N-ethylpyridazin-4-amine

Endpoint summary

Description of key information

Based on GLP-conform in vitro skin sensitisation Turnkey Testing, the following results were achieved:

- DPRA, OECD 442C (peptide reactivity): negative

- LuSens, OECD 442D (keratinocyte activation): negative

- h-CLAT, OECD 442E (dendritic cell activation): positive
Conclusively, the test substance is predicted not to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 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))

Version / remarks:

04 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L2018-006
- Expiration date of the batch: 01 Mar 2020
- Purity: 98.5% (tolerance +/- 1.0 %)
- Physical state / color: solid / white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: Because the test-substance preparation was incubated with the peptide shortly after preparation, no analysis of the test substance in the vehicle was performed.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the study period was guaranteed.

TEST SUBSTANCE SOLUBILITY
- After stirring and ultrasonication the test substance was soluble (colloid solution) in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 123.16 g/mol and a purity/contents of 98.5%.

OTHER SPECIFICS:
- Identity: confirmed
- Homogeneity: given
- Molecular weight: 123.16 g/mol
- Log KOW: 0.06 (calculated)
- Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system): The OECD toolbox did not indicate an alert for protein binding for the substance.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

- EXPERIMENTAL PROCEDURE
The test substance was dissolved in a suitable vehicle. Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (=NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

- TEST SUBSTANCE SOLUBILITY
Prior to the assay the solubility of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test-susbtance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

- SYNTHETIC PEPTIDES:
-Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
-Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

- CONTROLS
-Negative control (NC): vehicle control = acetonitrile
-Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as a 50 mM solution in acetonitrile
-Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide.

- SELECTION OF CONCENTRATIONS
The test substance was prepared at a 100 mM concentration considering a purity/contents of 98.5% and molecular weight of 123.16 g/mol. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

- PREPARATION OF PEPTIDE STOCK SOLUTIONS
Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.

- PREPARATION OF CALIBRATION SAMPLES (see table 1)
The calibration samples were prepared from the peptide stock solutions in 20% acetonitrile in the respective buffer (= dilution buffer). The analysis of the calibration samples was started before analysis of the test-substance samples.

- TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 123.16 g/mol and a purity/contents of 98.5%. After stirring and ultrasonication the test substance was soluble (colloid solution) in the vehicle.
Vehicle: Acetonitrile
Reason for the vehicle: The test substance was soluble (colloid solution) in acetonitrile.

- PREPARATION OF THE TEST-SUBSTANCE SAMPLES
The samples were prepared in triplicates for each peptide according to the pipetting scheme given in table 2. The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 ± 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

- PREPARATION OF THE VEHICLE CONTROLS
Several vehicle controls were prepared in triplicates in the same way as the test-substance samples described above but with the vehicle (deionized water) instead of the test substance:
One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

- PREPARATION OF THE CO-ELUTION CONTROL
One sample per peptide was prepared in the same way as the test-substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged and/or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

- MEASUREMENT OF PEPTIDE CONCENTRATIONS
The analyses of the samples were performed via HPLC under the conditions given in table 3.

- DATA EVALUATION
The integrated peak areas were transferred electronically into EXCEL data spreadsheets to carry out the necessary calculations. Some test substances or reaction products may co-eluate with the peptides. In these cases where proper integration and calculation of peptide depletion was not possible, the result for the respective peptide is reported as interference. For evaluation of peptide depletions peak areas at 220 nm are used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples (evaluation criteria see tables 4 and 5).

- ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² > 0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- In addition the positive control should cause depletion of both peptides comparable to historic data.

Positive control results:

Positive control (EGDMA):
Mean depletion (Cysteine-Peptide): 52.76% (+/- 2.72)
Mean depletion (Lysine-Peptide): 9.08% (+/- 0.94)
Mean of both depletions: 30.92%

Parameter:

other: Mean depletion (Cysteine-Peptide) [%]

Value:

1.32

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Parameter:

other: Mean depletion (Lysine-Peptide) [%]

Value:

-0.21

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Parameter:

other: Mean of both depletions [%]

Value:

0.66

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 6: Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide   mean depletion [%]      SD [%]		Lysine-Peptide   mean depletion [%]        SD [%]		mean of both depletions [%]
test substance	1.32	0.79	-0.21	0.37	0.66
PC:EGDMA in ACN	52.76	2.72	9.08	0.94	30.92

Table 7: Peptide depletion of NC, PC and the test substance for cysteine-peptide.

