment

- Determination of test concentrations: two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment.
- Choice for the highest test concentration: the highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

REPLICATES: 4 independent runs.

CELLS TREATMENT
- Preparation and Seeding of THP-1 Cells: 0.9 - 1 × 10^6 cells/well in a volume of 500 μl were seeded in a 24-well plate before the treatment. The treated THP-1 cells were incubated for 24 ± 0.5 hours.
- Observation: at the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
- Replicates: ach 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).
- Storage during staining analysis: all solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
- Incubation: 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: 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.

CELL VIABILITY
The cell viability was additionally detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD) in both h-CLAT runs.

ACCEPTANCE CRITERIA
The following acceptance criteria should be met when using the h-CLAT method:
- cell viability of medium control is adjusted to 100 % and the cell viability of the DMSO control should be more than 90 % in comparison to the medium control.
- cell viability of medium control and DMSO control should be more than 90% (if the cell viability was determined by setting an R1-Gate).
- 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 concentrations in each run.

Negative results are acceptable only for test items exhibiting a cell viability of < 90 % at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90 % the negative result should be discarded. In such case, it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/ml in saline (or medium or other solvents/vehicles), 1000 μg/ml in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90 %.

DOSE-RANGE FINDING - XTT TEST
- Test design: XTT test is based on the cleavage of the yellow tetrazolium salt XTT to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria.
- Preparation and seeding of THP-1 cells: on the day of the cytotoxicity experiment, directly before the application of the test item, solvent and medium control, a volume of 100 μl with a cell density of 0.9 - 1 × 10^6 THP-1 cells/ml was seeded in each well of a 96-well flat bottom plate.
- XTT Labelling Mixture: the XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.
- Maximum concentration: the highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.
- Concentrations: 8 concentrations of the test item were analysed.
- Replicates: all dose groups were tested in 7 replicates for the first XTT test and in 6 replicates for the second XTT test.
- Incubation period: 24 ± 0.5 hours.
- Observation: the cell cultures were microscopically evaluated for morphological alterations.
- Measurement: at the end of the incubation period, 50 μl of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader. The absorbance was measured at 450 nm (reference wavelength 690 nm).
- Evaluation of the XTT results: a decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance.
- Acceptability of the assay: the XTT test is considered to be acceptable if it meets the mean absorbance of the medium control is ≥ 0.5 and if the mean viability of the solvent control is ≥ 90 % in comparison to the medium control.

DEVIATION(S) TO THE OECD GUIDELINE
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry. The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

Run / experiment:

other: third and fourth run

Parameter:

other: % RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: fourth run

Parameter:

other: % RFI of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The RFI of CD86 and CD54 was not equal or greater than 150 % and 200 %, respectively at any dose in the first and second h-CLAT run. Since the histogram plot of the FL-3 channel in these negative evaluated h-CLAT runs did not show a cell viability < 90 % at the highest tested test item concentration in comparison to the calculated cell viability, two additional h-CLAT runs with adjusted test item concentrations were conducted.

The RFI of CD86 was greater than 150 % in the highest tested concentration of the third run and the RFI of CD86 and CD54 was equal or greater than 150 % and 200 %, respectively, in the highest tested concentration of the fourth independent run.
Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT. Since no clear dose response could be observed, a false-positive result cannot be fully excluded.

CONTROLS
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 CD54 RFI value of the positive control (2.0 µg/ml DNCB) in the third h-CLAT run did not exceed the positive criterion (CD54 ≥ 200 %). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/ml DNCB) in the third h-CLAT run exceeded the positive criteria.

CYTOTOXICITY
Cytotoxic effects were observed following incubation with test item starting with the concentration of 31.25 µg/ml up to the highest tested concentration (500 µg/ml) in the first XTT test and starting with the concentration of 62.5 µg/ml up to the highest tested concentration (500 µg/ml) in the second XTT test (threshold of cytotoxicity: < 75 %). A colour interference effect by the intrinsic colour of the test item was observed in the XTT test by a dose dependent increase of the chemical blank. After subtraction of the chemical blank value from the value of the test item treated cells, a mean CV75 value of 42.05 µg/ml for both XTT tests was calculated.

DOSE-RANGE FINDING - XTT TEST
The mean CV75 value of both XTT tests was estimated to be 59.6 μg/ml.
In the first run, the mean viability of the solvent control in comparison to the medium control was 104.8 %; the CV75 value of the first XTT test was 24.5 μg/ml.
In the second run, the mean viability of the solvent control in comparison to the medium control was 113.08 %; the CV75 value of the second XTT test was 42.05 μg/ml.

MAIN TEST (h-CLAT)

First run

Concentration	RFI (%)	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)^R1
	CD54 Antibody	CD86 Antibody
Medium Control	100.0	100.0	100.0	92.34
DMSO Control	100.0	100.0	100.0	93.07
Positive Control (DNCB), 2.0 µg/ml	270.5*	367.9*	74.7	82.33
Positive Control (DNCB), 3.0 µg/ml	406.4*	284.6*	62.5	78.67
Test Item, 14 µg/ml	73.4	55.3	87.6	94.29
Test Item, 17 µg/ml	97.1	90.7	83.8	94.33
Test Item, 20 µg/ml	91.9	76.6	82.4	93.98
Test Item, 24 µg/ml	96.5	85.2	78.4	93.21
Test Item, 29^#µg/ml	92.5	73.4	75.0	93.74
Test Item, 35^#µg/ml	106.4	105.1	71.6	93.83
Test Item, 42^#µg/ml	121.4	66.2	62.3	93.51
Test Item, 50^#µg/ml	107.5	76.2	59.9	92.55

* RFI value CD86 or CD54 fulfilled the positive criteria (CD ≥ 105 % and CD50 ≥ 200 %)

# medium yellow coloured

^R1 additional detected cell viability of the isotype control by setting an R1-gate (dead cells are gated-out by staining with 7-AAD).

