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Diss Factsheets

Administrative data

Description of key information

Three key elements of the AOP for skin sensitisation have been adressed in vitro. The results are shown below:

OECD 442 C: positive

OECD 442 D: negative

OECD 442 E: positive

Overall, there was indication for a positive response in two out of three key events. Therefore, the test material is considerd as sensitiser.

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:
2017-12-21 to 2018-01-20
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:
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 64.20%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
100
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:
6.46
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

4254.6284

0.5340

STD2

2648.5273

0.2670

2161.7087

0.2670

STD3

1327.5009

0.1335

1051.8925

0.1335

STD4

670.7400

0.0667

525.2503

0.0667

STD5

334.8562

0.0334

260.5259

0.0334

STD6

166.3505

0.0167

130.8325

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

0.0000

0.00*

100.00

100.00

0.00

0.00

0.0000

0.00*

100.00

0.0000

0.00*

100.00

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

1675.9452

0.2100

57.93

56.62

1.13

2.00

1753.5808

0.2197

55.98

1754.5223

0.2198

55.95

Test Item

3679.9751

0.4607

6.60

6.46

0.41

6.30

3703.4563

0.4637

6.00

3672.8149

0.4598

6.78

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 Prediction²

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

53.23

High Reactivity

sensitiser

100.00

High Reactivity

sensitiser

Positive Control

62.40

High Reactivity

sensitiser

68.17

Moderate Reactivity

sensitiser
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item showed high reactivity towards both peptides. The test item is considered as “sensitiser”.
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 was dissolved in dist. water, based on the results of the pre-experiments. Based on a molecular weight of 290 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 (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM 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 sample of the co-elution control of the positive 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 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 Cwater).

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was > 6.38% (53.23%). Based on the prediction model 1 the test item can be considered as sensitiser. A precipitation in the cysteine experiment was observed. 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 and the reactivity might be underestimated.

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 64.20%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-05 to 2018-03-20
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 (7.72 (experiment 1); 4.70 (experiment 2).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
4.17
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 2000 µM.
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
32.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
906.94
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:
2.18
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 2000 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
31.2
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:
other: 733.90
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

100.4

87.0

93.7

9.5

8.00

98.5

66.4

82.5

22.7

16.00

97.0

76.3

86.6

14.6

32.00

95.4

59.3

77.4

25.5

64.00

95.8

48.8

72.3

33.2

Test Item

0.98

87.2

100.6

93.9

9.5

1.95

90.8

102.8

96.8

8.5

3.91

90.5

101.5

96.0

7.8

7.81

92.0

98.1

95.0

4.3

15.63

92.2

96.9

94.6

3.3

31.25

89.0

100.6

94.8

8.2

62.50

79.4

87.8

83.6

5.9

125.00

66.6

62.5

64.6

2.9

250.00

51.9

50.1

51.0

1.2

500.00

37.5

44.0

40.7

4.6

1000.00

32.7

39.0

35.8

4.4

2000.00

32.2

31.2

31.7

0.7

 

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

1.28

1.23

1.23

1.25

0.03

 

8.00

1.44

1.35

1.29

1.36

0.07

 

16.00

1.82

1.66

1.50

1.66

0.16

*

32.00

2.52

2.75

1.68

2.32

0.57

*

64.00

7.62

(12.54)**

7.82

7.72

0.14

*

Test Item

0.98

1.33

1.15

1.37

1.28

0.12

 

1.95

1.05

1.03

0.96

1.01

0.05

 

3.91

1.15

1.04

1.08

1.09

0.06

 

7.81

1.13

1.15

1.04

1.11

0.06

 

15.63

1.09

1.10

1.19

1.13

0.06

 

31.25

1.33

1.43

1.29

1.35

0.07

 

62.50

1.32

1.37

1.13

1.27

0.13

 

125.00

1.25

1.30

1.16

1.24

0.07

 

250.00

1.46

1.30

1.26

1.34

0.11

 

500.00

1.50

1.56

1.11

1.39

0.25

 

1000.00

1.47

1.79

1.31

1.52

0.25

*

2000.00

4.92

4.87

2.71

4.17

1.26

*

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

**= an outlier according to Grubbs, Nalimov and Dixon

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

1.16

1.18

1.10

1.15

0.04

 