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean           SD
NC: ACN	-0.13	0.16	-0.03	0.00           0.15
test substance	0.42	1.66	1.89	1.32           0.79
PC:EGDMA in ACN	50.01	52.83	55.45	52.76          2.72

Table 8: Peptide depletion of NC, PC and the test substance for lysine-peptide.

Reaction with lysine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean SD
NC: ACN	-0.05	-0.09	0.14	0.00 0.12
test substance	-0.28	0.19	-0.53	-0.21 0.37
PC:EGDMA in ACN	9.67	9.58	8.00	9.08 0.94

Table 9: Historic control data of negative control / vehicle control (not including present study). Acetonitrile (Historic period: Jan 2017 - Feb 2019)

	C-peptide concentration [mM]	K-peptide concentration [mM]
Min	0.432	0.457
Max	0.564	0.548
Mean	0.492	0.504
SD	0.030	0.015
n	34	33

Table 10: Historic control data of positive control (not including present study). EGDMA, 50 mM in acetonitrile (Historic period: Jan 2017 - Feb 2019)   C-peptide concentration [mM]   C-peptide depletion [%] K-peptide concentration [mM]   K-peptide depletion [%] Min 0.080 46.26 0.395 6.79 Max 0.285 83.79 0.513 19.85 Mean 0.203 58.91 0.445 11.76 SD 0.046 8.17 0.020 2.78 n 32 32 30 30

Interpretation of results:

other: Non peptide binding/peptide binding

Conclusions:

No predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.
In the present study, the test substance shows minimal or no peptide binding and therefore minimal or no chemical reactivity under the test conditions chosen.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

DPRA:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50

(for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a coelution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm. The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM (colloid solution). The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 1.32%. The mean K-peptide depletion, caused by the test substance was determined to be -0.21%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.66%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model cited in chapter 3.10 it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March - May 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:

25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L2018-006
- Expiration date of the batch: 01 Mar 2020
- Purity: 98.5% (tolerance +/- 1.0 %)
- Physical state / color: solid / white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: Because the test-substance preparation was applied shortly after preparation, no analysis of the test substance in the vehicle was performed.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the study period was guaranteed.

TEST SUBSTANCE SOLUBILITY
- After stirring and ultrasonication the test substance was soluble in 4% DMSO in culture medium 3. Visual inspection of each dilution step was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium 3) to achieve the required 4x concentration of the highest concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution.

OTHER SPECIFICS:
- Identity: confirmed
- Homogeneity: given
- Molecular weight: 123.16 g/mol
- Log KOW: 0.06 (calculated)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

- SELECTION OF CONCENTRATIONS
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test-substance preparation as provided by the sponsor (0.5 μM up to 2030 μM corresponding to final test-substance concentrations of 0.5 μM up to 2000 μM taking a purity/contents of 98.5% into account) and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 1044 μM (test substance as provided by the sponsor).
The highest tested concentration in the 1st main experiment was 1.23-fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration (concentration selection for the 1st experiment [µM]: 503, 604, 725, 870, 1044, 1253, 1503, 1804). As no rel. viability below 70% was observed in the 1st experiment, higher concentrations were tested in further experiments (concentration selection for the 2nd and 3rd experiment [µM]: 567, 680, 816, 979, 1175, 1410, 1692, 2030).

- CELL LINE LuSens (Human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany)

- CONTROLS
- Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 μM (= 450 μg/mL) in 1% DMSO in culture medium 3
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 90.8 μM (= 18 μg/mL) in 1% DMSO in culture medium 3
- Vehicle control (VC): 1% DMSO in culture medium 3
- Blank control: culture medium 3 without cells
- Basal control: culture medium 3 with cells

- TEST-SUBSTANCE PREPARATIONS
The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium 3) to achieve the required 4x concentration of the highest concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate). The test-substance preparations were prepared by stirring and ultra-sonication.

- PREPARATION OF THE CELLS
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% relative humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 105 cells/mL cell suspension), using culture medium 2 for incubation for ca. 24 hours. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

- TEST-SUBSTANCE APPLICATION FOR MTT AND LUCIFERASE ASSAY
After cell adaption for ca. 24 hours culture medium 2 was aspirated and replaced with 150 μL culture medium 3. The test substance was prepared as described in section 3.5.2. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%). After test-substance application the plates were sealed with plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard
culture conditions for the exposure period of 48 ± 1 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.