Second run

Concentration	RFI (%)	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)^R1
	CD54 Antibody	CD86 Antibody
Medium Control	100.0	100.0	100.0	95.82
DMSO Control	100.0	100.0	100.0	95.75
Positive Control (DNCB), 2.0 µg/ml	248.3*	509.4*	76.5	85.53
Positive Control (DNCB), 3.0 µg/ml	367.2*	722.4*	68.5	81.53
Test Item, 14 µg/ml	71.6	86.7	84.6	95.59
Test Item, 17 µg/ml	80.2	74.5	89.1	95.14
Test Item, 20 µg/ml	81.9	74.9	87.2	96.30
Test Item, 24 µg/ml	81.9	76.9	81.3	95.66
Test Item, 29^#µg/ml	84.5	73.3	79.7	95.89
Test Item, 35^#µg/ml	91.4	67.8	74.8	95.86
Test Item, 42^#µg/ml	91.4	67.8	69.4	95.48
Test Item, 50^#µg/ml	135.3	138.8	47.8	90.05

* RFI value CD86 or CD54 fulfilled the positive criteria (CD ≥ 105 % and CD50 ≥ 200 %)

# medium yellow coloured

^R1additional detected cell viability of the isotype control by setting an R1-gate (dead cells are gated-out by staining with 7-AAD).

Third run

Concentration	RFI (%)	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)^R1
	CD54 Antibody	CD86 Antibody
Medium Control	100.0	100.0	100.0	94.87
DMSO Control	100.0	100.0	100.0	95.19
Positive Control (DNCB), 2.0 µg/ml	186.7^§	200.2*	81.6	89.07
Positive Control (DNCB), 3.0 µg/ml	225.4*	328.3*	77.1	86.22
Test Item, 24 µg/ml	87.3	56.5	60.9	95.02
Test Item, 29 µg/ml	87.3	54.8	61.3	95.44
Test Item, 35 µg/ml	93.4	50.4	55.9	93.92
Test Item, 41 µg/ml	80.1	63.1	51.5	92.45
Test Item, 50^#µg/ml	89.5	70.3	51.1	94.08
Test Item, 60^#µg/ml	85.6	79.1	47.4	92.70
Test Item, 72^#µg/ml	112.2	79.1	33.9	85.92
Test Item, 86^#µg/ml	122.1	164.8*	21.4	67.91

* RFI value CD86 or CD54 fulfilled the positive criteria (CD ≥ 105 % and CD50 ≥ 200 %)

§RFI value of CD54 did not exceed the positive criteria (CD54 ≥ 200 %).

# medium yellow coloured

^R1additional detected cell viability of the isotype control by setting an R1-gate (dead cells are gated-out by staining with 7-AAD).

Fourth run

Concentration	RFI (%)	RFI (%)	Cell Viability (%)	Cell Viability ISO (%)^R1
	CD54 Antibody	CD86 Antibody
Medium Control	100.0	100.0	100.0	95.39
DMSO Control	100.0	100.0	100.0	95.00
Positive Control (DNCB), 2.0 µg/ml	261.4*	515.9*	78.6	84.63
Positive Control (DNCB), 3.0 µg/ml	415.0*	511.6*	77.4	83.72
Test Item, 24 µg/ml	90.7	116.4	94.7	95.08
Test Item, 29 µg/ml	93.6	126.1	94.2	94.69
Test Item, 35 µg/ml	86.4	122.4	90.8	94.84
Test Item, 41 µg/ml	1.4	144.7	81.3	95.11
Test Item, 50^#µg/ml	75.0	109.0	72.3	94.92
Test Item, 60^#µg/ml	89.3	70.8	62.6	95.06
Test Item, 72^#µg/ml	100.0	64.5	51.0	94.39
Test Item, 86^#µg/ml	215.0*	269.4*	18.0	58.71

Shaded test groups: cell viability below 50 %, are excluded from the evaluation

* RFI value CD86 or CD54 fulfilled the positive criteria (CD ≥ 105 % and CD50 ≥ 200 %)

# medium yellow coloured

^R1additional detected cell viability of the isotype control by setting an R1-gate (dead cells are gated-out by staining with 7-AAD).

RESULTS OF XTT

First run

Test group	Concentration [µg/ml]	Cytotoxicity	Photometric Evaluation
			Mean Ab-sorbance*^§	Standard-Deviation	Chem. Blank^§	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.639	0.039	0.238	0.401	95.42
Solvent Control	-	no	0.649	0.038	0.229	0.42	100
Test item	3.91	no	0.649	0.053	0.261	0.388	92.32
	7.81	no	0.65	0.059	0.29	0.361	85.85
	15.63	no	0.682	0.053	0.333	0.349	82.98
	31.25	yes^#	0.751	0.054	0.461	0.289	68.86
	62.5	yes^#	0.888	0.052	0.669	0.219	52.19
	125	yes^#	1.133	0.032	1.044	0.089	21.11
	250	yes^#	1.629	0.013	1.646	-0.016	-3.85
	500	yes^#	2.861	0.079	2.889	-0.028	-6.61

§: Given absorbances are rounded values.

