Registration Dossier

Administrative data

Description of key information

Two key events of the skin sensitization AOP were addressed by in vitro experiments. In these experiments, no indication of sensitization potential was found. These data underpinned with a negative QSAR prediction on skin sensitization provide sufficient evidence to conclude that the test compound is not 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:
key study
Study period:
2017-12-07 to 2017-12-21
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 65.46%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0.51
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%]
Value:
11.24
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

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

4862.8555

0.5340

4663.5073

0.5340

STD2

2384.9666

0.2670

2361.9275

0.2670

STD3

1183.9525

0.1335

1192.6749

0.1335

STD4

582.0698

0.0667

602.5394

0.0667

STD5

301.6335

0.0334

302.3090

0.0334

STD6

159.0007

0.0167

151.3044

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

1453.8585

0.1612

68.18

67.81

0.64

0.94

1504.0075

0.1667

67.08

1453.6510

0.1612

68.18

Test Item

4573.3066

0.5044

0.00

0.51

0.88

173.21

4618.0737

0.5093

0.00

4472.1553

0.4932

1.52

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

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

3542.4495

0.4042

9.90

11.24

0.95

8.44

3497.0088

0.3990

11.05

3468.2253

0.3957

11.78

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

5.71

Minimal Reactivity

no sensitiser

0.51

Minimal Reactivity

no sensitiser

Positive Control

65.46

High Reactivity

sensitiser

67.81

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. However, 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 item was dissolved in isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 366 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. Precipitation was observed for the samples of the test item (including test item co-elution control) and for the positive control samples (including positive control co-elution control). Samples were not centrifuged prior to the HPLC analysis, but test item samples were pipetted into new vials since the precipitates were observed on the surface.

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. Precipitation was observed for the samples of the test item (including co-elution control). Phase separation was observed for the samples of the positive control (including the positive control co-elution control). Samples of the test item were centrifuged 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.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C isopropanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (5.71%).

Precipitation in both peptides was observed. Since it cannot be determined if the precipitate resulted from the test item or the peptides, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

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 both experiments 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 65.46%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-14 to 2018-02-05
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 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 (4.60 (experiment 1); 3.43 (experiment 2)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.54
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 500µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
130.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
7.63
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 15.63 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
126.5
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

105.2

83.5

94.4

15.3

8.00

108.8

87.0

97.9

15.4

16.00

114.3

96.3

105.3

12.7

32.00

123.0

100.6

111.8

15.9

64.00

167.6

111.7

139.6

39.5

Test Item

0.98

106.2

103.7

105.0

1.7

1.95

101.6

104.1

102.8

1.8

3.91

134.7

100.4

117.6

24.3

7.81

164.2

124.9

144.6

27.8

15.63

184.1

126.5

155.3

40.7

31.25

168.6

111.6

140.1

40.3

62.50

160.4

106.2

133.3

38.4

125.00

154.7

107.3

131.0

33.6

250.00

141.8

115.7

128.7

18.5

500.00

130.3

111.0

120.7

13.6

1000.00

118.1

104.8

111.5

9.4

2000.00

89.6

104.1

96.9

10.3

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

1.44

1.05

1.43

1.31

0.22

 

8.00

1.26

1.24

1.48

1.33

0.13

 

16.00

1.70

1.25

1.80

1.58

0.29

*

32.00

2.46

1.88

2.36

2.23

0.31

*

64.00

5.70

3.23

4.86

4.60

1.26

*

Test Item

0.98

1.30

1.22

0.93

1.15

0.20

 

1.95

1.27

1.12

0.99

1.13

0.14

 

3.91

1.45

1.39

0.73

1.19

0.40

 

7.81

2.06

1.51

0.98

1.52

0.54

 

15.63

1.54

1.61

1.07

1.41

0.29

 

31.25

1.68

1.30

1.04

1.34

0.32

 

62.50

2.01

1.51

0.98

1.50

0.52

 

125.00

1.69

1.25

1.23

1.39

0.26

 

250.00

1.51

1.48

1.03

1.34

0.27

 

500.00

1.87

1.41

1.34

1.54a)

0.29

*

1000.00

1.61

1.32

1.02

1.32

0.30

 

2000.00

1.24

1.03

0.84

1.04

0.20

 

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

a) Since the slight increase about the threshold of 1.5 was caused mainly by the value of the first replicate and no dose effect relationship was observed, the significant luciferase induction at this concentration was regarded as biological not relevant.

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

1.07

1.12

1.08

0.04

 

8.00

1.17

1.29

1.25

1.24

0.07

 

16.00

1.31

1.35

1.68

1.45

0.20

 

32.00

1.93

1.85

1.69

1.83

0.12

*

64.00

3.67

2.88

3.74

3.43

0.48

*

Test Item

0.98

0.96

1.42

0.96

1.11

0.27

 

1.95

1.14

1.03

1.09

1.08

0.06

 

3.91

1.17

1.20

1.32

1.23

0.08

 

7.81

1.00

1.22

1.20

1.14

0.12

 

15.63

1.29

1.82

1.22

1.45

0.33

 

31.25

1.11

1.34

1.32

1.26

0.13

 

62.50

1.20

1.25

1.69

1.38

0.27

 

125.00

1.19

1.22

1.71

1.37

0.29

 

250.00

1.20

1.39

1.73

1.44

0.27

 

500.00

1.19

1.31

1.63

1.38

0.23

 

1000.00

1.13

1.28

1.34

1.25

0.11

 

2000.00

1.01

0.92

1.25

1.06

0.17

 

* = 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

1.31

1.08

1.19

0.16

 

8.00

1.33

1.24

1.28

0.06

 

16.00

1.58

1.45

1.51

0.10

*

32.00

2.23

1.83

2.03

0.29

*

64.00

4.60

3.43

4.01

0.83

*

Test Item

0.98

1.15

1.11

1.13

0.02

 

1.95

1.13

1.08

1.10

0.03

 

3.91

1.19

1.23

1.21

0.03

 

7.81

1.52

1.14

1.33

0.27

 

15.63

1.41

1.45

1.43

0.03

 

31.25

1.34

1.26

1.30

0.06

 

62.50

1.50

1.38

1.44

0.09

 

125.00

1.39

1.37

1.38

0.01

 

250.00

1.34

1.44

1.39

0.07

 

500.00

1.54

1.38

1.46

0.12

 

1000.00

1.32

1.25

1.28

0.05

 

2000.00

1.04

1.06

1.05

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]

(7.63)a

n/a

-

-

Imax

1.54

1.45

1.49

0.07

IC30[µM]

n/a

n/a

-

-

IC50[µM]

n/a

n/a

-

-

n/a: not applicable

a) see discussion

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

10.1

pass

9.1

pass

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

≥ 1

3.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

13.39

pass

18.27

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.60

pass

3.43

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.
Executive summary:

In the present study the test material was dissolved in THF. Based on a molecular weight of 366 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 aconstant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. 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 1.54 was determined at a test item concentration of 500 µM. The corresponding cell viability was 130.3%. No further significant luciferase induction >1.5 was found in the tested concentration range. Since the slight increase about the threshold of 1.5 at the concentration of 500 µM was caused mainly by the value of the first replicate and no dose effect relationship was observed, the significant luciferase induction at this concentration was regarded as biological not relevant.Therefore, no EC1.5 value could be calculated.

In the second experiment, a 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, 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
Parameter:
other: Structural Alert for skin sensitisaton
Value:
0
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 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.