- VISUAL INSPECTIONS
Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 ± 1 hours in order to detect test-substance precipitates.

- LUCIFERASE ASSAY
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of One-Glo-preparation (= 100 μL One-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for ca. 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

- CELL VIABILITY ASSAY MTT
Cell culture medium was aspirated from all wells. The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and culture medium 3) was added to each well of the 96-well microtiter plate and incubated for at least further 2 hours in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

- STATISTICAL ANALYSES
For the statistical evaluation of luciferase fold induction the EXCEL-function T.TEST is used (see table 1).

- ACCEPTANCE CRITERIA
- A tested concentration is not further evaluated when relative viability is less than 70%.
- The cell viability of VC cells must yield at least 85%.
- The mean of the positive control EGDMA should achieve ≥ 2.50 fold-induction and the mean of the LA < 1.50 and the mean of the viability must be ≥70%.
- The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
- The mean of the basal expression of the cells must be <1.50 as compared to the solvent control.
- In addition, positive, negative and vehicle control data should lie within the range of the historic data.

- EVALUATION OF THE RESULTS
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments. A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration displaying viability below 70%). To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

Positive control results:

Positive control: EGDMA 90.8 µM
mean (fold induction): 5.42 (1st experiment) / 3.89 (2nd experiment) / 3.60 (3rd experiment)

Run / experiment:

other: 1-3

Parameter:

other: EC1.50

Remarks:

Calculation of an EC1.50 was not applicable as luciferase fold induction did not exceed 1.50.

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

- RESULTS OF PRELIMINARY CYTOTOXICITY ASSESSMENT (see table 2):
The final test substance concentrations were calculated considering a purity/contents of 98.5%. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 1044 μM (test substance as provided by the sponsor).

- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 2: Results of preliminary cytotoxicity assessment.

Concentration (final test substance) [µM]	Concentration (test substance) [µM]	mean OD570-690 of 3 replicates	mean rel Viability [%]
VC	VC	0.306	100
0.5	0.5	0.298	98
1	1	0.296	97
5	5	0.292	95
10	10	0.281	92
50	51	0.280	92
100	102	0.245	80
500	508	0.239	78
1000	1015	0.231	75
2000	2030	0.182	60

Table 3: Summary of LuSens Main Experiments

A total of 3 valid experiments were performed. However, in the 1st experiment the concentrations selected were too low (no viability < 70% or keratinocyte activating effect were observed) and the experiment is not useful for evaluation. Concentrations with fold inductions above 1.50 with rel. viability ≥70% and with statistical significance are indicated in bold and in grey when < 70% viability.

Concentration (test substance) [µM]	1^stexperiment fold induction rel. viability [%]			t-test		Concentration (test substance) [µM]	fold induction	2^ndexperiment rel. viability [%]		t-test
	mean	mean	p-value		markers		mean	mean	p-value		markers
503	1.02	84	0.125		n.s.	567	0.88	73	0.033		*
604	1.14	85	0.072		n.s.	680	0.93	75	0.136		n.s.
725	1.08	80	0.151		n.s.	816	0.86	72	0.066		n.s.
870	0.91	80	0.113		n.s.	979	0.83	70	0.000		**
1044	0.83	77	0.000		**	1175	0.96	71	0.319		n.s.
1253	0.75	75	0.000		**	1410	0.88	66	0.072		n.s.
1503	0.72	73	0.001		**	1692	0.99	68	0.463		n.s.
1804	0.60	70	0.005		**	2030	1.05	63	0.251		n.s.
VC	1.00	100	-		-	VC	1.00	100	-		-
EGDMA 90.8 µM	5.42	100	0.000		**	EGDMA 90.8 µM	3.89	75	0.000		**
LA 5000 µM	0.96	100	0.143		n.s.	LA 5000 µM	0.86	86	0.016		*
Concentration	3^rdexperiment fold induction rel. viability [%] t-test
(test substance)
[µM]	mean	mean	p-value		markers
567	0.71	86	0.000		**
680	0.72	68	0.004		**
816	0.64	84	0.000		**
979	0.63	80	0.000		**
1175	0.66	79	0.002		**
1410	0.63	75	0.006		**
1692	0.58	68	0.005		**
2030	0.57	72	0.000		**
VC	1.00	100	-		-
EGDMA 90.8 µM	3.60	91	0.000		**
LA 5000 µM	0.93	99	0.083		n.s.

