Registration Dossier

Administrative data

Description of key information

OECD 442 C: no prediction due to precipitation of the test material

OECD 442 D: negative

OECD 442 E: positive

As a worst case approach, the test item is considered to be 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:
2017-11-23 to 2018-01-20
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 65.38%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
0.55
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0.37
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

17.8700

0.5340

4254.6284

0.5340

STD2

9.0120

0.2670

2161.7087

0.2670

STD3

4.5180

0.1335

1051.8925

0.1335

STD4

2.2520

0.0667

525.2503

0.0667

STD5

1.1010

0.0334

260.5259

0.0334

STD6

0.5300

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

4.4470

0.1325

74.33

74.15

0.18

0.25

4.4770

0.1334

74.15

4.5100

0.1344

73.96

Test Item

16.9410

0.5052

0.26

0.55

0.25

46.16

16.8690

0.5031

0.68

16.8640

0.5029

0.71

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

4019.5232

0.5032

0.26

0.37

0.13

35.68

4016.2871

0.5028

0.34

4009.0984

0.5019

0.52

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

--

--

--

0.55

Minimal Reactivity

negative

Positive Control

65.38

High Reactivity

positive

74.15

Moderate Reactivity

positive

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 item was dissolved in DMF, based on the results of the pre-experiments. A maximum solubility of 37.5 mM was determined. However, in the main experiments stock solutions of 100 mM were prepared accidentally. It is assumed that not the whole amount of test item was dissolved. Since with this concentration the highest possible concentration was guaranteed, the experiments were not repeated. For reasons of simplicity the concentrations of the stock solutions are reported as 100 mM. Based on a molecular weight of 348.47 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 the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM stock 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 the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the co-elution control of the positive control sample.

A minor co-elution of the test item with the lysine peptide peak was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion and were not used for evaluation. The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide (0.55%). 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 65.38%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-14 to 2018-06-18
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 (7.40 (experiment 1); 3.90 (experiment 2) and 3.79 (experiment 3)).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
1.48
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 31.25 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
145.3
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:
1.64
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 15.63 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
108.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Value:
12.25
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Key result
Parameter:
other: luciferase activity
Run / experiment:
3
Value:
1.45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: concentration: 42.22 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
3
Value:
149.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5 [µM]
Run / experiment:
3
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.

Cytotoxicity

Results of the Cytotoxicity Measurement

Conc. [µM]

Cell Viability [%]

Exp 3

Exp 1

Exp 2

Mean

SD

Conc.

[µM]

Cell Viability [%]

Positive Control

4.00

99.3

106.9

103.1

5.4

4.00

105.8

8.00

102.3

114.7

108.5

8.8

8.00

99.1

16.00

97.6

104.6

101.1

4.9

16.00

112.3

32.00

97.9

113.5

105.7

11.0

32.00

118.6

64.00

90.6

108.0

99.3

12.3

64.00

122.5

Test Item

0.98

125.0

105.9

115.5

13.6

4.24

109.1

1.95

122.3

106.7

114.5

11.0

5.65

106.3

3.91

138.1

103.7

120.9

24.3

7.53

101.1

7.81

152.8

106.9

129.8

32.5

10.03

101.4

15.63

158.4

108.9

133.7

34.9

13.37

104.4

31.25

145.3

81.8

113.5

44.9

17.82

99.2

62.50

134.4

0.1

67.2

95.0

23.76

115.9

125.00

130.2

0.2

65.2

92.0

31.67

132.4

250.00

122.2

0.6

61.4

86.0

42.22

149.7

500.00

128.0

0.6

64.3

90.1

56.28

145.7

1000.00

123.3

0.4

61.9

86.9

75.02

130.2

2000.00

129.3

0.4

64.8

91.2

100.00

110.5

Conc.: Concentration; Exp: Experiment

Induction of Luciferase Activity Experiment 1

Experiment1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Positive Control

4.00

1.02

1.08

1.05

1.05

0.03

 

8.00

1.64

1.51

1.45

1.53

0.10

*

16.00

1.61

1.80

1.84

1.75

0.12

*

32.00

3.20

3.64

4.58

3.81

0.70

*

64.00

7.99

6.39

7.83

7.40

0.88

*

Test Item

0.98

1.20

1.17

1.16

1.18

0.02

 

1.95

1.17

1.12

1.18

1.15

0.03

 

3.91

1.25

1.29

1.33

1.29

0.04

 

7.81

1.40

1.34

1.33

1.35

0.04

 

15.63

1.44

1.35

1.34

1.38

0.05

 

31.25

1.68

1.38

1.39

1.48

0.17

 

