Registration Dossier

Administrative data

Description of key information

A GLP-compliant in vitro skin sensitization turnkey strategy was used to assess the skin sensitizing potential of the test substance. Based on the negative results of the Direct Peptide Reactivity Assay (according to OECD Guideline 442 C) and the Keratinocyte Activation Assay - LuSens (according to OECD Guideline 442 D) the h-CLAT (according to OECD Guideline 442 E) was waived. By applying the "2 out of 3"-approach the results of the DPRA and the LuSens are sufficient for final assessment. Therefore, the substance is not considered to be classified for skin sensitization.

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:
Mar - Apr 2019
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 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt Rheinland-Pfalz
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- Physical state / color: solid / yellowish
- Molecular weight: 196.2 g/mol
- Log Kow: 2.85 (calculated)

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

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 196.2 g/mol and a purity / contents of 99.6 %. After stirring and ultra-sonication the test substance was soluble in the vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solution
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
The test substance was dissolved in a suitable vehicle. Per run three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

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

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 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 (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.
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine-peptide
Value:
10.52
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 5: Summary of mean peptide depletions of Cysteine and Lysine

 

Cysteine-peptide

Lysine-peptide

1sttest run

Lysine-peptide

2ndtest run

Lysine-peptide

3rdtest run

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Mean depletion [%]

SD [%]

Test substance

10.52

2.13

0.66

0.41

interference

interference

PC: EGDMA in ACN

52.76

2.72

interference

9.08

0.94

9.70

0.75

Table 6: Historic control data of negative control / vehicle control (not including present study), Jan 2017 - Feb 2019

 

C-peptide concentration [mM]

K-peptide concentration [mM]

Min

0.432

0.457

Max

0.564

0.548

Mean

0.492

0.504

SD

0.030

0.015

n

34

33

Table 7: Historic control data of positive control (not including present study), Jan 2017 - Feb 2019

 

C-peptide concentration [mM]

C-peptide depletion [%]

K-peptide concentration [mM]

K-peptide depletion [%]

Min

0.080

46.26

0.395

6.79

Max

0.285

83.79

0.513

19.85

Mean

0.203

58.91

0.445

11.76

SD

0.046

8.17

0.020

2.78

n

32

30

Interpretation of results:
other: no peptide binding potential
Conclusions:
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.
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. For the C-containing peptide, one test run was performed. For the K-containing peptide, three test runs were performed. Per test run 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 C-containg peptide was present. For the K-peptide reaction co-elution was noticed using two different analysis methods. The mean C-peptide depletion, caused by the test substance was determined to be 10.52 %. The mean K-peptide depletion, caused by the test substance was determined to be 0.66 % in a first test run. However, although no co-elution was present, this reaction is considered to be formally invalid, as co-elution was noticed in the positive control due to insufficient separation of the analytic column. In two further test runs, using the standard and alternative analysis method and a new analytic column, co-elution was noticed in the samples of the test substance. Due to the interference observed 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:
Feb - Mar 2019
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
GLP compliance:
yes (incl. certificate)
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
- Batch No.of test material:
L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- Physical state / color: solid / yellowish
- Molecular weight: 196.2 g/mol
- Log Kow: 2.85 (calculated)

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

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 (DMSO) to achieve the required 100x concentration of the highest concentration (stock solution). Further concentrations were prepared as 100x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate) and were further diluted (1:25) in culture medium 3 to obtain 4x concentrations (dilution 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 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. Three independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested.

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

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

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

CELL VIABILITY ASSAY MTT
Cell culture medium was aspirated from all wells. 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.

EVALUATION OF RESULTS
A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70 % in at least two consecutive concentrations of two independent experiments.
A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 μM if molecular weight is applicable or 2000 μg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration
displaying viability below 70 %).
To be relevant for evaluation, the cell viability must be more than 70 % in at least three tested concentrations of an experiment.
Parameter:
other: mean fold induction
Run / experiment:
1st experiment
Value:
1.38
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: mean fold induction
Run / experiment:
3rd experiment
Value:
1.28
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: mean fold induction
Run / experiment:
4th experiment
Value:
1.28
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: rel. viability [%]
Run / experiment:
1st experiment
Value:
91.38
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: rel. viability [%]
Run / experiment:
3rd experiment
Value:
91.25
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: rel. viability [%]
Run / experiment:
4th experiment
Value:
97.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Summary of LuSens Main Experiments

Concentration (test substance)

[µM]

1stExperiment

3rdExperiment

4thExperiment

Mean fold induction

Mean rel. viability [%]

t-test

Mean fold induction

Mean rel. viability [%]

t-test

Mean fold induction

Mean rel. viability [%]

t-test

p-value

markers

p-value

markers

p-value

markers

560

672

807

968

1162

1394

1673

2008

1.21

1.16

1.13

1.23

1.53

1.38

1.66

1.76

93

96

92

93

89

90

88

90

0.055

0.088

0.018

0.000

0.001

0.000

0.000

0.002

n.s.

n.s.