* mean absorbance (absolute) of 7 wells

** relative absorbance (rounded values)

# medium yellow coloured

Second run

Test group	Concentration [µg/ml]	Cytotoxicity	Photometric Evaluation
			Mean Ab-sorbance*^§	Standard-Deviation	Chem. Blank^§	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.76	0.029	0.254	0.505	88.43
Solvent Control	-	no	0.815	0.026	0.243	0.572	100
Test item	3.91	no	0.851	0.11	0.277	0.574	100.46
	7.81	no	0.822	0.081	0.298	0.524	91.64
	15.63	no	0.939	0.084	0.361	0.578	101.16
	31.25	no	0.888	0.065	0.431	0.457	79.93
	62.5	yes^#	1.076	0.075	0.65	0.426	74.5
	125	yes^#	1.182	0.037	1.048	0.134	23.36
	250	yes^#	1.692	0.024	1.708	-0.016	-2.88
	500	yes^#	3.020	0.032	2.965	0.055	9.58

§: Given absorbances are rounded values.

* mean absorbance (absolute) of 7 wells

** relative absorbance (rounded values)

# medium yellow coloured

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75 % cell viability)

Interpretation of results:

other: the result will be evaluated within a weight of evidence approach

Conclusions:

The test item activated THP-1 cells under the tested conditions. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

The 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 test item dissolved in 0.2 % DMSO when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 31.25 µg/ml up to the highest tested concentration (500 µg/ml) in the first XTT test and starting with the concentration of 62.5 µg/ml up to the highest tested concentration (500 µg/ml) in the second XTT test (threshold of cytotoxicity: < 75 %). A colour interference effect by the intrinsic colour of the test item was observed in the XTT test by a dose dependent increase of the chemical blank. After subtraction of the chemical blank value from the value of the test item treated cells, a mean CV75 value of 42.05 µg/ml for both XTT tests was calculated.

The following concentrations of the test item were tested in the main experiments: 14, 17, 20, 24, 29, 35, 42 and 50 µg/ml

The test item with a negative log Pow was tested in 4 independent runs. The RFI of CD86 and CD54 was not equal or greater than 150 % and 200 %, respectively at any dose in the first and second h-CLAT run. Since the histogram plot of the FL-3 channel in these negative evaluated h-CLAT runs did not show a cell viability < 90 % at the highest tested test item concentration (as recommended in the OECD guideline 442E, evaluated by setting an R1-gate) in comparison to the calculated cell viability, two additional h-CLAT runs with adjusted test item concentrations were conducted.

The following concentrations of the test item were tested in the third and fourth main experiment (h-CLAT): 24, 29, 35, 41, 50, 60, 72 and 86 µg/ml

The RFI of CD86 was greater than 150 % in the highest tested concentration of the third run and the RFI of CD86 and CD54 was equal or greater than 150 % and 200 %, respectively, in the highest tested concentration of the fourth independent run. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT. Since no clear dose response could be observed, a false-positive result 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 CD54 RFI value of the positive control (2.0 µg/ml DNCB) in the third h-CLAT run did not exceed the positive criterion (CD54 ≥ 200 %). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/ml DNCB) in the third h-CLAT run exceeded the positive criteria.

Conclusion

The test item activated THP-1 cells under the tested conditions. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From June 11th to 20th, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Assessment of test item solubility
The solubility of test item was assessed at a concentration of 100 mM in acetonitrile and acetonitrile/water 50/50 v/v.

Preparation of peptide stock solution
Stock solution of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca. 20 ml aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 m. A buffer blank was prepared as well.

Preparation of stability controls and precision controls
Stability controls (Reference Control B, n=6) and precision controls (Reference Control A) of both peptides were prepared at a concentration of 0.5 mM. Reference Control A and Reference Control B were prepared with buffer and acetonitrile, Reference Control C (n=3) was prepared with buffer and acetonitrile/water 50/50 v/v.

Preparation of positive control solution and test item stock solution
The positive control chemical (cinnamic aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of test item was prepared in acetonitrile/water 50/50 v/v.

Preparation of positive control and cysteine peptide depletion samples and co-elution controls
Triplicate solutions each of test item and the positive control stocks were diluted with cysteine peptide to prepare final solutions containing 0.5 mM cysteine and 5 mM of either test item or the positive control. For the co-elution control, blank buffer solution was used in place of the cysteine stock solution.

Preparation of positive control and lysine peptide depletion samples and co-elution controls
Triplicate solutions each of test item and the positive control stocks were diluted with the lysine peptide to prepare solutions containing 0.5 mM lysine and 25 mM of either test item or the positive control. For the co-elution control, blank buffer solution bwas used in place of the lysine stock solution.

Incubation
The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25 °C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to intiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of each peptide in the presence of test item and the associated positive controls were quantified by HPLC using UV detection as detailed in the chromatographic section.

Calculations
The peak response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area response of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - 100 × peptide peak area in replicate depletion sample / mean peptide peak area of reference control samples B or C.

Positive control results:

- cysteine peptide depletion: mean depletion % = 71.7, SD % = 0.44 (samples prepared at a concentration of 376 µg/ml, 0.5 mM)
- lysine peptide depletion: mean depletion % = 60.4, SD % = 7.53 (samples prepared at a concentration of 388 µg/ml, 0.5 mM)

Run / experiment:

other: mean of 3 run

Parameter:

other: mean depletion cysteine %

Value:

17.2

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: mean of 3 run

Parameter:

other: mean depletion lysine %

Value:

93.3

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Solubility assessment
The solubility of test item in acetonitrile/water 50/50 v/v at a nominal concentration of 100 mM was confirmed. Test item was insoluble in acetonitrile at 100 mM.

Reactivity assessment
All analytical acceptance criteria for each peptide run were met. Applying the depletion model, reactivity is classed as high and the DPRA prediction is positive, hence the test item is predicted to be potential skin sensitizer by this assay. There was co-elution (in terms of a test item peak eluting alongside the lysine without getting baseline resolution) during the lysine assay, however integration of both the lysine peak and the test item peak was achieved and was consistent for each of the three samples so that the data could be used and a prediction based on the lysine assay made.

