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:
key study
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:
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:
direct peptide binding assay
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 63.50%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
0.21
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0
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

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:
key study
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:
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 (6.34 in experiment 1; 3.67 in experiment 2).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
2.15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
2.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Value:
43.43
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Value:
56.62
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
25.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
34.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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:
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 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:
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
Parameter:
other: Structural Alert for Skin Sensitisation
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 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.