2,5-dichloro-4-[[2-(dibutylamino)-4-methyl-6-[[2-(4-sulphophenyl)ethyl]amino]-5-pyrimidinyl]azo]benzenesulphonic acid, sodium salt

Endpoint summary

Description of key information

Not skin sensitising

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 October 04 to November 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

CELLS
- Source: THP-1 (monocytic leukeamia cell line) cells provided from American Type Culture Collection.
- Storage: cells were stored in liquid nitrogen and the assays were performed thanks to a master bank supplied by Biopredic International (Saint Gregoire - France).
- Culturing: cryopreserved cells have been thawed. Cells were cultured, at 37 °C under 5 % CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10 % Foetal Calf Serum, 100 units/ml penicillin and 100 pg/ml streptomycin.
- Seeding: THP-1 were routinely seeded every 3-4 days at the density of 0.15 to 0.2 x10^6 cells/ml.
- Preparation of test incolumun: for testing, THP-1 cells were seeded at a density of 0.2 x 10^6 and pre-cultured in culture flasks for 72 hours.
- Viability: the quality of each batch of THP-1 cells should be checked. Viability of the cells must be above 90 %.

MAIN EXPERIMENT
Test item
- Concentration range: 7.81 to 100 μg/ml. The maximum concentration tested was 100 μg/ml, depending on its poor solubility.
- Solvent: the test item was tested in a medium containing 0.2 % DMSO. (Dimethysulfoxide).

Procedures
Based on the cytotoxlcity assay the eight final test item concentrations were selected.
ln case of non-toxic concentration for the top dose used in preliminary test, the maximum concentration selected for activation test did not exceed 1000 µg/ml when the test item was dissolved or stably dispersed in Ethanol or DMSO, and 5000 µg/ml when the test item was dissolved in a saline vehicle.
Each experiment of activation test was performed on eight concentrations.
THP-1 cells were plated at 1 x 10^6 cells/ml/well in 24 well plates and treated for 24:05 hours with selected test item concentrations. After treatment cells were washed twice with FACS buffer. Then cells were stained for 30 min at 4 °C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse lgG1. Using the manufacturer's recommended dilutions, cells were incubated with above mAbs at 6 µl/3 x 10^5 cells /50 µl for the anti-human CD86 mAb, and 3 µl/3 x 10^§5 cells /50 µl for the anti-human CD54 mAb. FITC labelled-mouse lgG1 was used as an isotype control at a dilution of 3 µl/3 x 10^5 cells /50 µl. Then, the cells were stained also with 7-AAD for 30 min at 4 °C. After washing and resuspension with FACS buffer, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA and InCyte software, on 10000 living cells.

CONTROLS
Vehicle control: as the maximal dose for test item was obtained in the DMSO, the vehicle control for all of the assays in the study was the DMSO.
Negative control: Lactic Acid (LA) produced a negative response for both markers.
Reference controls: 2,4-dinitrochlorobenzene (DNCB), nickel sulfate (NiSO4) produced a positive response for both CD86 and CD54.

PRELIMINARY CYTOTOXICITY
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50 %, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.
Eight concentrations of test item have been prepared by a two-fold serial dilution from a maximum final concentration of 1000 µg/ml or lower, depending on its solubility limit.
In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at 2 x 10^6 cells/ml. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µl (1 x 10^6 cells/well) with various concentrations of test item (1:1 ratio) for 24±0.5 hours at 37 “C under 5 % CO2. After treatment cells were washed twice with phosphate-buffered containing 0.1 % (w/v) bovine serum albumin identified as FACS buffer. The cells were stained with 7-AAD (5 µg/ml final concentration). Then cells were analysed with flow cytometry using GUAVA and InCyte software to measure cell viability. The living cells (7-AAD-) gate was set in the 7-AAD negative area. 10^4 7-AAD- cells were counted as the living population.
According to the results the dose levels for the main study were selected.