Depletion of peptide in presence of test item

	Mean peak area of reference control (µV.sec)	Mean peak area of peptide in test samples (µV.sec)	Mean peptide depletion ( % )
Cysteine	B: 898360 (n=6)	728330 (n=3)	17.2
	C: 879920 (n=3)
Lysine	B: 744350 (n=6)	49581 (n=3)	93.3
	C: 742560 (n=3)

mean of cysteine and lysine% depletion	reactivity class	DPRA prediction
0 % ≤ mean % depletion ≤ 6.38 %	no or minimal reactivity	negative
6.38 % < mean % depletion ≤ 22.62 %	low reactivity	positive
22.62 % < mean % depletion ≤ 42.4 7 %	moderate reactivity
42.47 % < mean % depletion ≤ 100 %	high reactivity

Cysteine peptide depletion

Sample	peak area (µV.sec)	peptide conc.^1(µg/ml)	peptide depletion ( % )	mean depletion ( % )	SD ( % )
positive control	250163	104	72.22^2	71.7	0.44
	257948	107	71.32^2
	255201	106	71.62^2
test item	749975	314	14.83^3	17.2	2.35
	726121	304	17.53^3
	708884	296	19.43^3

CV = Coefficient of Variation

¹ Samples prepared at a concentration of 376µg/ml (0.5 mM)

² Calculated against a mean reference control B peak area of 898360 µV.sec(n=6)

³ Calculated against a mean reference control C peak area of 879920 µV.sec(n=3)

Lysine peptide depletion

sample	peak area (µV.sec)	peptide conc.^1 (µg/ml)	peptide depletion  ( % )	mean depletion  ( % )	SD ( % )
positive control^1	253561	141	65.92^2	60.4	7.53
	271548	151	63.52^2
	358374	200	51.92^2
test item	52501	26.6	92.93^3	93.3	0.36
	49060	24.6	93.43^3
	47181	23.6	93.63^3

Data presented for information only

CV = Coefficient of Variation

¹ Samples prepared at a concentration of 388 µg/ml (0.5 mM)

² Calculated against a mean reference control B peak area of 744350µV.sec(n=6)

³ Calculated against a mean reference control C peak area of 742560µV.sec(n=3)

Interpretation of results:

other: the results will be evaluated with a weight of evidence approach

Conclusions:

Significant depletion was confirmed for both peptides and with an overall mean depletion of 55.3 %, this categorizes the reactivity of test item as "high" and hence the DPRA prediction is positive.

Executive summary:

The study was run according to OECD guideline 442C (DPRA) to assess the reactivity and sensitizing potential of test item.

Solutions of test item were analysed by a validated analytical method, in the cysteine containing synthetic peptide and the lysine containing synthetic peptide.

Mean depletion was 55.3 %, thus categorizing the reactivity of test item as "high" and hence the DPRA prediction is positive, as based on this assay.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From January 28th to February 14th, 2019

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:

2018

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol no 155: KeratinoSens^tm

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

-Cell culture: the cells used in this assay were the transgenic dcell line KeratinoSens with a stable inserton of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37 ± 2 °C in a humified atmosphere containing 5 % in CO2 in air. Maintenance medium was 500 ml Dulbecco's Modified Eagles Medium cointaning Glutamax (DMEM), supplemented with 50 ml foetal bovine serum (FBS) and 5.5 ml Geneticin.

-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 9 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.

-Cell passage: actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells have reached 80-90 % confluence, the medium from each flask was removed, the cells washed twice with Dulbecco's phosphate buffered saline (DPBS) and harvested using trypsin-ECTA solution. Cultures were incubated at 37 ± 2 °C in a humified atmosphere containing 5 % CO2 in air until complete detachment and disaggregation of the monolayer had occured. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks were pooled at this point). The cells were resuspended and distributed into flasks cointaining 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 from frozen stock.

-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 diluited with maintenance medium without geneticin to give 1×10^5 viable cells/ml and 100 µl volumes pipetted into all wells except one well 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. One well 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 humified atmosphere of 5 % CO2 in air to allow the cells to attach.

-Preparation of positive control: cinnamic aldehyde 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 the following formula: V=5 × [((p/100) × w)/MW] - w/1000
where: V=volume of DMSO in ml to be added, p=purity of the chemical in %, MW=molecular weight of the chemical in g/mol, w=exact weight of the chemical added to the vial in mg.
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. Preparation of the positive control was shared with other studies performed in the same assay.

-Test item solubility: prior to commencing testing, the solubility of the test item in DMSO was assessed. The test item was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

-Preparation of the test item: a stock solution of the test item was prepared by weighing the test item into a tared glass container and diluiting to 200 mM in DMSO. Serial diluitions were prepared using DMSO to obtain 12 master concentrations. The master concentrations were then further diluited 25 fold in DMEM, supplemented with 5.0 ml foetal bovine serum to give working solutions. The working solutions were for treatment with a further 4 fold dilution factor so that the final concentrations of the test item ranged from 0.98 to 2000 µM (based on a dilution factor of 2).

Positive control results:

The EC(1.5) values of the positive control, cinnamic aldehyde, were 4.84 µM and 10.10 µM for 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 3.62 and 4.21 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Run / experiment:

other: Test 1

Parameter:

other: I(max)

Value:

1.13

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test 2

Parameter:

other: I(max)

Value:

1.02

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ASSESSMENT OF RESULTS
Data evaluation
The following parameter were calculated in the Keratino Sens test method:
- the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemicals and positive control;
- the EC1.5 value representing the concentration for which induction of Luciferase activity is above the 1.5 fold threshold (i.e. 50 % enhanced luciferase activity) was obtained;
- the IC50 and IC30 concentration values for 50 % and 30 % reduction of cellular viability.

