Registration Dossier

Administrative data

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 two positive results observed in the studies 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

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May - Aug 2018
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:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
in vitro turnkey testing strategy
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Purity test date: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test item
23.5 g/100 g (NH4)2C2H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- Physical state / color: liquid / colorless to yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: the test substance was soluble in deionized water

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 deionized water (considering a molecular weight of 575.74 g/mol and a purity / contents of 46.5 %). 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 vitro 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.

Controls:
Negative control (NC): vehicle control = deionized water
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA), prepared as 50 mM emulsion in deionized water
Co-elution control (SK): Sample prepared of the respective peptide buffer and the test substance but without peptide

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 cloudiness of the test substance 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.

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 substances and control samples.

Preparation of the test substance samples:
The samples were prepared in triplicates for each peptide according to the following pipetting scheme.
C-peptide: 750 µL C-peptide stock solution, 200 µL solvent (vehicle), 50 µL test substance preparation (or PC-preparation or solvent (VC)).
K-peptide: 750 µL K-peptide stock solution, 250 µL test substance preparation (or PC-preparation or solvent (VC)).
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 HPLC 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 wa 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: cysteine-peptide
Parameter:
other: mean peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: true value is negative (- 11.21)
Run / experiment:
other: lysine-peptide
Parameter:
other: mean peptide depletion [%]
Value:
0.77
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: precipitation after 24 h incubation time
Other effects / acceptance of results:
Solubility of the test substance samples with the peptides:
The test substance was dissolved in deionized water at a concentration of 100 mM. The samples of the test substance with the C-containing peptide were solutions at the time of preparation and after the 24-hour incubation time. The samples of the test substance with the K-containing peptide were emulsions at the time of preparation and precipitates were noted after the 24-hour incubation time.

Co-elution:
No co-elution of the test substance and peptides occurred as demonstrated by consistent values of the area ratios 220 nm / 258 nm and chromatograms of the co-elution control.

Mean peptide depletion:
Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable for the test substance.

Table 1: Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for cysteine-peptide

Reaction with cysteine-peptide

Peak area [mAU*s] at 220 nm

Peptide concnetration [mM]

Peptide depletion [%]

Sample 1

Sample 2

Sample 3

Sample 1

Sample 2

Sample 3

Mean

SD

Sample 1

Sample 2

Sample 3

Mean

SD

NC: H2O

400.2

392.6

398.4

0.496

0.486

0.493

0.492

0.005

- 0.79

1.14

- 0.35

0.00

1.01

Test substance

443.8

438.5

441.2

0.550

0.543

0.543

0.547

0.003

- 11.88

- 10.54

- 11.21

- 11.21

0.67

PC: EGDMA

70.2

117.9

67.8

0.083

0.142

0.080

0.102

0.035

83.18

71.03

83.79

79.33

7.20

Table 2: Area ratio 220 nm / 258 nm of NC, PC and the test substance for cysteine-peptide

Reaction with cysteine-peptide

Peak area [mAU*s] at 258 nm

Area ratio 220 nm / 258 nm

Sample 1

Sample 2

Sample 3

Sample 1

Sample 2

Sample 3

NC: H2O

12.7

13.6

12.6

31.6

28.8

31.7

Test substance

15.1

15.0

15.1

29.4

29.3

29.3

PC: EGDMA

n.a.

n.a.

n.a.

-

-

-

Table 3: Peak area, peptide concentration and peptide depletion of NC, PC and the test substance for lysine-peptide

Reaction with lysine-peptide

Peak area [mAU*s] at 220 nm

Peptide concnetration [mM]

Peptide depletion [%]

Sample 1

Sample 2

Sample 3

Sample 1

Sample 2

Sample 3

Mean

SD

Sample 1

Sample 2

Sample 3

Mean

SD

NC: H2O

408.7

407.9

407.4

0.516

0.515

0.514

0.515

0.001

- 0.18

0.01

0.16

0.00

0.17

Test substance

402.0

405.5

407.3

0.507

0.512

0.514

0.511

0.004

1.51

0.63

0.17

0.77

0.68

PC: EGDMA

362.3

368.0

366.7

0.456

0.463

0.462

0.460

0.004

11.47

10.03

10.35

10.61

0.76

Table 4: Area ratio 220 nm / 258 nm of NC, PC and the test substance for lysine-peptide