62.50

1.30

1.28

1.52

1.37

0.13

 

125.00

1.28

1.11

1.27

1.22

0.09

 

250.00

1.14

1.20

1.08

1.14

0.06

 

500.00

1.24

1.23

1.25

1.24

0.01

 

1000.00

1.22

1.18

1.07

1.16

0.08

 

2000.00

1.42

1.17

1.03

1.21

0.20

 

* = 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

Positive Control

4.00

1.22

1.16

1.04

1.14

0.09

 

8.00

1.42

1.26

1.19

1.29

0.12

 

16.00

1.64

1.74

1.56

1.65

0.09

*

32.00

1.89

2.27

2.23

2.13

0.21

*

64.00

3.27

4.16

4.28

3.90

0.55

*

Test Item

0.98

1.30

1.27

1.35

1.30

0.04

 

1.95

1.05

1.11

1.22

1.13

0.09

 

3.91

1.36

1.36

1.19

1.30

0.10

 

7.81

1.38

1.21

1.36

1.32

0.09

 

15.63

1.67

1.86

1.39

1.64

0.23

*

31.25

1.65

1.16

1.65

1.48

0.28

 

62.50

0.00

0.00

0.00

0.00

0.00

 

125.00

0.00

0.00

0.00

0.00

0.00

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

Induction of Luciferase Activity Experiment 3

Experiment 3

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Positive Control

4.00

1.21

1.28

1.49

1.32

0.15

 

8.00

1.11

1.37

1.29

1.26

0.13

 

16.00

1.15

1.44

1.35

1.32

0.15

 

32.00

1.83

1.70

2.13

1.88

0.22

*

64.00

3.77

3.63

3.97

3.79

0.17

*

Test Item

4.24

1.18

1.24

1.25

1.23

0.04

 

5.65

0.98

0.91

1.10

1.00

0.09

 

7.53

1.09

1.05

1.14

1.09

0.05

 

10.03

0.86

0.91

1.09

0.95

0.12

 

13.37

0.90

1.02

1.17

1.03

0.14

 

17.82

0.84

0.91

1.10

0.95

0.14

 

23.76

0.98

1.12

1.19

1.10

0.11

 

31.67

0.98

1.19

1.31

1.16

0.17

 

42.22

1.59

1.27

1.48

1.45

0.16

 

56.28

1.33

1.44

1.54

1.44

0.11

 

75.02

1.30

1.35

1.39

1.35

0.05

 

100.00

1.21

1.59

1.32

1.37

0.19

 

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

Induction of Luciferase Activity – Overall Induction

Conc. [µM]

Fold Induction

Exp 3

Exp 1

Exp 2

Mean

SD

Conc.

[µM]]

Fold Induction

Positive Control

4.00

1.05

1.14

1.09

0.06

4.00

1.32

8.00

1.53

1.29

1.41

0.17

8.00

1.26

16.00

1.75

1.65

1.70

0.07

16.00

1.32

32.00

3.81

2.13

2.97

1.18

32.00

1.88

64.00

7.40

3.90

5.65

2.47

64.00

3.79

Test Item

0.98

1.18

1.30

1.24

0.09

4.24

1.23

1.95

1.15

1.13

1.14

0.02

5.65

1.00

3.91

1.29

1.30

1.30

0.01

7.53

1.09

7.81

1.35

1.32

1.34

0.03

10.03

0.95

15.63

1.38

1.64

1.51

0.19

13.37

1.03

31.25

1.48

1.48

1.48

0.00

17.82

0.95

62.50

1.37

0.00

0.68

0.97

23.76

1.10

125.00

1.22

0.00

0.61

0.86

31.67

1.16

250.00

1.14

0.00

0.57

0.81

42.22

1.45

500.00

1.24

0.00

0.62

0.88

56.28

1.44

1000.00

1.16

0.00

0.58

0.82

75.02

1.35

2000.00

1.21

0.00

0.60

0.86

100.00

1.37

Conc.: Concentration; Exp: Experiment

Additional Parameters

Parameter

Exp. 1

Exp. 2

Exp. 3

Mean

SD

EC1.5[µM]

n.a.

12.25

n.a.

12.25

-

Imax

1.48

1.64

1.45

1.52

0.10

IC30[µM]

n.a.

35.75

n.a.

35.75

-

IC50[µM]

n.a.

43.40

n.a.