Table 4: Historic control data. Data shown of test period Mar 2017 until May 2019 (not including present study).

Negative Control (5000 µM (= 450 µg/mL))	fold induction	rel. viability [%]
Min	0.71	88
Max	1.13	140
Mean	0.93	102
SD	0.09	8
n	201	184

Positive Control EGDMA (90.8 µM (= 18 µg/mL))	fold induction	rel. viability [%]
Min	2.83	70
Max	8.64	122
Mean	5.33	87
SD	1.15	10
n	201	184

Vehicle Control (1% DMSO)	fold induction	rel. viability [%]
Min	1.00	100
Max	1.00	100
Mean	1.00	100
SD	0.00	0
n	201	184

Basal expression	fold induction	rel. viability [%]
Min	0.61	121
Max	1.32	179
Mean	0.93	141
SD	0.13	12
n	201	184

Interpretation of results:

other: no keratinocyte activating potential

Conclusions:

No predictions can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.
In the present study, the test substance does not have a keratinocyte activating potential.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

LuSens:

The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP)

was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 1044 μM (corresponding to test substance as provided by the sponsor). In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. The following results were observed: The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable as luciferase fold induction did not exceed 1.50. In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD442E "In vitro skin sensitisation assays addressing the key event on activation of dendritic cells on the adverse outcome pathway for skin sensitisation"

Version / remarks:

25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht; Kaiser-Friedrich-Straße 7, 55116 Mainz

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: L2018-006
- Expiration date of the batch: 01 Mar 2020
- Purity: 98.5% (tolerance +/- 1.0 %)
- Physical state / color: solid / white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: The h-CLAT was performed in an aqueous test system. Due to the use of culture medium as vehicle the verification of the stability of the test substance in the vehicle is not required.
- Solubility and stability of the test substance in the solvent/vehicle: The stability under storage conditions over the study period was guaranteed.

TEST SUBSTANCE SOLUBILITY
- After stirring the test substance was soluble in culture medium. Visual inspection of each dilution step was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2x concentration of the highest concentration (stock solution). Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution.

OTHER SPECIFICS:
- Identity: confirmed
- Homogeneity: given
- Molecular weight: 123.16 g/mol
- Log KOW: 0.06 (calculated)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

- PREPARATION OF THE CELLS
THP-1 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium supplemented with 10% fetal bovine serum (heat inactivated), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.05 mM 2-mercaptoethanol under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% relative humidity) until for 5 passages but not longer than passage 30 prior to testing.
Prior to use of the cells for a study, a reactivity check was performed with each new-thawed cells, as proposed in the OECD test guideline, using Nickel(II)sulfate hexahydrate, lactic acid and 1- chloro-2,4-dinitrobenzene in order to demonstrate qualification of the cells for the assay.
For substance incubation, cells were seeded in 24-well plates (500 μL of 2.0 x 106 cells/mL cell suspensions). Three independent, valid experiments were performed. In each experiment, duplicates of each test-substance concentration were tested; however, in the second experiment one sample could be evaluated only, due to a technical error.

- SELECTION OF CONCENTRATIONS
In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 11 concentrations of the test-substance preparation as provided by the sponsor (4 μg/mL up to 5076 μg/mL corresponding to final test substance ingredient concentrations of 4 μg/mL up to 5000 μg/mL taking the purity of 98.5% into account) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 23251 μg/mL (test substance as provided by the sponsor). The highest tested concentration in the 1st main experiment was 1.2-fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration (concentration selection for the main experiments [µg/mL]: 779, 935, 1122, 1346, 1615, 1938, 2326, 2791).

- CELL LINE: THP-1 cells (The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB-202).

- TEST SUBSTANCE PREPARATION
The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2x concentration of the highest concentration (stock solution). Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations in culture medium. The test-substance preparations were prepared by stirring.
Reason for the vehicle: The test substance was soluble in culture medium.
Form of application: Visual inspection of each dilution step was performed.

- TEST-SUBSTANCE APPLICATION
Treatment was performed by adding 500 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 106 cells/mL. After test-substance application the plates were sealed with plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 24 ± 0.5 hours.

- VISUAL INSPECTIONS
Each test-substance concentration was visually inspected directly after application and after the exposure period of 24 ± 0.5 hours in order to detect test-substance precipitates.