Fold luciferase activity induction was calculated and the overall maximal fold induction (Imax) was calculated as the average of the individual repetitions.
Fold induction = (Lsample - Lblank) / (Lsolvent-Lblank)
where: Lsample is the luminescence reading in the test chemical well, Lblank is the luminescence reading in the blank well containing no cells and no treatment, Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control.

EC1.5 was calculated by linear interpolation according and the overall EC1.5 was calculated as the geometric mean of the individual repetitions
EC1.5 = (Cb - Ca) × [(1.5 - Ia) / (Ib - Ia)] + Ca
where:
Ca is the lowest concentration in µM with >1.5 fold induction
Cb is the highest concentration in µM with <1.5 fold incution
Ia is the fold induction measured at the lowest concentration with >1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with <1.5 fold induction (mean of three replicate wells)

Viability = [(Vsample - Vblank) / (Vsolvent - Vblank) × 100
where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control

IC50 and IC30 were calculated by linear interpolation and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions
ICx = (Cb - Ca) × [(100 - x) - Va / (Vb - Va)] + Ca
where:
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in µM with >x % reduction in viability
Cb is the highest concentration in µM with Va is the % viability at the lowest concentration with >x % reduction viability
Vb is the % viability at the highest concentration with
For each concentration showing >= 1.5 fold luciferase activity induction, statistical significance was calculated (by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction was statistically significant (p<0.05). The lowest concentration with >= 1.5 fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30 % reduction in cellular viability at the EC1.5 determining concentration. Furthermore, it was checked that no significant cytotoxic effects occured at the lowest concentration leading to >=1.5 fold luciferase induction. Where statistically non-significant induction above 1.5 fold was observed followed by a higher concentration with a statistically significant induction above the threshold of 1.5 was obtained for a non-cytotoxic concentration.

Interpretation of results and prediction model
A Keratino Sens prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens prediction is considered negative:
- the Imax is >= 1.5 fold and statistically significantly different as compared to the solvent vehicle control (as determined by a two-tailed, unpaired Student's T-test);
- the cellular viability is >70 % at the lowest concentration with induction of luciferase activity >=1.5 fold (i.e. at the EC1.5 determining concentration);
- the EC1.5 value is <1000 µM;
- there is a dose-response increase for luciferase induction (or a biphasic response)
If in a given test, all of the first three conditions listed above are met, but a clear dose-response for the luciferase induction cannot be observed, then the result of that test should be considered inconclusive and further testing may be required. In addittion, a negative result obtained with concentrations <1000 µM and that do not reach citotoxicity (<70 % viability) at the maximal tested concentration should also be considered as incoclusive.

Test acceptance criteria
In order for an assay to be accepted the following criteria must be met:
- the luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 µM).
- the EC1.5 value of the positive con trol should be within two standard deviations of the historical mean of the testing facility. In addition, the average induction in the three replicates for cinnamic aldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
- the average coefficient of variation of the luminescence reading for the negative solvent control (i.e. DMSO) should be below 20 % in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.

RESULTS
The Imax for test item was 1.13 in test 1 and 1.02 in test 2. The Imax for both tests was <1.5 fold compared to the DMSO control and thus the EC1.5 could not be calculated.

The cellular viability fell below 70% in both tests. The IC30 value was 4.98 μM in test 1 and 8.06 μM in test 2 and the IC50 values were 10.12 μM and 14.28 μM in tests 1 and 2, respectively. There was no overall dose-response for luciferase induction.

The luciferase activity induction obtained with the positive control, cynnamic 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 EC(1.5) values of the positive control, cinnamic aldehyde, were 4.84 µM and 10.10 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 3.62 and 4.21 for test 1 and 2, 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) as 17.0 % and 14.1 % for test 1 and 2, respectively, which met the acceptance criterion of below 20 %.

Results for test item – 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	0.72	0.69	0.55	0.87	1.13	0.21	0	0	0.01	0.01	0.01	0.01
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	76.25	73.39	73.73	60.18	25.75	1.85	1.94	2.69	3.87	3.7	3.45	3.62
Imax	1.13
EC1.5(µM)	N/A
IC30(µM)	4.98
IC50(µM)	10.12

Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.33	2.14	1.79	2.23	3.62
Statistically significant	No	Yes	Yes	Yes	Yes
Viability (%)	98.89	115.72	104.03	114.71	106.72
Imax	3.62
EC1.5(µM)	4.84
IC30(µM)	N/A
IC50(µM)	N/A

Results for test item – 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	0.86	0.78	0.7	0.67	1.02	0.82	0	0	0	0	0	0.01
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	98.45	82.28	81.88	70.81	45.67	9.78	3.31	2.67	2.75	3.88	3.96	3.8
Imax	1.02
EC1.5(µM)	N/A
IC30(µM)	8.06
IC50(µM)	14.28

Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.32	1.42	1.73	2.15	4.21
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	97.64	100.63	99.18	102.25	102.9
Imax	4.21
EC1.5(µM)	10.1
IC30(µM)	N/A
IC50(µM)	N/A

Interpretation of results:

other: the result will be evaluated within a weight of evidence approach

Conclusions:

The test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens), supporting the prediction that the test item is not a skin sensitizer.

Executive summary:

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

The I_maxfor test item was 1.13 in test 1 and 1.02 in test 2. The I_maxfor both tests was <1.5 fold compared to the DMSO control and therefore the EC_1.5could not be calculated. The cellular viability fell below 70 % in both tests. The IC₃₀value was 4.98 µM in test 1 and 8.06 µM in test 2 and the IC₅₀values were 10.12 µM and 14.28 µM in tests 1 and 2, respectively. No overall dose-response for luciferase induction was seen.

All acceptance criteria for the positive control, cinnamic aldehyde, were met.