VALIDITY OF THE STUDY
Acceptability criteria for evaluating results induced by the positive control, the vehicles controls and the test item, were based on previous studies cited above and the guideline for h-CLAT (OECD 442E, 2016).
The study is considered valid if the following criteria are fully met:
- in the positive control (DNCB) RFl values of both CD86 and CD54 should be over the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %).
- in the positive control the cell viability should be more than 50 %
- in the vehicle control cell viability should be more than 90 %
- in the vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD54 ≥ 200 % and CD86 ≥ 150 %) compared to the medium control
- in the vehicle control the MFI ratio of both CD86 and CD54 to isotype control should be > 105 %
- in the test item cell viability should be more than 50 % in at least four tested concentrations in each run.

Run / experiment:

other: both two runs

Parameter:

other: % RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

cell viability > 50 %

Run / experiment:

other: both two runs

Parameter:

other: % RFI of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

cell viability > 50 %

Other effects / acceptance of results:

No "increase" of the CD54/86 expression compared with the negative control was noticed, for any dose of test item.
In both experiments, an absence of any increase of the CD54/86 expression was observed with the test item. None of the tested doses induced a 1.5 or 2 fold increase of CD86 and CD54 expression respectively compared to the negative control.

No contamination was noticed during the study.

PRELIMINARY CYTOTOXICITY
Based on the solubility test, the doses range for cytotoxicity test was the following (with two fold dilution factor): from 0.78 to 100 µg/ml.

VALIDITY OF THE STUDY
Test system was validated as all criteria were fully accepted.
Negative controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria.
Positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.
These results validated the experimental conditions.

	Concentration range*	Viability	CD54	CD86	MIT (μg/ml)
Run 1	7.81 to 100 μg/ml	> 50 %	RFI < 200	RFI < 150	-
Run 2		> 50 %	RFI < 200	RFI < 150

RFI of CD54/86 expression

Sample	Experiment 1			Experiment 2
	CD54	CD86	CV (%)	CD54	CD86	CV (%)
RPMI	100	100	98	100	100	98
DMSO	108	118	98	106	105	98
DNCB, 4 µg/ml	1301	517	77	914	410	81
Test iten (vehicle 0.2 % DMSO), 0.78 µg/ml	98	138	97	98	134	97
Test iten (vehicle 0.2 % DMSO), 1.56 µg/ml	88	132	97	91	126	97
Test iten (vehicle 0.2 % DMSO), 3.13 µg/ml	90	144	97	85	137	97
Test iten (vehicle 0.2 % DMSO), 6.25 µg/ml	88	122	97	90	145	97
Test iten (vehicle 0.2 % DMSO), 12.5 µg/ml	89	135	97	40	105	98
Test iten (vehicle 0.2 % DMSO), 25 µg/ml	74	118	98	50	109	98
Test iten (vehicle 0.2 % DMSO), 50 µg/ml	79	123	97	51	90	98
Test iten (vehicle 0.2 % DMSO), 100 µg/ml	58	89	97	53	71	98

PRELIMINARY CYTOTOXICITY

Dose level µg/ml	% cell viability
100	97
50.0	97
25.0	97
12.5	98
6.25	98
3.13	98
1.56	98
0.78	98

QUALITY CONTROL OF THE TEST SYSTEM

CV and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls

	CV (%)		Ratio CD54/IgG		Ratio CD86/IgG
Sample	Criteria	Result	Criteria	Result	Criteria	Result
RPMI	> 90 %	98	> 105	124	> 105	183
0.9 % NaCl	> 90 %	98	> 105	124	> 105	194
DMSO	> 90 %	98	> 105	128	> 105	206
NiSO4	> 50 %	81
DNCB	> 50 %	77
LA	> 50 %	98

RFI values obtained for vehicles and controls

Sample	RFI
	Criteria	Result CD54	Result CD86
0.9 % NaCl	CD54 ≤ 200 and CD86≤150 %	100	110
DMSO	CD54 ≤ 200 and CD86≤150 %	108	118
NiSO4	CD54 ≥ 200 % and CD86 2≥ 150 %	4984	365
DNCB	CD54 ≥ 200 % and CD86 2≥ 150 %	1301	517
LA	CD54 ≤ 200 and CD86≤150 %	193	116

Interpretation of results:

other: the test, quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, resulted to be negative

Conclusions:

The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell lineresulted to be negative.