- CELL STAINING AND FLOW CYTOMETRIC ANALYSIS
After visual inspection the cells were transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL buffer. Cells were incubated with 600 μL of 0.01% Globulins Chon fraction II,III at 4°C for 15 min to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots (approximately 0.3 x 106 cells/180 μL/group) in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 μL working antibody solution was added to each pellet. Working solutions of the antibodies were prepared (see table 1). Cell staining was performed at 4°C for ca. 30 min in the dark. After staining the cells were washed twice with 200 μL buffer and finally re-suspended in 200 μL buffer. Before analysis in flow cytometer the cells were stained with 5 μL of PI (50 μg/mL diluted in buffer) to yield a final concentration of 1.22 μg/mL PI.

- ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when relative viability is less than 50%.
- Cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86< 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥105%.
-The reactivity check of new thawed cells should produce the following result:
- Positive response in CD86 and CD54 for NiSO4 and DNCB
- Negative response in CD86 and CD54 for LA.
-In addition, positive, negative and vehicle control data should lie within the range of the historic data.

- EVALUATION OF THE RESULTS
A test substance is predicted to activate monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments. A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 5000 μg/mL for the vehicle culture medium or 1000 μg/mL for 0.2% DMSO in culture medium) or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested). To be relevant for evaluation, the cell viability must be more than 50% in at least four tested concentrations of an experiment.

- CONTROLS
Negative control (NC): Lactic acid (LA, CAS no.: 50-21-5), 1000 μg/mL in culture medium
Positive control (PC): 1-chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 μg/mL in 0.2% DMSO in culture medium
Vehicle control (VC): culture medium
Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1.

Positive control results:

Positive control: DNCB 4 µg/mL:
- RFI CD86 mean [%]: 290 (1st experiment) / 236 (2nd experiment) / 251 (3rd experiment)
- RFI CD54 mean [%]: 657 (1st experiment) / 843 (2nd experiment) / 548 (3rd experiment)
- Rel. Viability [%]: 59 (1st experiment) / 83 (2nd experiment) / 68 (3rd experiment)

Run / experiment:

other: 3

Parameter:

other: EC150% for CD86 [µg/mL]

Value:

1 055

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: EC200% for CD54[µg/mL]

Value:

1 024

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 2325 μg/mL (test substance as provided by the sponsor).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 2: h-CLAT. Results of preliminary cytotoxicity assessment. The final test substance concentrations were calculated considering the purity/contents of 98.5%.

Concentration (final test substance) [µg/mL]	Concentration (test substance) [µg/mL]	%PI negative cells replicate 1	%PI negative cells replicate 2	mean viability of duplicates	rel. viability mean [%]
VC	VC	97	97	97	100
3.9	4	98	97	97	100
7.8	8	97	97	97	100
16	16	97	97	97	100
31	32	97	97	97	100
63	63	97	98	97	100
125	127	97	97	97	100
250	254	97	97	97	100
500	508	97	97	97	99
1000	1015	96	95	95	98
2500	2538	72	67	69	71
5000	5076	2	5	3	3

Table 3: Summary of h-CLAT main experiments: mean values of RFI CD86, RFI CD54 and relative viability.

3 valid experiments were performed. However, in the second experiment one sample instead of two samples is available for each concentration, only, due to a technical error. RFI above 150% (CD86) or 200% (CD54) with rel. viability ≥50% are indicated in bold.

1^stexperiment				2^ndexperiment
Concentration(testsubstance)[µg/mL]	RFI CD86	RFI CD54	Viability rel. viability [%]	Concentration(testsubstance)[µg/mL]	RFI CD86	RFI CD54	Viability rel. viability [%]
	mean [%]	mean [%]			mean [%]	mean [%]
779	156	221	97	779	147	145	99
935	160	276	93	935	148	180	97
1122	146	331	88	1122	140	222	96
1346	135	321	89	1346	119	224	95
1615	161	372	80	1615	128	280	93
1938	153	314	75	1938	120	245	85
2326	119	286	69	2326	103	215	76
2791	85	207	67	2791	66	154	75
VC	100	100	100	VC	100	100	100
LA 1000 µg/mL	87	128	99	LA 1000 µg/mL	72	77	99
DNCB 4 µg/mL	290	657	59	DNCB 4 µg/mL	236	843	83
3^rdexperiment
Concentration	RFI CD86	RFI CD54	Viability
(test substance) [µg/mL]	mean [%]	mean [%]	rel. viability [%]
779	149	294	98
935	146	326	96
1122	152	385	93
1346	141	404	90
1615	134	414	81
1938	111	352	75
2326	93	348	74
2791	56	219	74
VC	100	100	100
LA 1000 µg/mL	76	137	99
DNCB 4 µg/mL	251	548	68

Table 4: Historic control data. Data shown of test period Sep 2018 until Feb 2019 (not including present study).