It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™).

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From February 19 to 25, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 19.9 – 23.9 g ( The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight).
- Housing: Grouped caging (4 animals/cage)
- Diet (e.g. ad libitum): ssniff® Rat/Souris-Elevage E complete ad libitum
- Water (e.g. ad libitum): tap water from watering bottles ad libitum
- Acclimation period: 14 days
- Indication of any skin lesions: Only healthy animals (and not showing any sign of skin lesion) were used

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: To:

Vehicle:

dimethyl sulphoxide

Concentration:

50 %, 25 %, 10 % and 5 % (w/v) as formulation (apparently solutions)

No. of animals per dose:

4 animals for treatment group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: soluble in DMSO and formulated at 50 %, 25 % and 10 % (w/v)
- Irritation: the effect on the ear thicknesses observed in the 50 % (w/v) dose group was not considered as obvious sign of a significant irritation.
- Systemic toxicity: No mortality, significant, treatment related effect on body weights or any other sign of systemic toxicity were observed.
- Ear thickness measurements: an increase of > 25 % of ear thickness (compared to the initial values) was observed in the 50 % (w/v) dose group for one of the two animals on Day 6 (30 % or 25 % increase was observed on the left or right ear, respectively). No significantly (> 25 %) increased ear thicknesses were observed for the other animals in this dose group or in the other dose groups on the days of measurement (Day 3 or Day 6).
- Erythema scores: No erythema or any other local effect was observed in any dose group during the test.

MAIN STUDY
The test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v).
An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.
The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 5.6) thus confirming the validity of the assay.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: the test item is considered as a skin sensitizer, if:
Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION: Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatment animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Parameter:

SI

Value:

6.1

Test group / Remarks:

Group treated at 50 % (w/v) test item concentration

Parameter:

SI

Value:

2.7

Test group / Remarks:

Group treated at 25 % (w/v) test item concentration

Parameter:

SI

Value:

1.9

Test group / Remarks:

Group treated at 10 % (w/v) test item concentration

Parameter:

SI

Value:

1.5

Test group / Remarks:

Group treated at 5 % (w/v) test item concentration

Parameter:

EC3

Value:

> 25

Test group / Remarks:

Due to the unreliable SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 %

Body Weight Measurement

Body weight decrease by > 5 % was observed in the following test groups: positive control group (1 of the 4 animals with 7 % decrease) and in the 25 % (w/v) dose group (1 of the 4 animals with 9 % decrease). No body weight decrease by > 5 % was observed in the other groups. In spite of these observations no significant, dose related effect on the body weights was believed during the test.

Clinical Observations, Signs of Irritation

No mortality was observed in any treatment group. The following symptoms of a possible systemic toxicity were observed in the 50 % (w/v) dose group (4 of the 4 animals): hunched back posture, increased respiratory rate on Day 2 (after the treatment); hunched back posture, narrow eyes and restlessness on Day 3 (after the treatment). All animals were symptom-free before the treatments on these days as well as on the other days. Loss of hair on the head was also observed in this dose group as a sign of a local or systemic effect. No local or systemic effects were observed in the other dose groups.

Irritation was monitored by erythema scoring in all test groups and by ear thickness measurements in the DMSO control group and in the test item treated groups. No erythema was observed in any test group during the test. Significantly increased ear thickness values (i. e. an increase of ≥ 25 % compared to the initial value) were observed in the 50 % (w/v) dose group both on Day 3 and on Day 6: the minimum or maximum increase was 15 % or 40 %, respectively. No significantly increased ear thicknesses were observed in the other groups.

Other Observations

Test item residuals were observed on the ears of animals. Amount of the residual was dose related and was not completely removed by normal animal grooming hence the residuals were observed from the first treatment to the end of the test (from Day 1 to Day 6).

Proliferation Assay

Visually larger lymph nodes compared to the relevant vehicle controls (AOO or DMSO) were observed in the positive control group and in the 50 % (w/v) dose group. The appearance of the lymph nodes was normal in both vehicle control groups (AOO or DMSO) and in the other test item treated groups.

Significant lymphoproliferative response (indicated by an SI ≥ 3) compared to the concurrent control (DMSO) was noted for Acid Yellow 218 at 50 % (w/v) concentration. No SI ≥ 3 was observed at the other test concentrations. The observed stimulation index (SI) values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. Dose-related response was observed.

Significance of the dose-response was evaluated by linear regression by using SI values of all four test concentrations and also by using three SI values (not including the 50 % (w/v) test concentration).

Significant dose-related response was observed (p = 0.019, r2 = 0.96) when all SI values were used. No statistical significance was observed when three concentrations were evaluated (p = 0.058, r2 = 0.99) although the response was dose-related.

Reliability of the Test

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.

A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 5.6). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.

Interpretation of Observations

The maximum dose selection was based on results of the formulation evaluation and the dose range finding test (DRF).

The test item was soluble in most of the standard LLNA vehicles. The maximum soluble concentration was 50 % (w/v) in Dimethyl sulfoxide (DMSO). Since no significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test up to this maximum concentration, the test item was examined in the main test as 50 %, 25 %, 10 % and 5 % (w/v) formulations in the selected vehicle.

Based on the positive control result the test was valid.

In this LLNA the observed stimulation index values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively.

No confounding effects of irritation or systemic toxicity were observed in the 25 %, 10 % or 5 % (w/v) dose groups hence the proliferation values related to these test concentrations are considered reliable.

Symptoms of a possible systemic toxic effect were observed at the 50 % (w/v) test concentration however it was not expected (based on the preliminary test results). The symptoms were not excessive, the animals recovered in all cases and no effect on the body weights were observed in this dose group. The significantly increased ear thicknesses may indicate irritation effect of the test item at 50 % (w/v) concentration although it is believed that the observed test item residuals could have effect on the outcome of the ear thickness measurements, especially on Day 3.

