Registration Dossier

Administrative data

Description of key information

Based on the results of an in chemico/in vitro test strategy the test item is peptide reactive (DPRA, OECD TG 442C) and does activate keratinocytes (LuSens, OECD TG 422D). Therefore, the substance is predicted to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
3 Dezember 2018 - 6 December 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04. Feb. 2015
Deviations:
yes
Remarks:
Please refer to "Other effects/acceptance of results"
Qualifier:
according to
Guideline:
other: EURL ECVAM (European Union Reference Laboratory for alternatives to animal test-ing): “DB-ALM Protocol n° 154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensi-tisation Testing.”
Version / remarks:
29. Jun. 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: SOP 118 00 875 edition 1, valid from 29. Oct. 2018, „Durchführung des DPRA-Tests nach OECD 442C
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Synthetic peptides:
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, are used.
Sequence Cys-Peptide (Cysteine): Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Sequence Lys-Peptide (Lysine): Ac-RFAAKAA-COOH (MW = 775.9 g/mol)

Instruments and Devices:
HPLC system
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
An ACE Excel SuperC18 150x3 mm column with 3 µm particles and pre-column Phenom-enex SecurityGuard C18, 4x3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline.

Heating chamber, Centrifuge, Fridge, Repeater pipette, pH-meter, Analytical scale, Precision scale, Pipette 100 – 1000 µL, Vortexer, Glass thermometer, Conductometer, Carbon analyser

Chemicals:
Water for chromatography
H2O, Honeywell, HPLC grade
Demineralised water
H2O, from ion exchange cartridge. Total organic carbon (TOC) < 1 ppm, conductivity < 0.1 S/cm
Acetonitrile for chromatography
CH3CN, ACN, Honeywell, HPLC grade
CH3CN, ACN, AppliChem, analysis grade
Trifluoroacetic acid
TFA, Merck, for spectroscopy
Ammonium hydroxide
NH3,25 %, p.a.
Ammonium acetate,
CH3COONH4, p.a, Sigma Aldrich
Sodium dihydrogen phosphate
NaH2PO4 * 1 H2O, p.a.
Disodium hydrogen phosphate
Na2HPO4 * 7 H2O, p.a.

Buffers:
100 mM Phosphate buffer (mix out of solution A + B)
Solution A: 1.38 g sodium dihydrogen phosphate monohydrate (monobasic) are dissolved in 100 mL demineralized water.
Solution B: 6.70 g disodium hydrogen phosphate heptahydrate (dibasic) are dissolved in 250 mL demineralized water.
Final 100 mM phosphate buffer is mixed out of 18 mL of solution A and 82 mL of solution B. The pH is adjusted to 7.503 with solution B.
100 mM Ammonium acetate buffer (batch no. 20181204)us) are dissolved in 200 mL demineralised water, pH is adjusted to 10.199 with 25 % ammonium hydroxide solution.

Positive control:
Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %),100 mM solution in acetonitrile for the cysteine peptide
2,3-Butanedione (CAS 431-03-8, >97 %), 100 mM solution in ace-tonitrile for the lysine peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments performed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution are prepared in triplicate (Sets A, B1, B2 and C, total 12 samples per peptide). Set A is analysed together with the peptide calibration standards, sets B1 and B2 are analysed at the start and end of the analysis sequence and are used as stability control for the peptide over the total analysis time. Set C is incubated and analysed together with the samples and is used for calculation of the peptide depletion.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Peptide stock solutions
The peptide stock solutions are freshly prepared for each assay.
0.667 mM Cys-Peptide solution was prepared by dissolving 22.5 mg of the peptide in 45 mL phosphate buffer, pH 7.5.
0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45 mL ammonium acetate buffer, pH 10.2.

Test item stock solution:
The test item stock solution is freshly prepared for each assay.
100 mM test item solution was prepared by dissolving 154.4 mg test item in 3 mL of the solvent acetonitrile for the Cys-peptide and 154.5 mg test item for the Lys-peptide, respectively. The solution was vortexed until the test item was dissolved.

Peptide calibration standards:
From each peptide stock solution the following calibration standards was prepared in the appropriate dilution buffer: 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM peptide. Blank dilution buffer was measured. Calibration samples were analysed before the samples containing the test item.