*

**

**

**

**

**

0.94

1.23

1.16

1.04

1.36

1.43

1.47

1.59

92

92

89

91

88

90

93

95

0.025

0.006

0.044

0.076

0.015

0.000

0.010

0.000

*

**

*

n.s.

*

**

**

**

1.09

1.17

1.04

1.19

1.27

1.34

1.48

1.64

101

100

98

103

96

95

92

95

0.033

0.022

0.313

0.007

0.012

0.011

0.000

0.006

*

*

n.s.

**

*

*

**

**

VC

EGDMA 90.8 µM

LA 5000 µM

1.00

5.43

0.90

100

96

103

-

0.000

0.001

-

**

**

1.00

4.99

1.05

100

108

98

-

0.000

0.172

-

**

n.s.

1.00

4.80

0.89

100

102

102

-

0.000

0.034

-

**

*

n.s. no statistical significance

*statistical significance for p≤0.05

** statistical significance for p≤0.01

Table 2: 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

51

102

509

1017

2035

VC

0.5

1

5

10

51

102

511

1021

2043

0.186

0.167

0.169

0.173

0.172

0.173

0.168

0.169

0.167

0.158

100

90

91

93

92

93

90

91

90

85

Table 3: Historic control data of LuSens. Data shown of test period Mar 2017 until Mar 2019 (not including present study).

 

Negative control LA

(5000 µM (= 450 µg/mL))

Positive control EGDMA

(90.8 µM (= 18 µg/mL))

Vehicle control

(1 % DMSO)

Basal expression

Fold induction

Min

0.71

2.83

1.00

0.61

Max

1.13

8.64

1.00

1.32

Mean

0.93

5.35

1.00

0.93

SD

0.09

1.15

0.00

0.13

n

197

197

197

197

Rel. Viability [%]

Min

88

70

100

121

Max

140

122

100

179

Mean

102

87

100

141

SD

8

10

0

12

n

180

180

180

180

Interpretation of results:
other: no indication of keratinocyte activation
Conclusions:
In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.
Executive summary:

The keratinocyte activating potential of 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. No cytotoxicity was observed. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. The following results were observed:

The test substance was soluble in DMSO (100 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 due to the overall negative result of the study.

In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

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

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

However, in the current case the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

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. For the C-containing peptide, one test run was performed. For the K-containing peptide, three test runs were performed. Per test run 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 C-containg peptide was present. For the K-peptide reaction co-elution was noticed using two different analysis methods. The mean C-peptide depletion, caused by the test substance was determined to be 10.52 %. The mean K-peptide depletion, caused by the test substance was determined to be 0.66 % in a first test run. However, although no co-elution was present, this reaction is considered to be formally invalid, as co-elution was noticed in the positive control due to insufficient separation of the analytic column. In two further test runs, using the standard and alternative analysis method and a new analytic column, co-elution was noticed in the samples of the test substance. Due to the interference observed 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 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. No cytotoxicity was observed. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 3 valid experiments were performed. The following results were observed:

The test substance was soluble in DMSO (100 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 due to the overall negative result of the study. In summary, after 48 hours of exposure to test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.

Table 1: Summary of individual results

Test Method

Test Result

Test Evaluation

Direct Peptide Reactivity Assay (DPRA)

10.52 % cysteine-peptide depletion. Calculation of mean peptide depletion is not applicable due interference of the test substance in the lysine peptide samples.

Negativea

Keratinocyte Activation Assay – LuSens

In at least two independent experiments no biologically relevant ARE-dependent luciferase activity induction was observed.

Negative

Dendritic Cell Line Activation Assay Human Cell Line Activation Test (h-CLAT)

Not determined

Not determined

a Evaluation 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 and does not activate keratinocytes. Applying the evaluation criteria the test substance is predicted not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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 testing strategy, the test results of two out of three in vitro testes were determined to be negative with the test substance. Therefore, the test substance is considered to be not classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.