Based on all these it is concluded that the proliferation value obtained at the 50 % (w/v) concentration was influenced by effects other than sensitisation hence is not reliable. On the other hand, only its magnitude is questionable hence it should not be excluded from the evaluation of the dose-response correlation.

The SI value of 2.7 observed at 25 % (w/v) concentration was close to the threshold value of 3. The response was clearly dose-related and biologically relevant. The lack of the statistical significance is probably a consequence of an inadequate number of data points in case of evaluation of three test concentration.

Based on the overall results the significantly increased lymphoproliferation at the 50 % (w/v) concentration as well as the dose-related response are considered as evidence that Acid Yellow 218 is a skin sensitizer.

Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.

Due to the unreliable SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 % hence the test item can be ranked among weak skin sensitizers according to the published data for classification of contact allergens

Interpretation of results:

other: Category 1B (indication of skin sensitising potential) based on CLP criteria

Conclusions:

Skin sensitizer

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of Acid Yellow 218 following dermal exposure in the Local Lymph Node Assay, according to the OECD guideline 429.

Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration.

The test item was soluble in most of the standard LLNA vehicles. The maximum soluble concentration was 50 % (w/v) in Dimethyl sulfoxide (DMSO).

No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test up to this maximum concentration. According to this the test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v).

An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 5.6) thus confirming the validity of the assay.

No mortality was observed during the test. No significant treatment related effect on body weights was observed in any test group. Body weight decrease by > 5 % were observed in some cases but without dose relevance and the mean body weights did not decrease significantly in any test groups. Although it was not expected, signs of systemic toxic effect of the test item (hunched back posture, increased respiratory rate, narrow eyes and restlessness) as well as loss of hair (on the head) were observed in the 50 % (w/v) dose group. The symptoms were not excessive, the animals recovered in all cases and no effect on the body weights were observed in this dose group.

Sign of irritation (significantly increased ear thickness values) was also observed in the 50 % (w/v) dose group. On the other hand, it is considered that the ear thickness measurement could have influenced by the test item residuals observed during the whole test on the ears of animals in the test item treated groups.

Significantly increased lymphoproliferation [indicated by a Stimulation Index (SI) ≥ 3] compared to the relevant control (DMSO) was noted for Acid Yellow 218 at 50 % (w/v) test concentration. No SI ≥ 3 was observed in the other dose groups. The observed stimulation index values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively.

It is concluded that the proliferation value obtained at the 50 % (w/v) concentration was influenced by effects other than sensitisation hence is not reliable. On the other hand, only its magnitude is questionable hence it should not be excluded from the evaluation of the dose-response correlation. The SI value of 2.7 observed at 25 % (w/v) was close to the threshold value of 3. Significance of the dose-response was evaluated by linear regression by using SI values of all four test concentrations and also by using three SI values (not including the 50 % (w/v) test concentration).

Significant dose-related response was observed (p = 0.019, r2 = 0.96) when all SI values were used. No statistical significance was observed when three concentrations were evaluated (p = 0.058, r2 = 0.99).

The response was clearly dose-related and biologically relevant. The lack of the statistical significance is considered as consequence of an inadequate number of data points in case of evaluation of three test concentrations.

Based on the overall results the significantly increased lymphoproliferation at the 50 % (w/v) concentration as well as the dose-related response are considered as evidence that Acid Yellow 218 is a skin sensitizer.

Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.

Due to the questionable magnitude of the SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 % hence the test item can be ranked among weak skin sensitizers (10 ≤ EC3 ≤ 100) according to the published data for classification of contact allergens.

Conclusion

In conclusion, under the conditions of the present assay, Acid Yellow 218 tested at the maximum feasible concentration of 50 % (w/v, based on the solubility) and also at concentrations of 25 %, 10 % or 5 % (w/v) as adequate homogeneous formulations (solution, prepared with Dimethyl sulfoxide as vehicle) was shown to have skin sensitization potential (skin-sensitizer) in the Local Lymph Node Assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitising potential of Acid Yellow 218 was at the beginning evaluated relying on a weight of evidence approach. In chemico and in vitro studies were run to assess 3 different key events of skin sensitisation.  However, results from this studies didn't gave clear response about the skin senstitization properties of the test substance. For this reason a LLNA study was carried out.

DPRA assay

To assess the first key event, i.e. covalent binding of electrophilic substances to nucleophilic centres in skin proteins, a DPRA was run, according to OECD guideline 442C. The percentage depletion over time of two synthetic peptides (containing respectively a cysteine and a lysine amino acid) from the peptide mixtures, following an approximate 24 hour (22-26 hours) incubation with the test item, was calculated by HPLC using UV detection.

With a mean depletion of 55.3 %, the reactivity of test item was considered as "high" and hence the DPRA prediction is positive, as based on this assay.

ARE-Nrf2 Luciferase Test (KeratinoSens)

pression associated with specific cell signalling pathways such as the antioxidant/electrophile response element  (ARE)-dependent  pathways, the ARE-Nrf2 Luciferase Test (KeratinoSens) was run, according to OECD guideline 442D.

Two tests were run and the maximal average fold induction of luciferase activity (I_max) was below the threshold value of 1.5 compared to control, thus no EC1.5 could be calculated. Specific values were 1.13 and 1.02. The cellular viability fell below 70 % in both tests. Concentration values for 30 % and 50 % reduction of cellular viability were calculated: IC₃₀ was 4.98 µM in test 1 and 8.06 µM in test 2 and IC₅₀ were 10.12 µM and 14.28 µM in tests 1 and 2, respectively.

