Registration Dossier

Administrative data

Description of key information

DPRA: negative

KeratinoSens: positive

hClat: positive

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:
weight of evidence
Study period:
2017-08-11 to 2017-09-09
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:
direct peptide reactivity assay (DPRA)
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 65.19%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0.06
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:
0.26
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

4835.2368

0.5340

4196.2754

0.5340

STD2

2314.6648

0.2670

2135.2583

0.2670

STD3

782.4597

0.1335

1032.7322

0.1335

STD4

525.4504

0.0667

516.1820

0.0667

STD5

266.4071

0.0334

257.2941

0.0334

STD6

135.9798

0.0167

130.6166

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

1381.2087

0.1629

71.03

71.19

0.17

0.24

1364.7275

0.1611

71.37

1374.1948

0.1621

71.18

Test Item

4794.6045

0.5384

0.00

0.06

0.09

154.04

4759.6314

0.5345

0.17

4766.8809

0.5353

0.01

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

1633.4226

0.2075

58.76

59.19

0.48

0.81

1595.8407

0.2028

59.71

1619.8287

0.2058

59.10

Test Item

3953.4578

0.5018

0.18

0.26

0.15

58.26

3953.9629

0.5018

0.17

3943.2253

0.5005

0.44

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

0.16

Minimal Reactivity

no sensitizer

0.06

Minimal Reactivity

no sensitizer

Positive Control

65.19

High Reactivity

sensitizer

71.19

Moderate Reactivity

sensitizer

Interpretation of results:
other: The result should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. The test item might 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 acetonitrile based on the results of the pre-experiments. Based on a molecular weight of 206.29 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 no turbidity or 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 positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item a turbidity 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 (small droplets) was observed for the samples of the positive control (including co-elution of the positive control) and for the samples of the test item (including co-elution control 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 precipitation and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. However, due to the phase separation observed in the lysine peptide samples and possible underestimation of the reactivity, the sensitising potential of the test item was predicted only from the peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cacetonitrile).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was 6.38% (0.06%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

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 65.19%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-09-20 to 2017-10-19
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 (4.55 in experiment 1; 6.13 in experiment 2)
Run / experiment:
other: 1
Parameter:
other: luciferase activity [fold induction]
Value:
6.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
95.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
6.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Run / experiment:
other: 2
Parameter:
other: luciferase activity [fold induction]
Value:
4.35
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
91
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Value:
9.72
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 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

98.9

100.0

99.5

0.7

8.00

102.4

103.0

102.7

0.4

16.00

104.5

107.8

106.2

2.3

32.00

112.8

116.2

114.5

2.4

64.00

119.1

120.9

120.0

1.3

Test Item

0.98

96.2

99.7

97.9

2.5

1.95

93.0

87.2

90.1

4.1

3.91

105.2

86.2

95.7

13.4

7.81

100.3

85.8

93.1

10.2

15.63

110.7

97.6

104.1

9.3

31.25

125.4

109.4

117.4

11.3

62.50

129.1

130.6

129.8

1.0

125.00

95.3

91.0

93.2

3.0

250.00

0.1

0.1

0.1

0.0

500.00

0.3

0.1

0.2

0.1

1000.00

0.1

0.1

0.1

0.0

2000.00

0.4

0.4

0.4

0.0

 

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

1.03

1.21

1.13

1.12

0.09

 

8.00

1.28

1.38

1.30

1.32

0.05

 

16.00

1.57

1.75

1.65

1.66

0.09

*

32.00

2.43

2.71

2.42

2.52

0.16

*

64.00

3.82

5.53

4.29

4.55

0.88

*

Test Item

0.98

1.00

1.02

1.03

1.02

0.02

 

1.95

1.27

1.26

1.13

1.22

0.08

 

3.91

1.23

1.56

1.14

1.31

0.22

 

7.81

1.65

1.74

1.50

1.63

0.12

*

15.63

1.90

2.40

1.72

2.01

0.35

*

31.25

1.87

2.25

1.84

1.99

0.23

*

62.50

3.04

3.24

2.37

2.88

0.45

*

125.00

5.61

8.14

4.92

6.23

1.70

*

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 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

1.27

1.22

1.00

1.16

0.15

 

8.00

1.33

1.45

1.25

1.34

0.10

 

16.00

1.76

1.66

1.37

1.60

0.20

*

32.00

2.50

2.69

1.87

2.35

0.43

*

64.00

6.26

6.46

5.66

6.13

0.42

*

Test Item

0.98

1.04

1.14

0.79

0.99

0.18

 

1.95

1.01

1.36

0.94

1.10

0.23

 

3.91

1.19

1.28

1.21

1.23

0.05

 

7.81

1.38

1.55

1.32

1.42

0.12

 

