Registration Dossier

Administrative data

Description of key information

OECD 442C: negative

OECD 442D: 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:
key study
Study period:
2017-10-19 to 2018-01-18
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))
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. certificate)
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 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 63.81% (experiment 1) and 68.63% (experiment 2).
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run (experiment 1)
Value:
0.12
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run (experiment 1)
Value:
13.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run (experiment 2)
Value:
0.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run (experiment 2)
Value:
4.43
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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 Experiment 1

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

4523.9644

0.5340

4169.8604

0.5340

STD2

2255.1311

0.2670

2102.4409

0.2670

STD3

1081.2599

0.1335

1026.2726

0.1335

STD4

538.3588

0.0667

515.4506

0.0667

STD5

264.6839

0.0334

262.0216

0.0334

STD6

131.3584

0.0167

131.2270

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Cysteine and Lysine Values of the Calibration Curve Experiment 2

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

17.870

0.5340

4663.5073

0.5340

STD2

9.012

0.2670

2361.9275

0.2670

STD3

4.518

0.1335

1192.6749

0.1335

STD4

2.252

0.0667

602.5394

0.0667

STD5

1.101

0.0334

302.3090

0.0334

STD6

0.530

0.0167

151.3044

0.0167

STD7

0.000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide Experiment 1

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

1320.4471

0.1577

70.95

71.10

0.18

0.25

1315.4908

0.1571

71.06

1304.7782

0.1559

71.29

Test Item

4600.2661

0.5437

0.00

0.12

0.20

173.21

4583.2500

0.5417

0.00

4529.0562

0.5353

0.35

Depletion of the Cysteine Peptide Experiment 2

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

4.447

0.1325

74.33

74.15

0.18

0.25

4.477

0.1334

74.15

4.510

0.1344

73.96

Test Item

17.353

0.5175

0.00

0.30

0.52

173.21

17.167

0.5120

0.89

17.396

0.5188

0.00

Depletion of the Lysine Peptide Experiment 1

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

1690.0214

0.2164

56.37

56.51

0.47

0.83

1699.1774

0.2175

56.14

1664.2920

0.2131

57.04

Test Item

3573.1260

0.4572

7.76

13.20

5.57

42.20

3372.0542

0.4315

12.95

3141.8584

0.4020

18.89

Depletion of the Lysine Peptide Experiment 2

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

1539.7554

0.1748

64.31

63.12

1.08

1.71

1602.9802

0.1820

62.84

1630.8385

0.1852

62.20

Test Item

4162.2075

0.4752

3.52

4.43

0.86

19.40

4118.6553

0.4702

4.53

4088.5010

0.4667

5.23

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%PPD13.89%

No orMinimalReactivity

Negative

13.89% <PPD23.09%

LowReactivity

Positive

23.09% <PPD98.24%

ModerateReactivity

98.24% <PPD100%

High Reactivity

Categorization of the Test Item Experiment 1

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

6.66

Low Reactivity

sensitiser

0.12

Minimal Reactivity

no sensitiser

Positive Control

63.81

High Reactivity

sensitiser

71.10

Moderate Reactivity

sensitiser

Categorization of the Test Item Experiment 2

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

2.36

Minimal Reactivity

no sensitiser

0.30

Minimal Reactivity

no sensitiser

Positive Control

68.63

High Reactivity

sensitiser

74.15

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 both peptides. The test item is 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.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-10-25 to 2017-11-10
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)
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on 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 (3.95 in experiment 1; 3.17 in experiment 2).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
1.31
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 62.50 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
114
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
1.42
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 62.50 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
104.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
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