Executive summary:

The substance was tested for skin sensitisation potential, following the method and procedures described into the OECD Guideline 442E. DMSO (Dimethysulfoxide) was used as an intermediate solvent; the maximum concentration tested was 100 μg/ml, depending on its poor solubility. Experiment was conducted in duplicate.

The RFI of CD86 resulted to be lower than 150 % at any tested concentration (with cell viability ≥ 50 %), in both runs; the RFI of CD54 resulted to be lower than 200 % at any tested concentration (with cell viability ≥ 50 %), in both runs.

Therefore, under the test conditions of the experimental human Cell Line Activation Test, the test item has been judged to be a non-sensitizer.

Conclusion

The test quantifing changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell lineresulted to be negative.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October from 30 to November 10, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted on February 04th, 2015

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

SUBSTANCES
- Test item: the test item was tested at 12 concentrations, according to a geometric progression of ratio 2 from 0.2 µg/ml to 400 µg/ml.
- Negative control: 6 wells of solvent control (1 % DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate.
- Positive control: 5 concentrations of cinnamaldehyde on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.

Repetitions
The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

CELLS
- Cells: KeratinoSens"'" (Givaudan) maintained according to the current working instruction IL 09.
- Culturing conditions: cells were cultured in maintenance medium at 37 °C, 5 % CO2.
- Mycoplasma: cells are exempt of mycoplasma. Assessment of mycoplasma was performed according to the current working instruction IL 07.

PROCEDURES
Cells seeding (first day)
The cells were trypsinized according to the current working instruction IL 09. Cells suspension were adjusted to a density of 8 x 10^4 cells/ml in seeding medium.
125 µl of the cell suspension at 8 x10^4 cells/ml (i.e. 10^4 cells per well) were distributed in three white plates for the induction measurement and two transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours ± 1 hour at 37 °C, 5 % CO2.
Note: the H12 wells were lefi without cells and allowed the measurement of blanks.

Preparation of test item dilutions (second day)
Preparation of the test item stock solution
Given the slight solubility of the test item it was diluted at 1.6 mg/ml (i.e. 4X) in treatment medium, 4 % DMSO instead of 40 mg/ml.

Preparation of the positive control stock solution
The positive control was prepared at 200 mM in DMSO according to the formula below, then diluted to the concentration of 6.4 mM.

Preparation of the 100 X plate (positive and negative control)
A 100-fold concentrated dilutions series was prepared in 96-well plate.

Positive control
100 µl of DMSO were distributed in row G from columns 7 to 10.200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl of the column 11 in the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

Negative control
100 µl of DMSO were distributed in row G columns l to 6 and 12 and in the well H12.

Preparation of the 4 X dilution plate
Test item
The test item was placed in one of the rows B to F.
100 µl of treatment culture medium 4 % DMSO were distributed fi'om columns 1 to 11. 200 µl of the 1.6 mg/ml stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl of the column 12 in the column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.

PARAMETERS MEASURED
Two parameters are measured: the luciferase induction and the cytotoxicity.

Positive and negative control
The 100 X DMSO plate was diluted 25 fold in a 4X plate.

Contact between the cells and the test and reference items (second day)
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 pl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37 °C, 5 % CO2).

Luciterase activity (dgy 4)
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS.
Then 100 µl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis.
The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

Cell viability assessment with MTT method (dav 4)
Afier 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS.
Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37 °C, 5 % CO2).
Afier this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10 % SDS one night in the dark (37 °C, 5 % CO2). Afier a 10 minutes homogenization, the absorbances were measured at 540 nm.

VALIDATION
To validate the test, it is essential to check the validity criteria for the test.

Positive Control
- the gene induction must be statistically significant above the threshold of 1.5 in at least one dose,
- the EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinnamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde 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.

Negative control
For each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20 %.
lf for one repetition the validity criteria are not met, a third repetition should be considered.

Run / experiment:

other: both repeatition 1 and 2

Parameter:

other: Imax

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: both repeatition 1 and 2

Parameter:

other: viability at the EC1.5 concentration

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Repetition 1: Imax is higher than 1.5. However, at the EC1.5 concentration (18.72 μg/ml) viability is lower than 70 %. The repetition is considered as negative.
Repetition 2: Imax is higher than 1.5. However, at the EC1.5 concentration (2.79 μg/ml) viability is lower than 70 %. The repetition is considered as negative.