It was concluded that test item is negative in this assay.

in vitro Human Cell Line Activation Test (h-CLAT)

To assess the third key event, i.e. the activation of dendritic cells, typically assessed by expression of specific cell surface markers, chemokines and cytokines, a hCLAT was run, according to OECD guideline 442E.

Test item was tested in the first and second main experiment (h-CLAT) at concentrations of: 14, 17, 20, 24, 29, 35, 42 and 50 μg/ml. The RFI of CD86 and CD54 was not equal or greater than 150 % and 200 %, respectively at any dose in the first and second h-CLAT run. However, as cell viability was not < 90 % at the highest tested test item concentration, as recommended in the OECD guideline 442E, 2 additional h-CLAT runs with adjusted test item concentrations were run. Test item concentrations in the third and fourth main experiment (h-CLAT) were: 24, 29, 35, 41, 50, 60, 72 and 86 μg/ml. The RFI of CD86 was greater than 150 % in the highest tested concentration of the third run and the RFI of CD86 and CD54 was equal or greater than 150 % and 200 %, respectively, in the highest tested concentration of the fourth independent run.

On these bases, the h-CLAT prediction is considered positive for the test item in this assay. Since no clear dose response could be observed, a false-positive result cannot be fully excluded.

Local Lymph Node Assay (LLNA)

The skin sensitization potential of Acid Yellow 218 was assessed following dermal exposure in the Local Lymph Node Assay, according to the OECD guideline 429.

Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration.

The test item was soluble in most of the standard LLNA vehicles. The maximum soluble concentration was 50 % (w/v) in Dimethyl sulfoxide (DMSO).

No significant adverse effects (systemic toxicity or irritation) were observed in the dose range finding test up to this maximum concentration. According to this the test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v).

An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 5.6) thus confirming the validity of the assay.

No mortality was observed during the test. No significant treatment related effect on body weights was observed in any test group. Body weight decrease by > 5 % were observed in some cases but without dose relevance and the mean body weights did not decrease significantly in any test groups. Although it was not expected, signs of systemic toxic effect of the test item (hunched back posture, increased respiratory rate, narrow eyes and restlessness) as well as loss of hair (on the head) were observed in the 50 % (w/v) dose group. The symptoms were not excessive, the animals recovered in all cases and no effect on the body weights were observed in this dose group.

Sign of irritation (significantly increased ear thickness values) was also observed in the 50 % (w/v) dose group. On the other hand, it is considered that the ear thickness measurement could have influenced by the test item residuals observed during the whole test on the ears of animals in the test item treated groups.

Significantly increased lymphoproliferation [indicated by a Stimulation Index (SI) ≥ 3] compared to the relevant control (DMSO) was noted for Acid Yellow 218 at 50 % (w/v) test concentration. No SI ≥ 3 was observed in the other dose groups. The observed stimulation index values were 6.1, 2.7, 1.9 and 1.5 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively.

It is concluded that the proliferation value obtained at the 50 % (w/v) concentration was influenced by effects other than sensitisation hence is not reliable. On the other hand, only its magnitude is questionable hence it should not be excluded from the evaluation of the dose-response correlation. The SI value of 2.7 observed at 25 % (w/v) was close to the threshold value of 3. Significance of the dose-response was evaluated by linear regression by using SI values of all four test concentrations and also by using three SI values (not including the 50 % (w/v) test concentration).

Significant dose-related response was observed (p = 0.019, r2 = 0.96) when all SI values were used. No statistical significance was observed when three concentrations were evaluated (p = 0.058, r2 = 0.99).

The response was clearly dose-related and biologically relevant. The lack of the statistical significance is considered as consequence of an inadequate number of data points in case of evaluation of three test concentrations.

Based on the overall results the significantly increased lymphoproliferation at the 50 % (w/v) concentration as well as the dose-related response are considered as evidence that Acid Yellow 218 is a skin sensitizer.

Chemicals can be classified according to their relative skin-sensitization potency using EC3 value (dose calculated to induce a stimulation index of 3) calculated by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve according to published method.

Due to the questionable magnitude of the SI value at the 50 % (w)v) test concentration no EC3 value for the test item was calculated in this LLNA. On the other hand, it is evident from the test results that the theoretical EC3 value is > 25 % hence the test item can be ranked among weak skin sensitizers (10 ≤ EC3 ≤ 100) according to the published data for classification of contact allergens.

In conclusion, under the conditions of the present assay, Acid Yellow 218 tested at the maximum feasible concentration of 50 % (w/v, based on the solubility) and also at concentrations of 25 %, 10 % or 5 % (w/v) as adequate homogeneous formulations (solution, prepared with Dimethyl sulfoxide as vehicle) was shown to have skin sensitization potential (skin-sensitizer) in the Local Lymph Node Assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), skin sensitiser means a substance that will lead to an allergic response following skin contact.

The skin sensitising potential of Acid Yellow 218 was at the beginning evaluated within a weight of evidence strategy, taking into account experimental findings related to different aspects of skin sensitisation.

Three different key events of skin sensitisation were assessed by 3 specific in vitro test methods:

- key event 1 of skin sensitisation - DPRA (OECD 442C): positive

- key event 2 of skin sensitisation - KeratinoSens test (OECD 442D): negative

- key event 3 of skin sensitisation - hCLAT (OECD 442E): positive, with no clear dose-response

As the results obtanied in this three studies are discordants and cannot be used to draw a valid conclusion a LLNA assay was carried out. The EC3 value calculated under test conditions was estimated to be > 25 % meaning that the substance is considered as a weak skin sensitizer.

Overall, Acid Yellow 218 is considered a skin sensitizer and has been classified as Skin. Sens. Cat. 1B according to the CLP Regulation (EC 1272/2008).