Dibenzyldimethylammonium chloride

Endpoint summary

Description of key information

A GLP-compliant in vitro skin sensitization turnkey testing strategy was conducted according to OECD Guidelines 442C, 442D and 442E to assess the skin sensitizing potential of the test substance. The three in vitro methods addressed key events of the adverse outcome pathway (AOP) for skin sensitization as defined by the OECD: protein reactivity (DPRA), activation of keratinocytes (LuSens) and activation of dendritic cells (h-CLAT). Based on the positive results observed in the LuSens and the h-CLAT it is concluded that the test substance shows a skin sensitizing potential in this in vitro skin sensitization turnkey test strategy.

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:

May - Jun 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 Rheinland-Pfalz

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: solid, white
- Molecular weight: 261.794 g/mol
- Log Kow: - 0.77 (information provided by the sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient; under N2
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in acetonitrile considering a molecular weight of 261.794 g/mol and a purity / contents of 91.1 %. After short stirring the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

Details on the study design:

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

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 THE TEST SUBSTANCE SAMPLES
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 (acetonitrile) 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.

Run / experiment:

other: mean of both depletions

Parameter:

other: Mean peptide depletion [%]

Value:

0.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

mean

Parameter:

other: C-peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

negative results were set to "0"

Run / experiment:

mean

Parameter:

other: L-peptide depletion [%]

Value:

0.08

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Table 5: 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	- 0.05	0.17	- 0.11	0.00	0.15
Test substance	- 0.29	- 0.34	0.28	- 0.12	0.34
PC: EGDMA in ACN	50.31	51.92	53.42	51.88	1.56

Table 6: Peptide depletion of NC, PC and the test substance for lysine-peptide

Reaction with cysteine-peptide	Peptide depletion [%]
	Sample 1	Sample 2	Sample 3	Mean	SD
NC: ACN	- 0.19	- 0.62	0.81	0.00	0.74
Test substance	0.00	0.15	0.08	0.08	0.07
PC: EGDMA in ACN	11.47	12.92	13.04	12.48	0.87

Table 7: Mean peptide depletions of Cysteine, Lysine and both peptides

	Cysteine-Peptide		Lysine-Peptide		Mean of both depletions [%]
	Mean depletion [%]	SD [%]	Mean depletion [%]	SD [%]
Test substance	- 0.12	0.34	0.08	0.07	0.04
PC: EGDMA in ACN	51.88	1.56	12.48	0.87	32.18

Conclusions:

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance was minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Executive summary:

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. 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 - 0.12 %.

The mean K-peptide depletion, caused by the test substance was determined to be 0.08 %.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.04 %.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model 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:

May - Jun 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:

14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: solid, white
- Molecular weight: 261.794 g/mol
- Log Kow: - 0.77 (information provided by the sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient; under N2
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- 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 according to the planned concentrations (master plate). The test substance preparations were prepared by stirring.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution

Details on the study design:

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

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.
Two 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. 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.

Run / experiment:

other: preliminary cytotoxicity assessment

Parameter:

other: CV75 [µM]

Value:

1 092

Vehicle controls validity:

valid

Run / experiment:

other: Experiment 1, plate 1

Parameter:

other: mean fold induction

Value:

3.19

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 1, plate 2

Parameter:

other: mean fold induction

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2, plate 1

Parameter:

other: mean fold induction

Value:

3.16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2, plate 2

Parameter:

other: mean fold induction

Value:

1.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

For calculation of mean fold induction the values with relative viability above 70 % were used.
The EC1.50 was calculated by linear regression from the results of the 171 µM and the 205 µM concentrations to be 187 µM.

Table 1: Results of preliminary cytotoxicity assessment

Concentration (final test substance) [µM]	Concentration (test substance) [µM]	Mean OD570-690of 3 replicates	Mean rel. viability [%]
VC 0.5 1 5 10 50 100 500 1000 2000	VC 0.5 1 5 11 55 110 549 1098 2195	0.330 0.303 0.315 0.327 0.326 0.328 0.299 0.308 0.247 0.171	100.0 92.0 95.4 99.2 99.0 99.5 90.6 93.5 74.8 52.0

Table 2: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 1, plate 1)

Concentration (test substance) [µM]	Fold induction		Rel. viability [%]		t-test
	Mean	SD	Mean	SD	p-value	Markers
613 735 882 1059 1270 1524 1829 2195	3.19 5.47 3.60 4.39 5.99 5.68 8.27 7.77	0.98 1.29 0.36 0.67 0.51 0.41 1.07 0.82	73 58 60 47 46 34 25 24	3 2 1 1 1 2 2 3	0.030 0.013 0.003 0.006 0.002 0.001 0.004 0.002	* * ** ** ** ** ** **
VC EGDMA 90.8 µM LA 5000 µM	1.00 4.94 0.78	0.14 0.17 0.02	100 95 101	4 9 6	- 0.021 0.000	- * **

Table 3: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 1, plate 2)

Concentration (test substance) [µM]	Fold induction		Rel. viability [%]		t-test
	Mean	SD	Mean	SD	p-value	Markers
142 171 205 246 295 355 425 510	1.51 1.78 1.47 1.91 3.18 2.93 3.10 3.07	0.22 0.17 0.12 0.21 0.67 0.55 0.40 0.45	101 90 94 93 88 84 78 73	5 3 2 2 3 3 3 2	0.027 0.006 0.007 0.008 0.015 0.013 0.006 0.007	* ** ** ** * * ** **
VC EGDMA 90.8 µM LA 5000 µM	1.00 5.55 0.87	0.10 0.66 0.09	100 93 97	3 2 3	- 0.000 0.012	- ** *