The test item induces the gene activity at concentrations very close to the cytotoxic levels in both repetitions. In this case the OECD guideline recommends supplementary assay with a narrower range using a dilution factor of 4/3 instead of 2.
In absence of confirmation and under the retained experimental conditions, the test item should be classified as not sensitizer. However, given the closeness between IC70 and EC1.5, this conclusion should be taken with care and further analyse should be considered.

POSITIVE CONTROL
Mean Imax: 2.53
EC1.5: 18.04 µM

Overall on test results

	Viability	Induction
	IC70 μg/ml	Imax	Linear EC1.5 μg/ml	EC1.5 Lin/Log μg/ml
Rep 1	18.14	2.68	18.72	17.65
Rep 2	1.94	2.32	2.79	2.69
Mean	-	2.50	-	-
Geometric mean	5.94		7.22	6.89

Mean viability percentage

Concentration μg/ml	0.2	0.39	0.78	1.56	3.1	6.3	13	25	50	100	200	400
Rep 1	94.00	97.22	93.33	86.08	82.32	77.89	75.87	62.85	18.93	0.81	1.21	2.15
Rep 2	69.24	78.09	75.70	72.71	61.59	23.92	-0.24	0.00	0.48	0.12	0.00	0.72
Viability	81.6	87.7	84.5	79.4	72.0	50.9	37.8	31.4	9.7	0.5	0.6	1.4

Mean induction

Concentration μg/ml	0.2	0.39	0.78	1.56	3.1	6.3	13	25	50	100	200	400
Rep 1	0.96	1.00	0.88	0.89	0.87	0.98	1.28	1.72	2.68	0.02	0.02	0.01
Rep 2	1.05	1.10	1.36	1.36	1.54	2.32	0.02	0.00	0.01	0.02	0.04	0.04
Induction	1.00	1.05	1.12	1.12	1.20	1.65	0.65	0.86	1.35	0.02	0.03	0.03
SD	0.07	0.07	0.34	0.34	0.47	0.95	0.89	1.22	1.89	0.00	0.02	0.02

Student t-test

Rep 1	0.74	0.867	0.203	0.243	0.155	0.784	0.044	0.004	0.035	0.000	0.000	0.000
Rep 2	0.612	0.420	0.008	0.003	0.004	0.037	0.000	0.000	0.000	0.000	0.000	0.000

Control solvent

Rep 1	13.5
Rep 2	8.1

Positive control - cinnamaldehyde

Concentration μg/ml	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Imax
Rep 1	1.03	1.30	1.35	1.73	2.27	22.17	2.27
Rep 2	1.03	1.24	1.55	1.90	2.79	14.69	2.79
Mean	1.03	1.27	1.45	1.82	2.53	18.04*	2.53

*geometric mean

Interpretation of results:

other: the substance results to not activate the Keap1-Nrf2-ARE, under the tested conditions

Conclusions:

The substance resulted to not activate the Keap1-Nrf2-ARE, under the tested conditions.

Executive summary:

The skin sensitisation potential of test substance was investigated by Keratinocytes test based on the signaling pathway Keap1-Nrf2-ARE coupled to the luciferase reporter gene. The experiment was conducted following the method and procedures outlined into the OECD guideline 442D. The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

During borh the repetitions performed, the Imax resulted to be higher than 1.5. However, in both the cases, at the EC1.5 concentration (18.72 and 2.79 μg/ml, respectively) viability resulted to be lower than 70 %. Therefore, the two repetitions were considered as negative.

Conclusion

The substance results to not activate the Keap1-Nrf2-ARE, under the tested conditions

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January from 23 to 31, 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

4 February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

FORMULATION OF TEST ITEM
Solubility of the test item was evaluated in acetonitrile and water at the concentration of 100 mM. The test item did not dissolve in acetonitrile, however it formed a homogenous solution with ultrapure water. Since ultrapure water is the second preferred vehicle according to the guideline, solubility in other accepted vehicles was not evaluated.