Reaction with lysine-peptide

Peak area [mAU*s] at 258 nm

Area ratio 220 nm / 258 nm

Sample 1

Sample 2

Sample 3

Sample 1

Sample 2

Sample 3

NC: H2O

14.2

14.3

14.3

28.8

28.5

28.5

Test substance

14.0

14.3

14.1

28.7

28.4

29.0

PC: EGDMA

12.6

12.9

12.8

28.9

28.4

28.8

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction can be made for skin sensitisation according to GHS criteria based on the results of this in vitro study alone.
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 deionized water. 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 co-elution 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 deionized water at a concentration of 100 mM. The samples of the test substance with the C-containing peptide were solutions at the time of preparation and after the 24 -hour incubation time. The samples of the test substance with the K-containing peptide were emulsions at the time of preparation and precipitates were noted after the 24 -hour incubation time.

The mean C-peptide depletion, caused by the test substance was determined to be - 11.21 %.

The mean K-peptide depletion, caused by the test substance was determined to be 0.77 %.

No co-elution of test substance and peptides was present. Although a distinct negative C-peptide depletion values is noticed, the absence of interference is clearly demonstrated by the co-elution control and the consistent area ratio 220 nm / 258 nm.

Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

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

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May - Aug 2018
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:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
in vitro turnkey testing strategy
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Purity: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test item
23.5 g/100 g (NH4)2C4H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- Physical state / color: liquid / colorless to yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: Culture medium was used as vehicle because good homogeneity of the preparation was achieved.

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): Visual inspection of each dilution step was performed.
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:
Test system: THP-1 cells (human monocytic leukemia cell line)

Controls:
Negative control (NC): Lactic acid (LA), 1000 µg/mL in culture medium
Positive control (PC): 1-chloro-2,4-dinitrobenzene (DNCB), 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.

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 10^6 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 10^6 cells/mL. After test substance application the plates were sealed with semi-permeable 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 10^6 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 darf. 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:
25
Vehicle controls validity:
valid
Run / experiment:
other: Experiment 1
Parameter:
other: mean RFI CD86 [%]
Value:
206.75
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 1
Parameter:
other: mean RFI CD54 [%]
Value:
588.875
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 1
Parameter:
other: mean rel. viability [%]
Value:
88.25
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2
Parameter:
other: mean RFI CD86 [%]
Value:
247.125
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2
Parameter:
other: mean RFI CD54 [%]
Value:
640.875
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2
Parameter:
other: mean rel. viability [%]
Value:
91.75
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Results of preliminary cytotoxicity assessment

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

Mean rel. Viability [%]

VC

4

8

16

31

63

125

250

500

1000

2500

5000

VC

8

17

34

67

134

269

538

1075

2151

5376

10753

94

92

84

55

44

23

27

34

24

9

11

35

96

93

86

62

34

20

25

30

22

6

26

33

95

92

85

58

39

22

26

32

23

8

19

34

100

97

89

61

41

23

27

34

24

8

20

36

Table 2: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 1st experiment

Concentration (test substance) [µg/mL]

RFI CD86

RFI CD54

Viability

Mean [%]

SD [%]

Mean [%]

SD [%]

Rel. Viability [%]

SD of viability

8

10

12

14

17

21

25

30

183

196

201

193

201

211

253

216

34

56

56

40

38

50

73

91

445

496

514

486

750

588

763

669

1

15

122

33

17

13

57

115

98

96

95

95

89

85

79

69

1

1

1

1

1

1

2

3

VC

LA 1000 µg/mL

DNCB 4 µg/mL

100

72

260

12

21

15

100

139

814

12

21

70

100

99

77

1

1

4

Table 3: RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 2nd experiment

Concentration (test substance) [µg/mL]

RFI CD86

RFI CD54

Viability

Mean [%]

SD [%]

Mean [%]

SD [%]

Rel. Viability [%]

SD of viability

8

10

12

14

17

21

25

30

206

218

246

232

247

242

310

276

22

22

5

39

18

26

24

6

468

543

522

650

786

673

753

732

25

10

19

12

32

64

83

116

98

97

97

95

93

90

85

79

0

0

0

1

1

1

1

3

VC

LA 1000 µg/mL

DNCB 4 µg/mL

100

83

388

21

9

32

100

142

751

5

9

131

100

100

82

0

0

1

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction can be made for skin sensitisation 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 25 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:

At concentrations used in the main experiment 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.