Test item samples:
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item solution), the Lys-peptide sam-ples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item solution) using the stock solutions described. A final volume of 1 mL per sample was prepared for each sample.

Incubation:
The positive control, solvent control and test item samples are incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 24 ± 2 h. Samples appearing turbid or where precipitation is visible by the unaided eye are centrifuged (benchtop centrifuge, 10 min at 4500 rpm) and only the clear supernatant is used for measurement.

Measurements
HPLC system with UV/VIS-Detector

Evaluation of results:
Evaluation criteria of results according to the cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 6.38 ≤ 22.62 low reactivity positive
0- ≤ 6.385 minimal or no reactivity negative

Evaluation criteria of results according to the cysteine 1:10 prediction model.
mean Cys peptide depletion [%] Reactivity Evaluation
> 98.24 - ≤ 100 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 13.89 ≤ 23.09 low reactivity positive
0 - ≤ 13.89 minimal or no reactivity negative

Acceptance criteria
The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
The mean peptide depletion value for the positive control 2,3- Butane-dione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
The standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion.
Positive control results:
The mean Cys-Peptide depletion of the positive control was 80.01%.
Key result
Parameter:
other: mean peptide depletion Cys peptide (%)
Run / experiment:
1st run
Value:
78.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The ten proficiency chemicals listed in the guideline were tested using the analysis method described. All ten proficiency chemicals showed the expected DPRA prediction and eight out of the ten chemicals showed depletion values consistent with the classification reported in the OECD guideline.

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

Deviations from the Guideline
The mean percent area ratio 220 nm/258 nm of the positive control 2,3 Butanedione in experiment 1 is 120 %. This is marginal out of the given range 90-110 % and shows a peak impurity and co-elution. This was considered uncritical, because the value of peptide-depletion was reported as “co-elution – percent depletion estimated”, it is not an acceptance criterium for the study and the results of the Lys-peptide were not considered for the evaluation with the Cysteine 1:10 prediction model.


Table 1: Historical Data

Parameter

Depletion [%]

Depletion [%]

Peptide

Cys- Peptide

Lys- Peptide

Mean

82.34

35.35

Standard
Deviation

8.28

6.80

Range 2σ

65.77 – 98.91

21.76-48.95

Study

80.01

13.58

Table 2: Calculated peptide depletion values for the Cys-Peptide

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

77.46

80.01

2.28

Positive control Rep. 2

80.72

Positive control Rep. 3

81.85

Test item Rep. 1

74.29

78.11

3.77

Test item Rep. 2

78.21

Test item Rep. 3

81.83

Table 3: Calculated peptide depletion values for the Lys-Peptide

Sample name

Depletion [%]

Single

Mean

SD

Positive control Rep. 1

12.48

13.58 **

1.25

Positive control Rep. 2

13.32

Positive control Rep. 3

14.95

Test item Rep. 1

0 (-39.12) *

0.00 ***

0.00

Test item Rep. 2

0 (-38.23) *

Test item Rep. 3

0 (-34.79) *

* Note: Negative depletion values were considered as “zero” when calculating the mean.
** Note: The area ratio 220 nm/258 nm was marginal out of range. The value is reported as “co-elution – percent depletion estimated”
*** Note: Due to co-elution the results of the Lys-peptide were not considered when using the Cysteine 1:10 prediction model for the evaluation.

Interpretation of results:
other: peptide depletion
Conclusions:
The DPRA prediction is “positive” with moderate reactivity according to the Cysteine 1:10 model. It can be stated that in this study and under the experimental conditions reported, the test item possesses a moderate skin sensitisation potential
Executive summary:

A study according OECD TG 442C was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated 23 h at 25 °C for the Cys peptide and 22.5 h for the Lys-peptide, respectively. The peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously. One valid experiment was performed.

For the Lys-peptide co-elution occurred with the test item. Therefore, the evaluation was carried out with the Cysteine 1:10 prediction model.

The mean peptide depletion in the Cys-peptide assay was 78.11 %. All acceptance criteria were fulfilled; therefore, the test was considered valid. The DPRA prediction for the test item was positive with reactivity class moderate according to the Cysteine 1:10 prediction model.

