Registration Dossier

Administrative data

Description of key information

OECD 442 C: no prediction due to precipitation of the test material

OECD 442 D negative

QSAR prediction: negative

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:
2017-11-16 to 2017-12-23
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))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide reactivity assay (DPRA)
Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions:
20.32 mg cysteine peptide were pre-weighed in a vial and dissolved in a defined volume (39.475 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
22.01 mg lysine peptide were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (41.50 mL) to reach a concentration of 0.667 mM.
- Preparation of the test chemical solutions: The test item was pre-weighed into a glass vial and was dissolved in DMF. A stock solution with a concentration of 100 mM was prepared.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive control: Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution control: Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide.
Reference control A was prepared using acetonitrile.
Reference control B was prepared using acetonitrile.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item.

INCUBATION
- Incubation conditions: The reaction solutions were left in the dark at 25 +/- 2.5 °C for 24 +/- 2 h.
- Precipitation noted:
For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. Precipitation was observed for the samples of the test item (including the co-elution control of the test item).
For the 100 mM solution of the test item precipitation was observed when diluted with the lysine peptide solution. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Precipitation was also observed for the samples of the test item (including the co-elution control of the test item).

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer). A serial dilution of the peptide stock solution (0.667 mM) using the respective dilution buffer) was performed, resulting in 7 calibration solutions (0.534 mM, 0.267 mM, 0.134 mM, 0.067 mM, 0.033 mM, 0.017 mM, 0.000 mM)

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm
Vehicle / solvent:
other: DMF
Positive control:
cinnamic aldehyde
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.50%.
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
cysteine depletion
Value:
0.21 %
At concentration:
100 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
mean
Parameter:
lysine depletion
Value:
0 %
At concentration:
100 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Outcome of the prediction model:
no or minimal reactivity [in chemico]
Other effects / acceptance of results:
Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

5221.6792

0.5340

4311.3857

0.5340

STD2

2648.5273

0.2670

2143.2170

0.2670

STD3

1327.5009

0.1335

1043.3549

0.1335

STD4

670.7400

0.0667

519.3511

0.0667

STD5

334.8562

0.0334

259.5146

0.0334

STD6

166.3505

0.0167

130.4601

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1609.4324

0.1633

68.11

68.17

0.05

0.08

1604.4860

0.1628

68.21

1605.4248

0.1629

68.19

Test Item

4991.4937

0.5090

0.00

0.21

0.33

157.69

4966.8535

0.5064

0.04

4939.1538

0.5036

0.60

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1589.2585

0.1982

59.95

58.82

0.99

1.68

1662.4030

0.2073

58.11

1650.5492

0.2058

58.41

Test Item*

4080.3315

0.5063

0.00

0.00

0.00

n/a

4120.3213

0.5113

0.00

4055.7935

0.5033

0.00

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% PPD 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2
(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

-

-

-

0.21

Minimal Reactivity

no sensitiser

Positive Control

63.50

High Reactivity

sensitiser

68.17

Moderate Reactivity

sensitiser

Interpretation of results:
study cannot be used for classification
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation, the prediction model does not apply and a prediction cannot be made.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Executive summary:

In the present study the test material, the test item, was dissolved in DMF, based on the results of the pre-experiments.

Based on a molecular weight of 340 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control of the test item). Samples were centrifuged at 400g for 5 minutes prior to the HPLC analysis.

For the 100 mM solution of the test item precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Precipitation was also observed for the samples of the test item (including the co-elution control of the test item). Samples of the test item were centrifuged at 400g for 5 minutes prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Co-elution of the test item with the lysine peptide peak was observed. Therefore sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC CDMF). However, precipitation was observed in the cysteine experiment as well. Since it cannot be determined if the precipitate resulted from the test item or the peptide, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of cysteine peptide was  6.38% (0.21%).