8.00

1.16

1.10

1.09

1.12

0.04

 

16.00

1.65

1.69

1.49

1.61

0.11

*

32.00

2.23

1.88

1.93

2.01

0.19

*

64.00

4.84

4.51

4.75

4.70

0.17

*

Test Item

0.98

1.39

1.39

1.32

1.37

0.04

 

1.95

0.86

1.03

0.92

0.94

0.09

 

3.91

1.10

1.07

0.96

1.04

0.07

 

7.81

0.96

1.11

1.08

1.05

0.08

 

15.63

1.00

1.04

0.93

0.99

0.06

 

31.25

1.01

1.00

0.98

1.00

0.01

 

62.50

1.13

1.06

1.07

1.09

0.04

 

125.00

1.17

1.14

1.02

1.11

0.08

 

250.00

0.97

1.07

0.97

1.00

0.05

 

500.00

1.35

1.27

1.24

1.29

0.05

 

1000.00

1.69

1.78

1.75

1.74

0.05

*

2000.00

1.95

2.01

2.59

2.18

0.35

*

* = 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

1.25

1.15

1.20

0.07

 

8.00

1.36

1.12

1.24

0.17

 

16.00

1.66

1.61

1.63

0.04

*

32.00

2.32

2.01

2.16

0.22

*

64.00

7.72**

4.70

6.21

2.14

 

Test Item

0.98

1.28

1.37

1.32

0.06

 

1.95

1.01

0.94

0.97

0.05

 

3.91

1.09

1.04

1.07

0.04

 

7.81

1.11

1.05

1.08

0.04

 

15.63

1.13

0.99

1.06

0.10

 

31.25

1.35

1.00

1.17

0.25

 

62.50

1.27

1.09

1.18

0.13

 

125.00

1.24

1.11

1.17

0.09

 

250.00

1.34

1.00

1.17

0.24

 

500.00

1.39

1.29

1.34

0.07

 

1000.00

1.52

1.74

1.63

0.15

*

2000.00

4.17

2.18

3.18

1.40

 

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

** = Mean out of replicate 1 and 3. 2 replicate is an outlier according to Grubbs, Nalimov and Dixon.

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

906.94

733.90

820.42

122.36

Imax

4.17

2.18

3.18

1.40

IC30[µM]

108.55

106.47

107.51

1.47

IC50[µM]

282.69

254.91

268.80

19.64

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

13.1

pass

9.6

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

11.72

pass

14.23

pass

Induction PC at 64 µM

2.00 < x < 8.00

7.72*

pass

4.70

pass

* = Mean out of replicate 1 and 3. 2 replicate is an outlier according to Grubbs, Nalimov and Dixon.

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 DMSO. Based on a molecular weight of 290 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, a max luciferase activity (Imax) induction of 4.17 was determined at a test item concentration of 2000.00 µM. The corresponding cell viability was 32.2%. The lowest tested concentration with a significant luciferase induction >1.5 (1.52) was found to be 1000.00 µM. The corresponding cell viability was <70% (32.7%).Thecalculated EC1.5 was<1000 µM (906.94).

In the second experiment, a max luciferase activity (Imax) induction of 2.18 was determined at a test item concentration of 2000.00 µM. The corresponding cell viability was 31.2%. The lowest tested concentration with a significant luciferase induction >1.5 (1.74) was found to be 1000.00 µM. The corresponding cell viability was <70% (39.0%).The calculated EC1.5 was < 1000 µM (733.90).

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.

The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment one of the technical replicate exceeded the threshold with a fold induction of 12.54. On the one hand, the positive control clearly induces the luciferase induction in the KerationSens™ cell line, showing the capability of the test system to predict sensitisation. On the other hand, the results in this first experiment are clearly negative, indicating no oversensitivity of the test system. The single value of the positive control exceeding the upper threshold was therefore considered to be an outliner and not biological relevant. Furthermore all other controls confirmed the validity of the study.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on the study design:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (273% experiment 1; 348% experiment 2) and 200% for CD54 (250% experiment 1; 371% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1 and 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: 1 and 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item did upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered as 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:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided data, the test item is considered to be classified as skin sensitizer Cat. 1 and labelled with H317 (May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008.