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]		rel. viability mean [%]
Min	61	90		94		99
Max	100	174		98		101
Mean	82	128		96		100
SD	10	22		1		1
n (experiments)			17

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]		rel. viability mean[%]
Min	184	194		55		57
Max	356	1095		88		91
Mean	240	472		72		74
SD	35	240		9		9
n (experiments)			17

Vehicle Control (culture medium)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]
Min	79	71		95
Max	121	129		98
Mean	100	100		96
SD	12	12		1
n (experiments)			17

Vehicle Control (DMSO)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]		rel. viability mean[%]
Min	75	67		94		99
Max	152	165		98		101
Mean	115	116		97		100
SD	16	21		1		0
n (experiments)			17

Interpretation of results:

other: positive indication of skin sensitisation

Conclusions:

No prediction can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.
According to the results of the present study, the test substance induces dendritic cell activation, which is indicative of skin sensitisation.

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

h-CLAT:

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose, the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 2325 μg/mL (corresponding to test substance as provided by the sponsor).

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid experiments were performed although the result of the 2nd experiment confirmed the result of the 1st experiment, as one sample instead of two samples was available in the 2nd experiment, only, due to a technical error. The following results were observed:

The test substance was soluble in culture medium (2 x stock preparations and final concentrations). No precipitates were noticed in any concentration after 24 hours.

The EC150% (the concentration resulting in a RFI of 150%) for CD86 was calculated to be 1055 μg/mL (experiment 3). The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 1024 μg/mL (experiment 2).

Calculation of EC150% for CD86 and EC200% for CD54 was not applicable for the other experiments.

In summary, after 24 hours of exposure to the test substance, CD86 and CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

DPRA:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at 100 mM in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50

(for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, in order to detect possible interference of the test substance with the peptides, a coelution control was performed and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm. The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM (colloid solution). The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present. The mean C-peptide depletion, caused by the test substance was determined to be 1.32%. The mean K-peptide depletion, caused by the test substance was determined to be -0.21%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.66%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model cited in chapter 3.10 it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

LuSens:

The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP)

was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 1044 μM (corresponding to test substance as provided by the sponsor). In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. The following results were observed: The test substance was soluble in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable as luciferase fold induction did not exceed 1.50. In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

h-CLAT:

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose, the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration-response curve to be 2325 μg/mL (corresponding to test substance as provided by the sponsor).

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid experiments were performed although the result of the 2nd experiment confirmed the result of the 1st experiment, as one sample instead of two samples was available in the 2nd experiment, only, due to a technical error. The following results were observed:

The test substance was soluble in culture medium (2 x stock preparations and final concentrations). No precipitates were noticed in any concentration after 24 hours.

The EC150% (the concentration resulting in a RFI of 150%) for CD86 was calculated to be 1055 μg/mL (experiment 3). The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 1024 μg/mL (experiment 2).

Calculation of EC150% for CD86 and EC200% for CD54 was not applicable for the other experiments.

In summary, after 24 hours of exposure to the test substance, CD86 and CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Table 1: Summary of individual test results.

Test Method	Test Result	Test Evaluation
Direct Peptide Reactivity Assay (DPRA)	1.32% mean peptide depletion (-0.21% cysteine-peptide depletion; 0.66% lysine- peptide depletion).a	Negative
Keratinocyte Activation Assay - LuSens	In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.	Negative
Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)	In at least two independent experiments an induction of the expression of CD86 (above 150%) and CD54 (above 200%) were observed at sufficiently non-cytotoxic (cell viability ≥ 50%) concentration.	Positive

a Negative depletions were considered to be “zero” for calculation of mean peptide depletion.

Based on the results summarized in Table 1, the test substance is not peptide reactive, does not activate keratinocytes and activates dendritic cells.

Applying the evaluation criteria ("2 out of 3"), the test substance is predicted not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Negative responses were observed in a direct peptide binding assay (OECD442C) and in an activation of keratinocytes (LuSens) assay (OECD442D). Positive response was observed in an activation of dendritic cells (h-CLAT) assay (OECD442E). The test substance is therefore predicted not to be a skin sensitizer (Evaluation criteria "2 out of 3").