PROCEDURES
- Vials preparation: the vials were capped, vortexed to mix and placed to the HPLC autosampler.
- Incubation: 24 ± 2 hours at 25 ± 2.5 °C in the dark.
- Analysis: the batches were consisted of 2 parts, i.e one part with the A reference controls, the calibration standards and the co-elution controls (these samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part); the other part contained the B and C reference controls, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended.
- Replicates: samples were prepared and analysed in triplicates in batches.

POSITIVE CONTROL
- Test Substance: cinnamaldehyde.
- Stock solution: 100 mM solutions of cinnamaldehyde in acetonitrile were prepared just before use. The needed amount of test chemical was calculated (0.0668 g ± 10 %) based on the molecular weight and purity of the substance. 0.0699 g cinnamaldehyde was weighted for the positive stock solution used for the cysteine peptide depletion determination and 0.0647 g cinnamaldehyde was weighted for the stock solution used for lysine peptide depletion determination.

HPLC CONDITIONS
- HPLC system: SHIMADZU LC2030
- Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 µm)
- Column temperature: 30 °C
- Sample temperature: 25 °C
- Detector: 220 nm (258 nm)
- Injection volume: 7 µl
- System equilibration: 50 % phase A and 50 % phase B for 2 hours at 30 °C and running the gradient twice before injecting the first sample.
- Run time: 20 min
- Flow conditions: gradient flow.
- Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water.
- Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile.
- Mobile Phase C – 50 - 50 % (v/v) acetonitrile and ultrapure water.

BUFFET SOLUTIONS
Phosphate buffer pH 7.5 ± 0.05
- 18 (v/v) % 0.1 M sodium phosphate monobasic (of Sodium Phosphate Monobasic Monohydrate (NaH2PO4 x H2O) in purified water)
- 82 (v/v) % 0.1 M sodium phosphate dibasic (of Sodium Phosphate Dibasic Heptahydrate (Na2HPO4 x 7H2O) in purified water)
- pH was adjusted with either the monobasic or dibasic solution

Ammonium acetate buffer pH 10.2 ± 0.05
- 0.1 M Ammonium Acetate (CH3CO2NH4) in purified water
- pH was adjusted with Ammonium Hydroxide (NH4OH) drop by drop

PEPTIDE STOCK SOLUTIONS
Cysteine peptide stock solution, 0.667 mM, 0.501 mg/ml: the previously calculated amount of the peptide (0.01044 g) was pre-weighted and 20 ml of pH 7.5 phosphate buffer was added in the volumetric glass right before beginning the assay.
Lysine peptide stock solution, 0.667 mM, 0.518 mg/ml: the previously calculated amount of the peptide (0.01152 g) was pre-weighted and 20 ml of pH 10.2 acetate buffer was added in a volumetric glass right before beginning of the assay.

TEST CHEMICAL STOCK SOLUTION
100 mM solutions of the test chemical in the appropriate solvent were prepared just before use. The needed amount of test chemical was calculated (0.3337 g ± 10 %) and weighted based on the single average molecular weight. 0.3656 g test chemical was weighted for the stock solution used for the cysteine peptide depletion determination and 0.3644 g test chemical was weighted for the stock solution used for lysine peptide depletion determination.

CALIBRATION SOLUTIONS
Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20 % acetonitrile:buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.

EVALUATION CRITERIA
By using the cysteine 1:10 only prediction model, the threshold of 13.89 % mean percent peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers in the framework of Integrated Approaches to Testing and Assesment (IATA). On the basis of the cysteine 1:10 only prediction model, chemicals assigned to the minimal reactivity class should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity class should be classified as sensitisers.

- less/equal to 13.89 %: no or minimal reactivity - negative
- between 13.90 % and 23.09 %: low reactivity - positive
- between 23.1 % and 98.24 %: moderate reactivity - positive
- equal/more than 98.25 %: high reactivity - positive

Positive control results:

Obtained mean % cysteine peptide depletion: 69.56 %
Obtained mean % lysine peptide depletion: 55.93 %
Mean % obtained peptide depletion: 62.75

Parameter:

other: mean % cysteine and percent lysine depletion

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

moderate or high reactivity class

Parameter:

other: % cysteine depletion

Value:

66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Parameter:

other: % lysine depletion

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

due to co-elution

Other effects / acceptance of results:

The percent cysteine peptide depletion with the test item was 66.00 %, while the percent lysine depletion could not be determined with great confidence since co-elution was observed with lysine peptide. The maximum standard deviation for the test chemical replicates was 0.81 % for the percent cysteine depletion.
The mean percent cysteine and percent lysine depletion values were not calculated for the test chemical due to its co-elution.