Relative fluorescence intensity and concurrent relative viability determined in the main experiments are summarized in the following table.

1stExperiment

2ndExperiment

Concentration (test substance) [µg/mL]

RFI CD86

RFI CD54

Viability

Concentration (test substance) [µg/mL]

RFI CD86

RFI CD54

Viability

Mean [%]

Mean [%]

Rel. Viability [%]

Mean [%]

Mean [%]

Rel. Viability [%]

8

10

12

14

17

21

25

30

183

196

201

193

201

211

253

216

445

496

514

486

750

588

763

669

98

96

95

95

89

85

79

69

8

10

12

14

17

21

25

30

206

218

246

232

247

242

310

276

468

543

522

650

786

673

753

732

98

97

97

95

93

90

85

79

VC

LA 1000 µg/mL

DNCB 4 µg/mL

100

72

260

100

139

814

100

99

77

VC

LA 1000 µg/mL

DNCB 4 µg/mL

100

83

388

100

142

751

100

100

82

Calculation of an EC150 % (the concentration resulting in a RFI of 150 %) for CD86 and an EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was not applicable as fold inductions above 150 % / 200 % were obtained in all evaluable concentrations.

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:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May - Aug 2018
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:
February 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
in vitro turnkey testing strategy
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 170492F01
- Purity: 100.0 area-% (HPLC fingerprint)
- Content:
21.4 g/100 g Test item
23.5 g/100 g (NH4)3C4H4O6
30 g/100 g Tartrate
6.7 g/100 g Ammonium
9.1 g/100 g Antimony
53.5 g/100 g Water
- Physical state / color: liquid / colorless to yellowish, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

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 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): Visual inspection of each dilution step was performed.
Details on the study design:
Skin sensitisation (In vitro test system) - Details on study design:
Test system: LuSens cell line; human transgenic keratinocyte cell line derived from HaCaT cells

Controls:
Negative control (NC): DL-Lactic acid, 5000 µM (= 450 µg/mL) in 1 % DMSO in culture medium 3
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA), 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

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, c. 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 10^5 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 L culture medium 3. The test substance was prepared and 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 semi-permeable 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:
457
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
not applicable
Run / experiment:
other: Experiment 1
Parameter:
other: mean relative viability [%]
Value:
84.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2
Parameter:
other: mean relative viability [%]
Value:
87
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 1
Parameter:
other: mean fold induction
Value:
61.575
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: Experiment 2
Parameter:
other: mean fold induction
Value:
45.37
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

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

1

2

11

22

108

215

1075

2151

4301

0.350

0.415

0.430

0.427

0.434

0.390

0.338

0.071

0.002

0.008

100

119

123

122

124

111

96

20

0

2

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

Concentration (test substance) [µM]

Fold induction

Rel. Viability [%]

t-test

Mean

SD

Mean

SD

p-value

220

265

317

381

457

549

658

790

40.67

60.92

82.48

107.72

117.83

85.78

54.31

30.23

4.91

5.08

6.69

7.12

7.55

8.57

6.81

2.33

92

80

77

66

56

43

33

27

3

5

5

4

4

2

3

2

0.003

0.001

0.001

0.001

0.001

0.002

0.003

0.001

VC

EGDMA 90.8 µM

LA 5000 µM

1.00

4.95

0.88

0.09

0.32

0.15

100

77

88

7

0

5

-

0.000

0.077

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

Concentration (test substance) [µM]

Fold induction

Rel. Viability [%]

t-test

Mean

SD

Mean

SD

p-value

106

128

153

184

220

265

317

381

16.43

18.41

18.33

24.67

34.78

41.89

74.31

95.84

0.63

1.51

0.91

1.96

3.45

1.66

3.03

8.15

101

82

91

90

85

77

73

66

3

5

2

3

2

3

2

2

0.000

0.001

0.000

0.001

0.002

0.000

0.000

0.001

VC

EGDMA 90.8 µM

LA 5000 µM

1.00

5.41

0.87

0.13

0.36

0.18

100

97

104

4

7

6

-

0.000

0.084

Interpretation of results:
study cannot be used for classification
Conclusions:
No prediction can be made for skin sensitisation 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 457 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.