According to the evaluation criteria in the guideline, if a precipitation or phase separation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.50%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-11-14 to 2017-12-04
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)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details of test system:
Keratinoses transgenic cell line [442D]
Details on the study design:
442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item was dissolved in dimethyl sulfoxide. A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial.
- Preparation of the test chemical serial dilutions: Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells.
- Preparation of the positive controls: Cinnamic aldehyde was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM.
- Preparation of the solvent, vehicle and negative controls: DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 2
- Test chemical concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM
- Application procedure: After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 μL test item exposure medium. 50 μL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
- Exposure time: 48 h +/- 1 h
- Study evaluation and decision criteria used:

The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:

- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 μM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 μM is considered as inconclusive.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): Passage number: 5; 1E+4 cells were seeded in each well
- Incubation conditions: 24 +/- 1 h in assay medium at 37 °C +/- 1 °C and 5% CO2
- Washing conditions: After exposure cells were washed once with DPBS.
- Precipitation noted: none

LUCIFERASE ACTIVITY MEASUREMENTS
- Plate used: white 96-well plates (flat bottom)
- Lysate preparation: After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement.

DATA EVALUATION
- Cytotoxicity assessment: In the first experiment, a max luciferase activity (Imax) induction of 2.15 was determined at a test item concentration of 125 μM. The corresponding cell viability was 25.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.69) was found to be 62.50 μM. The corresponding cell viability was <70% (28.0%).
In the second experiment, a max luciferase activity (Imax) induction of 2.11 was determined at a test item concentration of 250 μM. The corresponding cell viability was 34.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.57) was found to be 62.50 μM. The corresponding cell viability was <70% (28.2%).
- Prediction model used: see “Study evaluation and decision criteria used”


Vehicle / solvent control:
DMSO
Negative control:
not applicable
Positive control:
cinnamic aldehyde [442D]
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (6.34 in experiment 1; 3.67 in experiment 2).
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
Imax [442D]
Value:
2.15
Cell viability:
25.1%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
Imax [442D]
Value:
2.11
Cell viability:
34.1%
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Value:
43.43 µM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Value:
56.62 µM
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Outcome of the prediction model:
negative [in vitro/in chemico]
Other effects / acceptance of results:
Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

   Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

94.4

101.8

98.1

5.2

8.00

97.6

111.0

104.3

9.5

16.00

107.1

107.4

107.2

0.2

32.00

112.4

118.5

115.4

4.3

64.00

116.5

125.8

121.1

6.6

Test Item

0.98

102.7

107.8

105.3

3.7

1.95

112.3

107.5

109.9

3.4

3.91

115.4

113.5

114.5

1.4

7.81

61.6

108.1

84.8

32.9

15.63

35.7

63.6

49.6

19.7

31.25

28.9

35.6

32.3

4.7

62.50

28.0

28.2

28.1

0.1

125.00

25.1

30.0

27.6

3.4

250.00

26.5

34.1

30.3

5.4

500.00

26.6

36.8

31.7

7.2

1000.00

28.7

35.9

32.3

5.1

2000.00

28.7

37.4

33.0

6.2

Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.00

1.15

1.17

1.11

0.09

 

8.00

0.94

0.96

1.12

1.00

0.10

 

16.00

1.25

1.30

1.31

1.28

0.03

 

32.00

1.50

1.68

1.94

1.71

0.22

*

64.00

5.44

5.09

8.50

6.34

1.88

*

Test Item

0.98

0.93

1.16

1.09

1.06

0.12

 

1.95

1.04

0.77

0.77

0.86

0.16

 

3.91

0.83

1.18

1.28

1.09

0.23

 

7.81

1.00

1.07

1.31

1.13

0.16

 

15.63

1.00

1.19

1.44

1.21

0.22

 

31.25

1.11

1.53

1.49

1.38

0.23

 

62.50

1.59

1.56

1.93

1.69

0.21

 

125.00

2.18

2.03

2.25

2.15

0.11

*

250.00

2.15

1.81

2.25

2.07

0.23

*

500.00

1.61

1.75

1.63

1.66

0.08

*

1000.00

1.05

1.41

1.18

1.21

0.18

 

2000.00

1.21

1.20

1.34

1.25

0.08

 

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.05

1.23

1.08

1.12

0.10

 

8.00

1.20

1.18

1.17

1.18

0.02

 

16.00

1.42

1.38

1.49

1.43

0.06

 

32.00

2.20

1.65

2.07

1.97

0.29

*

64.00

3.91

3.04

4.08

3.67

0.56

*

Test Item

0.98

0.91

0.95

0.81

0.89

0.07

 