The test item was assigned to the moderate reactivity class, classifying the test chemical into the sensitizer reactivity category.

SYSTEM SUITABILITY
Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior analysis. The mean peptide concentration of A reference control sample replicates was 0.54 mM for the cysteine peptide and 0.50 mM for the lysine peptide.

A standard calibration curve was generated for both cysteine and lysine peptides using serial dilutions standards from the peptide stock solutions. Calibration standard points were analysed by linear regression. Means of the peak areas versus the concentrations of both peptides showed good linearity with r2 = 0.9997 for the cysteine peptide and r2 = 0.9998 for the lysine peptide, covering the concentration range from 0.534 mM to 0.0167 mM. The back-calculated values for all the nominal concentrations of both peptides were within the acceptance criteria, not more than 8 % for the LOQs and not more than 11 % for the other calibration standards. Thus, all standards were accepted.

Reference control B replicates were included in the sequence to verify the stability of the peptide over time and reference control C replicates were used to verify that the solvent of the test item did not impact the percent peptide depletion. The mean cysteine peptide concentration of the reference control C replicates was 0.51 mM and the mean lysine peptide concentration of the reference control C replicates were 0.50 mM, which were within the acceptable 0.50 ± 0.05 mM range. Moreover the CV % for the nine reference control B and C replicates in acetonitrile and water respectively were much smaller than the acceptable 15 % for both peptides, since it was 3 % and 0 % for cysteine and lysine peptides.

All validity criteria were within acceptable limits and therefore the study can be considered valid.

CO-ELUTION
The test chemical did not absorbed at 220 nm significantly when tested with the cysteine peptide since the co-elution control area compared to the reference control area at similar retention time was only 2 %. Thus, there was no co-elution of the test chemical observed with the cysteine peptide.
However, the test chemical absorbed significantly at 220 nm when tested with the lysine peptide. The co-elution control area compared to the reference control area at similar retention time was more than 10 %. Thus, it can be concluded that there was co-elution of the test chemical observed when tested with the lysine peptide.

Cysteine peptide depletion values for the positive control and the test item

Test vessel	Peptide peak area at 220 nm	Peptide conc. calculated (mM)	Peptide depletion
			%	DS %
Reference ultra pure water, replicate 1	2097213	0.52	-	-
Reference ultra pure water, replicate 2	2080053	0.52	-
Reference ultra pure water, replicate 3	2079414	0.52	-
Test item, replicate 1	697521	0.17	66.74	0.81
Test item, replicate 2	725062	0.18	65.14
Test item, replicate 3	704370	0.18	66.13
Reference acetonitrile, replicate 1	2124325	0.53	-	-
Reference acetonitrile, replicate 2	1976461	0.49	-
Reference acetonitrile, replicate 3	2076242	0.52	-
Cinnamaldehyde, replicate 1	636637	0.16	70.03	0.86
Cinnamaldehyde, replicate 2	621147	0.15	68.57
Cinnamaldehyde, replicate 3	621254	0.15	70.08

Lysine peptide depletion values for the positive control and the test item

Name, replicate number	Peptide peak area at 220 nm	Peptide conc. calculated (mM)	Peptide depletion
			%	SD
ref C upw, rep I	2449062	0.49	-	-
ref C upw, rep II	2431885	0.49	-
ref C upw, rep III	2434854	0.49	-
Acid Yelow 236, rep I	255083	0.05	89.59	0.21
			co-elution
Acid Yelow 236, rep II	244060	0.05	89.96
			co-elution
Acid Yelow 236, rep III	253310	0.05	89.6
			co-elution
ref C acn, rep I	2479560	0.5	-	-
ref C acn, rep II	2457066	0.49	-
ref C acn, rep III	2468556	0.5	-
CINNAMALDEHYDE, rep I	1075585	0.21	56.62	0.22
CINNAMALDEHYDE, rep II	1089294	0.22	55.67
CINNAMALDEHYDE, rep III	1098347	0.22	55.51