43.40

-

Exp: Experiment; n.a.: not applicable

Acceptance Criteria

Criterion

Range

Exp 1

pass/fail

Exp 2

pass/fail

Exp 3

pass/fail

CV DMSO Solvent Control

< 20%

10.3

pass

11.5

pass

9.3

pass

CV THF Solvent Control

 

10.3

pass

15.0

pass

9.6

pass

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

≥ 1

4.0

pass

3.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

7.72

pass

12.71

pass

21.19

pass

Induction PC at 64 µM

2.00 < x < 8.00

7.40

pass

3.90

pass

3.79

pass

Exp: Experiment

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:
other: 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.
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 348.47 g/mol a stock solution of 200 mM was prepared for experiment 1 and 2. In order to verify the first and second experiment, a third experiment with adapted concentrations was performed. For experiment 3 a stock solution of 10 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 for experiment 1 and 2 and a constant dilution factor of 1:1.33 for experiment 3. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

Experiment 1 and 2:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Experiment 3:

100, 75.02, 56.28, 42.22, 31.67, 23.76, 17.82, 13.37, 10.03, 7.53, 5.65, 4.24 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, nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated.

In the second experiment, a max luciferase activity (Imax) induction of 1.64 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 108.9%. No furthersignificant luciferase induction > 1.5 was found in the tested concentration range.The calculated EC1.5 was < 1000 µM (12.25 µM).

In the third 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 vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-08-28 to 2018-10-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on 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 all three experiments. The threshold of 150% for CD86 (242% experiment 1; 351% experiment 2, 370% experiment 3) and 200% for CD54 (251% experiment 1; 252% experiment 2, 292% experiment 3) were clearly exceeded.
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1
Value:
132
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 250 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1
Value:
117
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 69.77 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2
Value:
189
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 208.33 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2
Value:
122
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 83.72 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
3
Value:
152
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 173.61 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
3
Value:
129
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 83.72 µg/mL
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.

Results of the Cell Batch Activation Test (Batch 1)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

87.5

373

>150

88.1

358

>200

yes

pass

NiSO4

100 µg/mL

83.5

295

>150

82.1

603

>200

yes

pass

LA

1000 µg/mL

95.7

81

≤150

95.8

100

≤200

no

pass

Results of the Cell Batch Activation Test (Batch 2)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

83.4

364

>150

82.3

419

>200

yes

pass

NiSO4

100 µg/mL

79.1

351

>150

77.9

688

>200

yes

pass

LA

1000 µg/mL

96.2

77

≤150

95.9

105

≤200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

93.40

Solvent Control

THF

--

95.10

Test item

C8

3.91

95.30

C7

7.81

95.60

C6

15.63

96.10

C5

31.25

96.30

C4

62.50

94.90

C3

125.00

95.50

C2

250.00

94.90

C1

500.00

94.70

Calculated CV75 [µg/mL]

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

No SD

CD54 and CD86 Expression Experiment 1

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

93.2

93.6

92.5

3139

1213

684

2455

529

92

92

459

177

Solvent Control 2 (THF)

0.20%

92.6

93.2

92.7

3075.0

1289.0

714.0

2361

575

100

100

431

181

Solvent Control 1 (DMSO)

0.20%

92.5

91.9

91.9

3337

1245

668

2669

577

100

100

500

186

DNCB

4.00

78.9

79.2

78.5

7147

2123

675

6472

1448

242

251

1059

315

Test item

250

90.3

90.2

90.0

3884

1414

776

3108

638

132

111

501

182

208.33

91.5

91.3

91.1

3527

1378

771

2756

607

117

106

457

179

173.61

90.3

90.4

90.3

3652

1377

790

2862

587

121

102

462

174

144.68

91.1

92.1

91.7

3541

1454

785

2756

669

117

116

451

185

120.56

91.7

91.4

91.5

3194

1359

767

2427

592

103

103

416

177

100.47

92.1

91.6

90.8

3326

1424

775

2551

649

108

113

429

184

83.72

91.7

91.6

91.3

3276

1398

991

2285

407

97

71

331

141

69.77

91.5

92.0

91.3

3403

1448

778

2625

670

111

117

437

186

CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

IgG Isotype

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

95.2

95.2

94.3

2540

1237

551

1989

686

90

113

461

225

Solvent Control 2 (THF)

0.20%

95.3

95.0

94.8

2264

1220

595

1669

625

100

100

381

205

Solvent Control 1 (DMSO)