100.2

99.2

99.7

0.7

8.00

112.2

104.1

108.2

5.7

16.00

124.3

106.0

115.1

12.9

32.00

129.6

111.4

120.5

12.9

64.00

135.1

112.8

124.0

15.7

Test Item

0.98

93.4

99.5

96.4

4.3

1.95

88.1

92.9

90.5

3.4

3.91

101.8

103.2

102.5

0.9

7.81

99.6

99.8

99.7

0.1

15.63

108.6

101.1

104.8

5.3

31.25

112.0

105.4

108.7

4.7

62.50

114.0

104.3

109.1

6.9

125.00

113.9

109.0

111.4

3.5

250.00

113.8

105.1

109.4

6.2

500.00

106.3

104.0

105.1

1.6

1000.00

107.6

98.3

102.9

6.6

2000.00

113.2

96.9

105.1

11.5

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

1.12

1.18

1.22

1.17

0.05

8.00

1.31

1.29

1.18

1.26

0.07

16.00

1.47

1.55

1.47

1.50

0.04

32.00

2.12

1.98

2.17

2.09

0.10

*

64.00

3.92

3.82

4.10

3.95

0.14

*

Test Item

0.98

0.83

0.88

0.88

0.86

0.03

1.95

0.99

0.87

0.84

0.90

0.08

3.91

1.05

0.96

0.99

1.00

0.05

7.81

1.12

1.14

1.04

1.10

0.05

15.63

1.10

1.04

1.12

1.08

0.04

31.25

1.08

1.02

1.23

1.11

0.11

62.50

1.17

1.23

1.52

1.31

0.19

125.00

1.22

1.11

1.22

1.18

0.07

250.00

1.46

1.32

1.13

1.30

0.17

500.00

1.20

1.07

1.23

1.17

0.09

1000.00

0.95

1.16

1.05

1.05

0.11

2000.00

0.92

0.92

0.99

0.94

0.04

* = 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

1.38

1.32

1.31

1.34

0.04

8.00

1.33

1.35

0.78

1.16

0.33

16.00

1.68

1.69

1.62

1.66

0.04

*

32.00

2.15

2.19

2.13

2.16

0.03

*

64.00

4.07

4.17

1.27

3.17

1.65

Test Item

0.98

1.16

1.12

0.50

0.93

0.37

1.95

1.25

1.14

0.31

0.90

0.51

3.91

1.16

1.09

0.17

0.81

0.55

7.81

1.16

1.10

0.85

1.04

0.17

15.63

1.17

1.34

1.16

1.22

0.10

31.25

1.47

1.09

1.33

1.30

0.19

62.50

1.50

1.50

1.25

1.42

0.15

125.00

1.22

1.38

0.93

1.18

0.23

250.00

0.97

1.22

0.68

0.95

0.27

500.00

0.95

1.26

1.15

1.12

0.15

1000.00

0.95

0.71

1.17

0.94

0.23

2000.00

0.91

0.81

1.02

0.91

0.10

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

Induction of Luciferase Activity – 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

1.17

1.34

1.25

0.12

 

8.00

1.26

1.16

1.21

0.07

 

16.00

1.50

1.66

1.58

0.12

*

32.00

2.09

2.16

2.13

0.05

*

64.00

3.95

3.17

3.56

0.55

*

Test Item

0.98

0.86

0.93

0.89

0.05

 

1.95

0.90

0.90

0.90

0.00

 

3.91

1.00

0.81

0.90

0.13

 

7.81

1.10

1.04

1.07

0.04

 

15.63

1.08

1.22

1.15

0.10

 

31.25

1.11

1.30

1.20

0.13

 

62.50

1.31

1.42

1.36

0.08

 

125.00

1.18

1.18

1.18

0.00

 

250.00

1.30

0.95

1.13

0.25

 

500.00

1.17

1.12

1.14

0.03

 

1000.00

1.05

0.94

1.00

0.08

 

2000.00

0.94

0.91

0.93

0.02

 

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

 

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n/a

n/a

n/a

n/a

Imax

1.31

1.42

1.36

0.08

IC30[µM]

n/a

n/a

n/a

n/a

IC50[µM]

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%

7.8

pass

15.8

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

16.09

pass

13.42

pass

Induction PC at 64 µM

2.00 < x < 8.00

3.95

pass

3.17

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:
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.
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.
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 for classification 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.