Reference control A replicates for peptides

	Peptide peak area at 220 nm	Peptide conc. calculated (mM)	Peptide peak area		Peptide concentration
			Mean	CV %	Mean (mM)	SD	CV %
cysteine, replicate 1	2174926	0.54	2174130	1 %	0.54	0.0031	1 %
cysteine, replicate 2	2185922	0.55
cysteine, replicate 3	2161542	0.54
lysine, replicate 1	2494195	0.50	2492515	0 %	0.5	0.0006	0 %
lysine, replicate 2	2489269	0.50
lysine, replicate 3	2494082	0.50

Reference control B and C (acetonitrile) replicates for cysteine peptide

Name, replicate number	Peptide peak area at 220 nm	Peptide conc. calculated (mM)	Peptide peak area		Peptide concentration
			Mean	CV %	Mean (mM)	SD	CV %
For cysteine peptide
Reference control B, replicate 1	2136073	0.53	2100248	0 %	0.52	0.0142	3 %
Reference control B, replicate 2	2153229	0.54
Reference control B, replicate 3	2166313	0.54
Reference control B, replicate 4	2075080	0.52
Reference control B, replicate 5	2089711	0.52
Reference control B, replicate 6	2104799	0.53
Reference control C, replicate 1	2124325	0.53
Reference control C, replicate 2	1976461	0.49
Reference control C, replicate 3	2076242	0.52
For lysine peptide
Reference control B, replicate 1	2486559	0.50	2474902	0 %	0.5	0.0021	0 %
Reference control B, replicate 2	2479902	0.50
Reference control B, replicate 3	2487972	0.50
Reference control B, replicate 1/2	2465063	0.49
Reference control B, replicate 2/2	2468658	0.50
Reference control B, replicate 3/2	2480782	0.50
Reference control C, replicate 1	2479560	0.50
Reference control C, replicate 2	2457066	0.49
Reference control C, replicate 3	2468556	0.50

Interpretation of results:

other: potential skin sensitiser

Conclusions:

Potential skin sensitiser.

Executive summary:

The study was undertaken to evaluate the skin sensitization potential of the test item in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides.

At the beginning of the assay the solubility of the test chemical was assessed and ultrapure water was chosen as the appropriate solvent. Test chemical stock solutions were prepared in ultrapure water at the concentration of 100 mM. Cysteine and lysine peptide stock solutions were prepared at the concentrations of 0.501 mg/ml and 0.518 mg/ml with sodium phosphate buffer (pH=7.5) and ammonium acetate buffer (pH=10.2) respectively. Calibration standards were made by serial dilutions from these peptide stock solutions. Positive control stock solutions were prepared in acetonitrile at the concentration of 100 mM. The test chemical stock solutions were combined with the peptide stock solutions (1:10 and 1:50 ratio with cysteine and lysine peptides respectively) as reaction samples. Positive controls, reference controls and co-elution controls were assembled with the peptide stocks. The vials were placed to the HPLC autosampler for 24 ± 2 h incubation at 25 ± 2.5 °C in the dark. High performance liquid chromatography (HPLC) analysis of the batch of reaction samples started after the incubation period. Concentrations of the peptides following reaction time were determined by HPLC with gradient elution and UV detection at 220 nm.

Cysteine and lysine depletion values were to be used to categorize the test chemical in one of the four classes of reactivity. Since co-elution was observed with lysine peptide, the cysteine 1:10 only prediction model was used, where the threshold of 13.89 % mean percent peptide depletion supported the discrimination between skin sensitisers and non-sensitisers in the framework of Integrated Approaches to Testing and Assesment (IATA).

The positive control replicates showed the expected percent peptide depletion values within acceptable limits (69.56 % with cysteine peptide and 62.75 % mean percent peptide depletion). The back-calculated values of the reference control replicates were within the expected molarity concentration range. The experiment was considered to be valid.