 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
16 July 2018 - 27 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
25. June 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS. ENV/JM/MONO(2015)6.
Version / remarks:
22. May 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: EU-Method B.60 of the Commission Regulation (EU) No. 2017/735: “In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method”
Version / remarks:
14. Feb. 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Preparation:
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (DMEM (Dulbecco´s Modified Eagle Medium)). The test item was insoluble in medium but soluble in DMSO at the required concentration (200 mM). Therefore, DMSO was used as solvent. Since the final concentration of the solvent during treatment is limited to 1 %, a stock solution containing 200 mM (CRFT) and 12.5 mM (experiment I and II) test item in DMSO was prepared. Subsequent dilution to 1% finally yielded a maximum concentration of 2000 µM in the pre-test and 125 µM in the experiments. For that, the stock solution was first used to prepare a geometric series of solutions (CRFT: factor 2; main experiments: factor 1.2) on a master plate. Afterwards all concentrations were further diluted (1:25) in medium no. 3 on a dilution plate. Another 1:4 dilution was achieved by adding 50 µL of each concentration of the dilution plate to the corre-sponding wells of the test plate containing the cells as well as 150 µL medium no. 3. In the end, the total dilution factor was 1:100. The stock solution as well as the dilutions were freshly prepared on the day of treatment.

Controls:
Negative Control: DL-Lactic acid
Positive Control: EGDMA (Ethylene glycol dimethylacrylate)
Solvent Control: DMSO

Cell Cultures:
The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen to allow a continuous stock of cells (mycoplasma contamination free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells. After thawing the cells were cultivated in DMEM (9 % FCS (Fetal calf serum) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.

Cytotoxicity Range Finder Test:
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. A reduction of the viability below 70 % is defined as a cytotoxic effect. In the CRFT the following 12 nominal concentrations of the test item were tested: 0.98 µM, 1.95 µM, 3.91 µM, 7.81 µM, 15.63 µM, 31.25 µM, 62.5 µM, 125 µM, 250 µM, 500 µM, 1000 µM, 2000 µM

Experimental Parameters of Experiment I and II:
Experimental Performance
Experiment I and II were performed in the same way. Experiment II serves only to confirm the results of experiment I. At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %) cells per mL. 120 µL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement). Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 25 h and 15 min in experiment I and 25 h in experiment II.
The treatment procedure was performed on both 96 well plates identically: After the incubation time the medium was removed from the cells and 150 µL medium no. 3 were added to each well. Afterwards 50 µL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 µL MTT working solution were added to each well. The plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 µL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow colour) to its insoluble formazan (purple colour) in living cells and therefore indicates the amount of living cells. After the measurement of the colour change, the values were transferred in a validated spreadsheet for the calculation of the viability.
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Afterwards 100 µL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 µL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 µL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.

Positive control results:
The positive control induced a clear effect with an induction value of 4.8 fold in comparison to the solvent control.
Key result
Parameter:
other: fold Luciferase induction
Remarks:
50.2 µM
Run / experiment:
1st experiment
Value:
3.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
60.3 µM
Run / experiment:
1st experiment
Value:
4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
72.3 µM
Run / experiment:
1st experiment
Value:
5.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
50.2 µM
Run / experiment:
2nd experiment
Value:
3.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
60.3 µM
Run / experiment:
2nd experiment
Value:
3.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: fold Luciferase induction
Remarks:
72.3 µM
Run / experiment:
2nd experiment
Value:
4.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use, the validity of the LuSens test was demonstrated in a proficiency study. 22 proficiency chemicals indicated by the OECD 442 D (status: Feb. 2015) and the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PRO-POSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIF-ERASE TEST METHODS) were tested. The 10 proficiency chemicals which are indicated by the current version of the OECD 442D (status: 25. June 2018) were all included in the proficiency study.
From 22 proficiency chemicals more than 80 % of the results were correctly categorized. From the 10 of the proficiency chemicals of the current OECD 442D (chemicals of the current version of the OECD 442 D (status: 25. June 2018) 80 % of the results were correctly categorized.
Therefore, the proficiency of the LuSens test was demonstrated.