0.20%

95.4

94.9

94.3

2777

1168

562

2215

606

100

100

494

208

DNCB

4.0

77.0

77.2

75.8

8350

2099

569

7781

1530

351

252

1467

369

Test item

250.00

93.50

92.80

93.80

3571

1335

643

2928

692

175

111

555

208

208.33

92.40

92.10

93.00

3818

1267

666

3152

601

189

96

573

190

173.61

93.00

93.50

92.90

3513

1356

648

2865

708

172

113

542

209

144.68

92.90

92.70

92.80

2847

1275

655

2192

620

131

99

435

195

120.56

94.10

94.40

93.10

2766

1306

662

2104

644

126

103

418

197

100.47

94.10

93.40

92.90

2712

1103

658

2054

445

123

71

412

168

83.72

94.40

94.60

94.70

2453

1396

633

1820

763

109

122

388

221

69.77

93.40

93.20

93.00

3221

1353

656

2565

697

154

112

491

206

CD54 and CD86 Expression Experiment 3

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

IgG Isotype

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

95.2

94.9

94.6

1666

937

628

1038

309

85

65

265

149

Solvent Control 2 (THF)

0.20%

96.5

95.8

95.9

1689

911

504

1185

407

100

100

335

181

Solvent Control 1 (DMSO)

0.20%

95.2

95.2

95.1

1713

971

496

1217

475

100

100

345

196

DNCB

4.0

83.3

84.2

84.4

5085

1972

586

4499

1386

370

292

868

337

Test item

250.0

90.7

94.2

93.6

2174

1034

568

1606

466

136

115

383

182

208.33

93.7

93.2

92.3

2184

989

573

1611

416

136

102

381

173

173.61

92.8

92.0

92.5

2415

1069

618

1797

451

152

111

391

173

144.68

93.8

93.5

93.2

2261

960

576

1685

384

142

94

393

167

120.56

94.2

94.2

93.7

2080

1004

553

1527

451

129

111

376

182

100.47

93.5

93.5

93.5

2108

1016

567

1541

449

130

110

372

179

83.72

93.4

94.1

94.1

2169

1098

575

1594

523

135

129

377

191

69.77

94.4

94.6

94.0

2087

1075

593

1494

482

126

118

352

181

Acceptance Criteria

Acceptance Criterion

Range

Exp. 1

pass/fail

Exp. 2

pass/fail

Exp. 3

pass/fail

cell viability solvent controls [%]

>90

91.9

-

93.6

pass

94.3

-

95.4

pass

94.6

-

96.5

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

8

pass

RFI of positive control of CD86

≥150

242

pass

351

pass

370

pass

RFI of positive control of CD54

≥200

251

pass

252

pass

292

pass

RFI of DMSO solvent control of CD86

<150

109

pass

111

pass

117

pass

RFI of DMSO solvent control of CD54

<200

109

pass

88

pass

154

pass

RFI of THF solvent control of CD86

<150

96

pass

84

pass

114

pass

RFI of THF solvent control of CD54

<200

109

pass

91

pass

132

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

459

pass

461

pass

265

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

500

pass

494

pass

345

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

431

pass

381

pass

335

pass

MFI ratio CD54/IgG1for medium control [%]

>105

177

pass

225

pass

149

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

186

pass

208

pass

196

pass

MFI ratio CD54/IgG1 for THF control [%]

>105

181

pass

205

pass

181

pass

Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

96.2

1.6

216

number of test doses with viability >50%

-

-

555

RFI of positive control of CD86

358.9

90.1

36

RFI of positive control of CD54

435.7

285.8

36

RFI of solvent control (THF) of CD86

102.9

17.4

36

RFI of solvent control (THF) of CD54

102.0

15.4

36

MFI ratio IgG1/CD86 for medium control [%]

285.6

124.3

36

MFI ratio IgG1/CD86 for THF control [%]

270.8

105.0

36

MFI ratio IgG1/CD54 for medium control [%]

308.6

137.1

36

MFI ratio IgG1/CD54 for THF control [%]

158.2

28.4

36

Interpretation of results:
other: The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item did upregulate the cell surface marker CD86 in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
The data generated with this method may be not sufficient to conclude on 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. For the dose finding assay stock solutions with concentrations ranging from 250 mg/mL to 1.95 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

500, 416.67, 347.22, 289.35, 241.13, 200.94, 167.45, 139.54 µg/mL

In all experiments precipitation of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 90.3% (CD86), 90.2% (CD54) and 90.0% (isotype IgG1 control) in the first experiment, to 93.5% (CD86), 92.8% (CD54) and 93.8% (isotype IgG1 control) in the second experiment and to 90.7 % (CD86), 94.2 % (CD54) and 93.6 % (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was upregulated to 175%, 189%, 172% (250 µg/mL to 173.61 µg/ml) and 154% (69.77 µg/mL) in the second experiment and to 152% (173.61 µg/mL) in the third experiment. In contrast, the expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments.

Since one of the cell surface markers exceeded the threshold in two independent experiments the test item is considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The provided information indicates need for classification for skin sensitisation class 1 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.