1.95

0.83

0.99

0.96

0.92

0.08

 

3.91

0.80

0.91

1.07

0.93

0.13

 

7.81

1.17

1.27

1.07

1.17

0.10

 

15.63

0.94

1.02

1.44

1.13

0.27

 

31.25

1.09

1.29

1.25

1.21

0.11

 

62.50

1.44

1.59

1.67

1.57

0.12

*

125.00

1.57

2.15

1.76

1.83

0.30

*

250.00

2.23

1.87

2.24

2.11

0.21

*

500.00

1.98

1.52

1.86

1.79

0.24

*

1000.00

1.64

1.47

1.94

1.68

0.24

*

2000.00

1.58

1.54

1.68

1.60

0.07

*

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity – Overall Induction

Overall Induction

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.11

1.12

1.11

0.01

 

8.00

1.00

1.18

1.09

0.13

 

16.00

1.28

1.43

1.36

0.10

 

32.00

1.71

1.97

1.84

0.19

*

64.00

6.34

3.67

5.01

1.89

 

Test Item

0.98

1.06

0.89

0.97

0.12

 

1.95

0.86

0.92

0.89

0.05

 

3.91

1.09

0.93

1.01

0.12

 

7.81

1.13

1.17

1.15

0.03

 

15.63

1.21

1.13

1.17

0.05

 

31.25

1.38

1.21

1.29

0.12

 

62.50

1.69

1.57

1.63

0.09

*

125.00

2.15

1.83

1.99

0.23

*

250.00

2.07

2.11

2.09

0.03

*

500.00

1.66

1.79

1.72

0.09

*

1000.00

1.21

1.68

1.45

0.33

 

2000.00

1.25

1.60

1.43

0.25

 

* = significant induction according to Student’s t-test, p<0.05

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

43.43

56.62

50.03

9.33

Imax

2.15

2.11

2.13

0.03

IC30[µM]

7.20

14.50

10.85

5.16

IC50[µM]

11.31

23.20

17.26

8.41

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

17.5

pass

12.5

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

24.15

pass

18.10

pass

Induction PC at 64 µM

2.00 < x < 8.00

6.34

pass

3.67

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.3

3.3

41

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.3

0.6

41

EC1.5 PC

7 < x < 34 µM

20.4

6.7

41

Induction PC at 64 µM

2.00 < x < 8.00

3.3

1.1

41

 

Interpretation of results:
other: The outcome should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test item was dissolved in DMSO.

Based on a molecular weight of 340 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was preparedby serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 2.15 was determined at a test item concentration of 125 µM. The corresponding cell viability was 25.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.69) was found to be 62.50 µM. The corresponding cell viability was <70% (28.0%).The calculated EC1.5was < 1000 µM (43.43 µM).

In the second experiment, a max luciferase activity (Imax) induction of 2.11 was determined at a test item concentration of 250 µM. The corresponding cell viability was 34.1%. The lowest tested concentration with a significant luciferase induction >1.5 (1.57) was found to be 62.50 µM. The corresponding cell viability was <70% (28.2%).The calculated EC1.5was<1000 µM (56.62 µM).

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction in the non-cytotoxic dose range.

Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to the QMRF and QPRF files provided under the section attached justification.
Qualifier:
according to guideline
Guideline:
other: ECHA Guidance on IR/CSA R.6 QSARs and grouping of chemicals
Principles of method if other than guideline:
Estimates the skin sensitising properties of chemicals using structural alert relationships.
GLP compliance:
no
Specific details on test material used for the study:
see QPRF
Key result
Group:
test chemical
Remarks on result:
no indication of skin sensitisation
Remarks:
No structural alert for skin sensitisation.
Interpretation of results:
other: Derek result: Nothing to report
Conclusions:
Using Derek Nexus v5.0, the skin sensitising potential of the test item was estimated to be absent (Nothing to report). The substance is within the applicability domain of the model. Thus, the estimation can be regarded as accurate.
Executive summary:

The skin sensitising properties were estimated using Derek Nexus v5.0. The skin sensitising potential of the test item was estimated to be absent (Nothing to report) based on the described QSAR method (Derek, 2017).

The adequacy of a prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information the test item is not classified for skin sensitisation according to the EU Regulation (EC ) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.