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

RANGE FINDING TEST:
No cytotoxic effect was observed at the controls as well as the test item concentrations 0.98 µM to 62.5 µM. The viability values were all above 77 %. A cytotoxic effect was determined at 125 µM up to 2000 µM.

In the Luciferase assay, all of the tested non cytotoxic concentrations induced a statistically significant increase in luciferase induction equal or above the threshold of 1.5 fold in comparison to the solvent control.

Table 1     Results of experiment I

 

 

Induction of Luciferase

Viability of the Cells

Parameter

Concentration

Induction

Standard

Deviation

Standard

Deviation

Relative

Viability

Standard

Deviation

Standard

Deviation

[µM]

fold

 

[%]

[%]

 

[%]

Solvent Control

-

1.0

0.06

6.23

100.0

2.78

2.78

Growth Control

-

1.2

0.08

6.52

143.9

3.43

2.38

Negative Control

5000

0.9

0.05

5.98

105.8

2.76

2.61

Positive Control

120

4.8

0.27

5.75

86.1

2.63

3.06

Test item

16.8

1.8

0.22

11.96

98.3

2.01

2.05

Test item

20.2

1.8

0.15

8.52

95.6

0.98

1.02

Test item

24.2

1.9

0.17

8.78

94.5

1.16

1.23

Test item

29.1

2.1

0.14

6.41

91.9

1.72

1.87

Test item

34.9

2.6

0.26

9.81

90.2

1.16

1.29

Test item

41.9

3.0

0.34

11.11

89.8

0.83

0.92

Test item

50.2

3.5

0.26

7.28

85.9

1.77

2.06

Test item

60.3

4.0

0.32

7.96

78.9

2.18

2.76

Test item

72.3

5.1

0.38

7.50

71.3

1.10

1.54

Test item

86.8

6.2*

0.35

5.56

61.3

1.62

2.64

Test item

104.2

6.6*

0.39

5.84

46.1

2.51

5.44

Test item

125.0

5.8*

0.67

11.45

30.5

1.72

5.65

* = Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.

Table 2     Results of experiment II

 

 

Induction of Luciferase

Viability of the Cells

Parameter

Concentration

Induction

Standard

Deviation

Standard

Deviation

Relative

Viability

Standard

Deviation

Standard

Deviation

[µM]

fold

 

[%]

[%]

 

[%]

Solvent Control

-

1.0

0.10

10.18

100.0

3.36

3.36

Growth Control

-

1.2

0.07

5.39

145.1

3.24

2.23

Negative Control

5000

0.9

0.07

7.55

104.4

2.94

2.81

Positive Control

120

4.7

0.22

4.68

84.3

0.90

1.07

Test item

16.8

1.5

0.06

4.45

98.6

6.36

6.44

Test item

20.2

1.6

0.07

4.46

97.0

2.57

2.65

Test item

24.2

1.8

0.18

9.79

96.6

1.71

1.77

Test item

29.1

2.0

0.10

4.92

94.7

2.13

2.25

Test item

34.9

2.4

0.19

7.86

90.0

1.81

2.01

Test item

41.9

2.7

0.17

6.18

88.0

0.77

0.88

Test item

50.2

3.2

0.18

5.76

84.9

4.32

5.09

Test item

60.3

3.9

0.27

7.04

80.1

4.94

6.17

Test item

72.3

4.9

0.65

13.32

71.4

0.35

0.49

Test item

86.8

6.1*

0.30

4.91

59.0

2.20

3.73

Test item

104.2

6.7*

0.37

5.55

48.1

2.51

5.22

Test item

125.0

6.3*

0.64

10.11

30.6

0.37

1.20

* = Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.

Table 3        Acceptability of experiment I and II

Criteria

Found in

experiment I

Found in

experiment II

The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.

Positive controlFold induction:

4.8

Relative viability: 86.1 %

Positive controlFold induction:

4.7

Relative viability:  84.3 %

The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70 %.

Negative control:

Fold induction:

0.9

Relative viability:

105.8 %

Growth control:

Fold induction:

1.2

Relative viability:

143.9 %

Negative control:

Fold induction:

0.9

Relative viability:

104.4 %

Growth control:

Fold induction:

1.2

Relative viability:

145.1 %

The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.