Table 4: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 2, plate 1)

Concentration (test substance) [µM]	Fold induction		Rel. viability [%]		t-test
	Mean	SD	Mean	SD	p-value	Markers
613 735 882 1059 1270 1524 1829 2195	3.16 3.10 3.87 7.70 8.73 8.71 8.78 8.27	0.41 0.48 0.60 1.61 0.60 0.77 0.15 1.60	73 68 66 54 51 38 30 36	0 3 1 2 2 1 2 1	0.005 0.008 0.007 0.009 0.001 0.002 0.000 0.008	** ** ** ** ** ** ** **
VC EGDMA 90.8 µM LA 5000 µM	1.00 5.89 1.05	0.12 0.74 0.29	100 89 97	4 2 5	- 0.000 0.361	- ** n.s.

Table 5: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 2, plate 2)

Concentration (test substance) [µM]	Fold induction		Rel. viability [%]		t-test
	Mean	SD	Mean	SD	p-value	Markers
142 171 205 246 295 355 425 510	1.02 1.33 1.69 1.54 1.71 1.87 1.90 2.04	0.09 0.10 0.05 0.36 0.15 0.54 0.04 0.13	86 81 87 85 86 93 83 81	3 5 4 2 4 15 2 5	0.384 0.005 0.000 0.058 0.004 0.053 0.000 0.001	n.s. ** ** n.s. ** n.s. ** **
VC EGDMA 90.8 µM LA 5000 µM	1.00 5.90 0.85	0.15 1.32 0.09	100 93 97	9 4 2	- 0.001 0.011	- ** *

Conclusions:

Based on the results of this study, the test substance shows as a positive indication of keratinocyte activation.
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

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 1092 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 2 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.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 187 μM (experiment 2). For the 1st experiment, calculation of an EC1.50 was not applicable.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was 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 has a keratinocyte activating potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May - Jun 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E

Version / remarks:

25 Jun 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 17 Jan 2020
- Physical state / color: solid, white
- Molecular weight: 261.794 g/mol
- Log Kow: - 0.77 (information provided by the sponsor)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient; under N2
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- 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 according to the planned concentrations in culture medium. The test substance preparations were prepared by stirring.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution

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). Two independent, valid experiments were performed. In each experiment, duplicates of each test-substance concentration were tested.

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.
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.

Run / experiment:

other: preliminary cytotoxicity assessment

Parameter:

other: CV75 [µg/mL]

Value:

755

Vehicle controls validity:

valid

Run / experiment:

other: Experiment 1

Parameter:

other: EC200% [µg/mL]

Value:

386

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: EC150% [µg/mL]

Value:

532

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: EC200% [µg/mL]

Value:

492

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Table 2: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of first experiment

Concentration (test substance) [µg/mL]	RFI CD86		RFI CD54		Viability
	Mean [%]	SD [%]	Mean [%]	SD [%]	Rel. viability [%]	SD of viability
253 303 364 437 524 629 755 906	96 112 126 112 141 127 115 91	1 7 3 3 2 10 20 18	171 171 182 241 258 283 290 342	29 29 37 40 65 30 57 152	99 98 96 93 89 82 72 65	1 1 1 1 2 3 5 6
VC LA 1000 µg/mL DNCB 4 µg/mL	100 67 302	18 8 7	100 153 667	47 25 56	100 100 84	0 0 6

Table 3: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of second experiment

Concentration (test substance) [µg/mL]	RFI CD86		RFI CD54		Viability
	Mean [%]	SD [%]	Mean [%]	SD [%]	Rel. viability [%]	SD of viability
253 303 364 437 524 629 755 906	107 126 126 132 149 167 152 89	1 23 11 4 17 8 12 22	148 143 157 174 215 276 320 760	33 26 34 23 33 1 5 367	98 97 95 91 86 78 66 58	0 1 1 2 3 4 2 6
VC LA 1000 µg/mL DNCB 4 µg/mL	100 81 386	6 11 94	100 128 1121	24 18 239	100 100 64	0 0 11

Conclusions:

Based on the results of this study, the test substance shows as a positive indication of dendritic cell activation.
No prediction can be made for skin sensitization according to GHS criteria based on the results of this in vitro study alone.

Executive summary:

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 755 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 2 valid experiments were performed. 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 532 μg/mL (experiment 2). For the 1st experiment, calculation of an EC150 % was not applicable.

The EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was calculated to be 386 μg/mL (experiment 1) and 492 μg/mL (experiment 2), respectively.

In summary, after 24 hours of exposure to the test substance 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:

adverse effect observed (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. 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 - 0.12 %.

The mean K-peptide depletion, caused by the test substance was determined to be 0.08 %.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.04 %.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model 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 1092 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 2 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.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated to be 187 μM (experiment 2). For the 1st experiment, calculation of an EC1.50 was not applicable.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was 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 has 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 755 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 2 valid experiments were performed. 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 532 μg/mL (experiment 2). For the 1st experiment, calculation of an EC150 % was not applicable.

The EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was calculated to be 386 μg/mL (experiment 1) and 492 μg/mL (experiment 2), respectively.

In summary, after 24 hours of exposure to the test substance 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.

Based on the results and applying the evaluation criteria, the test substance is not peptide reactive, activates keratinocytes and activates dendritic cells. Applying the evaluation criteria, the test substance is predicted to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. In an In Vitro Skin Sensitization Turnkey Test Strategy the test results of two out of three in vitro tests were determined to be positive with the test substance. Therefore, the test substance is considered to be classified for skin sensitization Cat. 1 under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.