Relative luciferase fold induction and concurrent relative viability determined in the main experiments are summarized in the following table.

Table 1: Summary of LuSens Main Experiments

Concentration (test substance) [µM]

1stExperiment

Concentration (test substance) [µM]

2ndExperiment

Fold induction

Rel. Viability [%]

t-test

Fold induction

Rel. Viability [%]

t-test

Mean

Mean

p-value

Mean

Mean

p-value

220

265

317

381

457

549

658

790

40.67

60.92

82.48

107.72

117.83

85.78

54.31

30.23

92

80

77

66

56

43

33

27

0.003

0.001

0.001

0.001

0.001

0.002

0.003

0.001

106

128

153

184

220

265

317

381

16.43

18.41

18.33

24.67

34.78

41.89

74.31

95.84

101

82

91

90

85

77

73

66

0.000

0.001

0.000

0.001

0.002

0.000

0.000

0.001

VC

EGDMA 90.8 µM

LA 5000 µM

1.00

4.95

0.88

100

77

88

-

0.000

0.077

VC

EGDMA 90.8 µM

LA 5000 µM

1.00

5.41

0.87

100

97

104

-

0.000

0.084

Calculation of an EC 1.50 (the concentration resulting in a 1.50 -fold luciferase induction) was not applicable as fold inductions above 1.50 were obtained in all evaluable concentrations.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In Vitro Skin Sensitisation Turnkey Testing Strategy

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 deionized water. 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 co-elution 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 test substance was dissolved in deionized water at a concentration of 100 mM. The samles of the test substance with the C-containing peptide were solutions at the time of preparation and after the 24 -hour incubation time. The samples of the test substance with the K-containing peptide were emulsions at the time of preparation and precipitates were noted after the 24 -hour incubation time.

The mean C-peptide depletion, caused by the test substance was determined to be - 11.21 %.

The mean K-peptide depletion, caused by the test substance was determined to be 0.77 %.

No co-elution of test substance and peptides was present. Although a distinct negative C-peptide depletion value is noticed, the absence of interference is clearly demonstrated by the co-elution control and the consistent area ratio 220 nm / 258 nm.

Due to the insolubility of the test substance in the K-peptide samples calculation of mean peptide depletion is not applicable and the cysteine 1:10 prediction model is used for evaluation.

Based on the observed results and applying the cysteine 1:10 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 457 µM (corresponding to the 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. Calculation of an EC1.50 (the concentration resulting in a 1.50 -fold luciferase induction) was not applicable as fold inductions above 1.50 were obtained in all evaluable concentrations.

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 25 µg/mL (corresponding to the 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:

At concentrations used in the main experiment 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. Calculation of an EC150 % (the concentration resulting in a RFI of 150 %) for CD86 and and EC200 % (the concentration resulting in a RFI of 200 %) for CD54 was not applicable as fold inductions above 150 % / 200 % were obtained in all evaluable concentrations.

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)

- 11.21 % cysteine-peptide depletion; 0.77 % lysine-peptide depletion. Calculation of mean peptide depletion is not applicable due to insolubility of the test substance in the lysine peptide samples.a

Negative

Keratinocyte Activation Assay – LuSens

In at least two consecutive concentrations of two independent experiments ARE-dependent luciferase activity induction above 1.50-fold of statistic significance at test substance concentrations that did not reduce cell viability below 70 % was observed.

Positive

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

aEvaluation based on 1:10 Cysteine prediction model.

Based on the results summarized in the table above 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 as a skin sensitizer Cat. 1 under Regulation (EC) No. 1272/2008, as amended for the tenth time in Regulation (EC) No. 2017/776.