6.23 %

10.18 %

At least 3 test concentrations must be within viability limits, i.e. have relative viability of at least 70 %.

9 concentrations are analysable

9 concentrations are analysable

At least one concentration should be cytotoxic, i.e. have a cell viability < 70 %, or the maximum concentration of 2000 µM (2000 µg/mL) should have been tested

yes

yes

All validity criteria were met. Therefore, the study is valid.

Table 4      Historical Data of the Negative Control and the Positive Control (status: 15. Jul. 2018)

 

Σ

Mean induction value

Standard

deviation

Range

Study

 

 

 

 

 

Experiment I

Experiment II

Positive Control

(EGDMA)

158

5.5

1.541

2.1 – 14.7

4.8

4.7

Negative control

(DL-Lactic acid)

180

1.0

0.079

0.8 – 1.3

0.9

0.9

Interpretation of results:
other: activation of keratinocytes
Conclusions:
In conclusion, it can be stated that under the experimental conditions of this study, the test item was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).
Executive summary:

An in vitro study according OECD TG 442D was performed to investigate the potential ofthe test item to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II:

16.8 µM, 20.2 µM, 24.2 µM, 29.1 µM, 34.9 µM, 41.9 µM, 50.2 µM, 60.3 µM, 72.3 µM, 86.8 µM, 104.2 µM, 125 µM

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

Since all acceptability criteria of the assay were met the study is valid.

In experiment I and II cytotoxic effects were observed at the concentrations 86.6 µM to 125 µM. Those concentrations were excluded from the evaluation of the luciferase induction.

Finally the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction in experiment I and II: 16.8 µM, 20.2 µM, 24.2 µM, 29.1 µM, 34.9 µM, 41.9 µM, 50.2 µM, 60.3 µM, 72.3 µM

A statistically significant increase ≥ 1.5 foldin luciferase induction in comparison to the solvent control was measured in all non-cytotoxic test item concentrations. Therefore, both experiments are clearly positive.

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

For the evaluation of the skin sensitisation potential of the test substance a weight of evidence approach was used. 

Studies

DPRA

A study according OECD TG 442C was performed in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile and the respective peptide was incubated 23 h at 25 °C for the Cys peptide and 22.5 h for the Lys-peptide, respectively. The peptide concentration after the incubation was measured using HPLC-UV. Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously. One valid experiment was performed.

For the Lys-peptide co-elution occurred with the test item. Therefore, the evaluation was carried out with the Cysteine 1:10 prediction model.

The mean peptide depletion in the Cys-peptide assay was 78.11 %. All acceptance criteria were fulfilled; therefore, the test was considered valid. The DPRA prediction for the test item was positive with reactivity class moderate according to the Cysteine 1:10 prediction model.

LuSens

An in vitro study according OECD TG 442D was performed to investigate the potential of the test item to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II:

16.8 µM, 20.2 µM, 24.2 µM, 29.1 µM, 34.9 µM, 41.9 µM, 50.2 µM, 60.3 µM, 72.3 µM, 86.8 µM, 104.2 µM, 125 µM

None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control.

The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.

Since all acceptability criteria of the assay were met the study is valid.

In experiment I and II cytotoxic effects were observed at the concentrations 86.6 µM to 125 µM. Those concentrations were excluded from the evaluation of the luciferase induction.

Finally the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction in experiment I and II: 16.8 µM, 20.2 µM, 24.2 µM, 29.1 µM, 34.9 µM, 41.9 µM, 50.2 µM, 60.3 µM, 72.3 µM

A statistically significant increase ≥ 1.5 foldin luciferase induction in comparison to the solvent control was measured in all non-cytotoxic test item concentrations. Therefore, both experiments are clearly positive.

Conclusion

Based on the results of an in chemico/in vitro test strategy the test item is peptide reactive (DPRA, OECD TG 442C) and does activate keratinocytes (LuSens, OECD TG 422D). Therefore, the 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 (EC) No 1272/2008. Based on available data on skin sensitisation, the test item is classified as skin sensitising Cat 1 (H317) according to Regulation (EC) No 1272/2008 (CLP), as amended for the 12th time in Commission Regulation (EU) 2019/521.