The percent cysteine peptide depletion value of test item was 66.00 %, while with lysine peptide co-elution occurred. Therefore, the 1:10 cysteine only prediction model was used for the discrimination between sensitisers and non-sensitisers based on the threshold of 13.89 %. The percent cysteine peptide depletion value of the test item far exceeds the mentioned threshold.

Conclusion

Results obtained from in chemico Direct Peptide Reactivity Assay with the test item indicated that the substance is a potential skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Investigation on the skin sensitisation potential has been performed with the integrated evaluation of the following studies: in chemico Direct Peptide Reactivity Assay (DPRA), in vitro Keratinocytes test (KeratinoSens) and in vitro Human Cell Line Activation Test (h-CLAT).

DIRECT PEPTIDE REACTIVITY ASSAY – DPRA

The study was undertaken to evaluate the skin sensitization potential of the test item in chemico. The Direct Peptide Reactivity Assay (DPRA) proposed the molecular initiating event of the skin sensitisation Adverse Outcome Pathway (AOP), namely protein reactivity, by quantifying the reactivity of the test chemical towards cysteine and lysine model synthetic peptides.

The percent cysteine peptide depletion value of test item was 66.00 %, while with lysine peptide co-elution occurred. Therefore, the 1:10 cysteine only prediction model was used for the discrimination between sensitisers and non-sensitisers based on the threshold of 13.89 %. The percent cysteine peptide depletion value of the test item far exceeds the mentioned threshold.

KERATINOSENS™ KERATINOCYTES TEST

The skin sensitisation potential of test substance was investigated by Keratinocytes test based on the signaling pathway Keap1-Nrf2-ARE coupled to the luciferase reporter gene. The experiment was conducted following the method and procedures outlined into the OECD guideline 442D. During both the repetitions performed, the Imax resulted to be higher than 1.5. However, in both the cases, at the EC1.5 concentration (18.72 and 2.79 μg/ml, respectively) viability resulted to be lower than 70 %. Therefore, the two repetitions were considered as negative.

HUMAN CELL LINE ACTIVATION TEST (h-CLAT).

The substance was tested for skin sensitisation potential, following the method and procedures described into the OECD Guideline 442E. The RFI of CD86 resulted to be lower than 150 % at any tested concentration (with cell viability ≥ 50 %), in both runs; the RFI of CD54 resulted to be lower than 200 % at any tested concentration (with cell viability ≥ 50 %), in both runs. Therefore, under the test conditions of the experimental human Cell Line Activation Test, the test item has been judged to be a non-sensitizer.

DISCUSSION AND CONCLUSION

[details about the irritating potential conclusion are given in the document attached]

Since each investigation (as protein binding, keratinocyte and dendritic cells activation) represents only one key event of the skin sensitisation Adverse Outcome Pathway, information generated with test methods developed to address specific key events may not be sufficient to conclude on the presence or absence of skin sensitisation potential of chemicals; therefore, an Integrated Approaches to Testing and Assessment is recommended.

Based on the study outcomes, Acid Yellow 236 results to be able to bind skin proteins; however, the possibility of this interaction does not entail that the subsequent events, involved in the skin sensitisation induction, occur.

The integration of the information deriving from in chemico study and QSAR estimation suggests that the parent compound contains functional groups, which could be able to bind the skin proteins. However, metabolization of the parent compound, would require conditions, which are not likely to be satisfied for skin contact. In addition, the assessment of the possible critical metabolite does not rise any reason of concern for skin sensitisation.

The test conducted with Acid Yellow 236 investigating the capability of the substance to induce a response in keratinocyte gave negative results, as well as the test investigating the possibility of the test item to activate dendritic cells.

In conclusion, although Acid Yellow 236 is characterized by functional groups, which may act as stressors, the Integrated Approaches to Testing and Assessment show that there are no reasons to expect a skin sensitisation potential for Acid Yellow 236.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

Based on the Integrated Approaches to Testing and Assessment, there are no reasons to expect a skin sensitisation potential for Acid Yellow 236.

In conclusion, Acid Yellow 236 does not comply to the definition as skin sensitizer, as indicated in the CLP Regulation (EC) No 1272/2008.