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Description of key information

For the assessment of skin sensitisation, in vitro methods have been applied. The following results have been obtained:

OECD 442 C: negative

OECD 442 D: negative

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:
key study
Study period:
2018-07-23 to 2018-08-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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 on the study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (6.43 (experiment 1); 7.85 (experiment 2)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.07
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 62.50 µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
86.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.17
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 125 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
96.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
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

86.4

113.7

100.0

19.4

8.00

92.4

116.6

104.5

17.1

16.00

96.0

117.3

106.6

15.0

32.00

86.6

122.4

104.5

25.3

64.00

84.1

109.9

97.0

18.2

Test Item

0.98

100.9

102.6

101.8

1.2

1.95

97.9

103.6

100.8

4.0

3.91

91.5

109.4

100.5

12.6

7.81

91.2

102.9

97.1

8.3

15.63

88.3

100.3

94.3

8.5

31.25

88.5

96.5

92.5

5.7

62.50

86.2

104.1

95.2

12.7

125.00

80.7

96.3

88.5

11.0

250.00

81.8

102.1

92.0

14.3

500.00

85.9

98.4

92.1

8.8

1000.00

68.5

86.5

77.5

12.8

2000.00

63.9

84.9

74.4

14.8

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

1.62

1.18

1.36

1.39

0.22

 

8.00

1.27

1.20

1.14

1.20

0.07

 

16.00

1.49

1.63

1.53

1.55

0.08

*

32.00

2.20

1.84

2.24

2.09

0.22

*

64.00

7.00

6.59

5.69

6.43

0.67

*

Test Item

0.98

1.08

1.02

1.01

1.04

0.04

 

1.95

0.99

0.94

1.04

0.99

0.05

 

3.91

1.12

1.07

1.03

1.07

0.04

 

7.81

1.01

1.01

1.11

1.04

0.06

 

15.63

1.04

0.92

1.12

1.03

0.10

 

31.25

0.94

0.97

0.95

0.96

0.02

 

62.50

1.16

0.94

1.12

1.07

0.12

 

125.00

1.05

0.86

1.02

0.97

0.10

 

250.00

0.87

1.08

0.92

0.95

0.11

 

500.00

0.82

0.82

0.83

0.82

0.01

 

1000.00

0.82

0.78

0.67

0.76

0.08

 

2000.00

0.60

0.73

0.61

0.65

0.07

 

* = 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

1.19

1.24

1.47

1.30

0.15

 

8.00

1.26

1.30

1.56

1.37

0.16

 

16.00

1.52

1.51

1.90

1.65

0.22

*

32.00

2.38

2.29

2.99

2.55

0.38

*

64.00

6.77

6.61

10.16

7.85

2.00

*

Test Item

0.98

0.94

0.89

1.19

1.01

0.16

 

1.95

0.98

0.87

1.04

0.97

0.09

 

3.91

0.88

1.01

1.15

1.01

0.13

 

7.81

1.18

0.89

1.03

1.03

0.14

 

15.63

0.98

1.01

1.09

1.03

0.05

 

31.25

0.87

0.89

1.11

0.96

0.13

 

62.50

0.94

1.07

0.99

1.00

0.07

 

125.00

1.07

0.95

1.48

1.17

0.28

 

250.00

0.85

0.77

1.18

0.93

0.22

 

500.00

0.82

0.71

1.16

0.90

0.24

 

1000.00

0.77

0.67

0.95

0.80

0.14

 

2000.00

0.60

0.53

0.84

0.66

0.16

 

* = 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

1.39

1.30

1.34

0.06

 

8.00

1.20

1.37

1.29

0.12

 

16.00

1.55

1.65

1.60

0.07

*

32.00

2.09

2.55

2.32

0.33

*

64.00

6.43

7.85

7.14

1.00

*

Test Item

0.98

1.04

1.01

1.02

0.02

 

1.95

0.99

0.97

0.98

0.02

 

3.91

1.07

1.01

1.04

0.04

 

7.81

1.04

1.03

1.04

0.01

 

15.63

1.03

1.03

1.03

0.00

 

31.25

0.96

0.96

0.96

0.00

 

62.50

1.07

1.00

1.04

0.05

 

125.00

0.97

1.17

1.07

0.14

 

250.00

0.95

0.93

0.94

0.01

 

500.00

0.82

0.90

0.86

0.05

 

1000.00

0.76

0.80

0.78

0.03

 

2000.00

0.65

0.66

0.65

0.01

 

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5

n.a.

n.a.

n.a.

n.a.

Imax

1.07

1.17

1.12

0.07

IC30

955.91

n.a.

n.a.

n.a.

IC50

n.a.

n.a.

n.a.

n.a.

n.a. = not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

9.4

pass

16.4

pass

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

≥ 1

3.0

pass

3.0

pass

EC1.5 PC

7 < x < 34 µM

14.84

pass

11.73

pass

Induction PC at 64 µM

2.00 < x < 8.00

6.43

pass

7.85

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.6

3.5

96

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

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96

Interpretation of results:
GHS criteria not met
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.


Executive summary:

In the present study the test material was dissolved in DMSO. Based on a molecular weight of 214.23 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 prepared by 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, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-25 to 2018-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group
Details on the study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
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 65.38%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable because of methodological limitations
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

15.0360

0.5340

14.1920

0.5340

STD2

7.5920

0.2670

7.1540

0.2670

STD3

3.7630

0.1335

3.5770

0.1335

STD4

1.8500

0.0667

1.7790

0.0667

STD5

0.8890

0.0334

0.8770

0.0334

STD6

0.4300

0.0167

0.4310

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

4.1310

0.1469

70.73

71.32

0.53

0.74

4.0270

0.1433

71.47

3.9870

0.1418

71.75

Test Item

14.7510

0.5228

0.00

0.00

0.00

--

14.3960

0.5102

0.00

14.3970

0.5102

0.00

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

5.3010

0.1990

60.30

59.44

0.84

1.41

5.4220

0.2036

59.39

5.5240

0.2074

58.63

Test Item*

1.8350

0.0688

86.39

87.57

1.07

1.22

1.5540

0.0582

88.47

1.6370

0.0614

87.86

* A peak in the co-elution control of the test item was observed at the retention time of the lysine peptide Therefore, co-elution of test item and peptide occurred and the given peak areas were not used for evaluation.

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

--

--

--

0.00

Minimal Reactivity

no sensitiser

Positive Control

65.38

High Reactivity

sensitiser

71.32

Moderate Reactivity

sensitiser

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might 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 material was dissolved in dist. water : acetonitrile 1:1 (v/v), based on the results of the pre-experiments. Based on a molecular weight of 214.23 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 stock 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. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

Co-elution of the test item with the lysine peptide peak was observed. Therefore, the given peak areas cannot be used for evaluation.

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 Cdist. water : acetonitrile 1:1 (v/v)).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.00%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

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 65.38%.

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 there is no need to classified according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.