15.63

1.84

2.01

1.43

1.76

0.30

*

31.25

2.08

2.32

2.04

2.15

0.15

*

62.50

2.58

2.90

2.14

2.54

0.38

*

125.00

4.33

4.65

4.07

4.35

0.29

*

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 – 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

1.12

1.16

1.14

0.03

 

8.00

1.32

1.34

1.33

0.02

 

16.00

1.66

1.60

1.63

0.04

*

32.00

2.52

2.35

2.44

0.12

*

64.00

4.55

6.13

5.34

1.12

*

Test Item

0.98

1.02

0.99

1.00

0.02

 

1.95

1.22

1.10

1.16

0.08

 

3.91

1.31

1.23

1.27

0.06

 

7.81

1.63

1.42

1.52

0.15

*

15.63

2.01

1.76

1.88

0.17

*

31.25

1.99

2.15

2.07

0.12

*

62.50

2.88

2.54

2.71

0.24

*

125.00

6.23

4.35

5.29

1.32

*

250.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

 

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

6.23

9.72

7.98

2.47

Imax

6.23

4.35

5.29

1.32

IC30[µM]

158.25

153.90

156.07

3.07

IC50[µM]

184.49

181.39

182.94

2.20

 

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

12.7

pass

10.5

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

12.29

pass

12.96

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.55

pass

6.13

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:
other: The result should be considered in the context of integrated approach such as IATA.
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.
Executive summary:

In the present study the test material was dissolved in DMSO

Based on a molecular weight of 206.29 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 exposurecells were lysed and luciferase activity was assessed by luminescence measurement.

In the firstexperiment, a max luciferase activity (Imax) induction of 6.23 was determined at a test item concentration of 125 µM. The corresponding cell viability was 95.3%. The lowest tested concentration with a significant luciferase induction >1.5 (1.63) was found to be 7.81 µM. The corresponding cell viability was >70% (100.3%).The calculated EC1.5was<1000 µM (6.23 µM).

In the second experiment, a max luciferase activity (Imax) induction of 4.35 was determined at a test item concentration of 125 µM. The corresponding cell viability was 91.0%. The lowest tested concentration with a significant luciferase induction >1.5 (1.76) was found to be 15.63 µM. The corresponding cell viability was >70% (97.6%).The calculated EC1.5was<1000 µM (9.72 µM).

A 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 sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-09-21 to 2018-01-09
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 assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017
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 (259% experiment 1; 373% experiment 2; 254% experiment 3) and 200% for CD54 (244% experiment 1; 375% experiment 2; 335% experiment 3) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
152
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
158
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
259
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
268
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
761
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 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 Dose Finding Assay

Sample

Experiment 1

Experiment 2

Concentration applied [µg/ml]

Cell Viability [%]

Concentration applied [µg/ml]

Cell Viability [%]

Medium Control

0.00

97.30

0.00

96.80

Solvent Control

0.00

96.70

0.00

97.10

Test item

7.81

95.90

7.81

95.30

15.63

95.50

15.63

94.30

31.25

94.90

31.25

93.20

62.50

91.60

62.50

92.70

125.00

81.20

125.00

70.20

250.00

5.40

250.00

6.50

500.00

4.40

500.00

2.90

1000.00

9.30

1000.00

5.40

Calculated CV75 [µg/mL]

132.29

107.82

Mean CV75 [µg/mL]

120.05

SD CV 75 [µg/mL]

17.31

 

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

-

94.4

94.6

93.7

1617

1349

724

893

625

74

90

223

186

Solvent Control

0.20%

94.6

94.1

94.4

1865

1360

663

1202

697

100

100

281

205

DNCB

4.00

83.5

82.3

80.8

3736

2325

627

3109

1698

259

244

596

371

Test item

144.07

11.4

10.1

11.6

1448

1446

918

530

528

44

76

158

158

120.06

46.4

47.6

47.3

1264

1668

610

654

1058

54

152

207

273

100.05

66.0

66.2

66.1

1664

1710

653

1011

1057

84

152

255

262

83.37

77.8

79.4

79.5

1843

1588

725

1118

863

93

124

254

219

69.48

84.6

84.3

83.3

1911

1528

769

1142

759

95

109

249

199

57.90

90.6

90.1

90.6

1894

1356

724

1170

632

97

91

262

187

48.25

90.8

91.5

90.8

1758

1279

714

1044

565

87

81

246

179

40.21

90.4

91.3

89.9

1745

1283

705

1040

578

87

83

248

182

 

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

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

96.4

95.9

96.1

1594

1277

637

957

640

94

101

250

200

Solvent Control

0.20%

96.7

96.7

96.6

1693

1311

679

1014

632

100

100

249

193

DNCB

4.0

81.2

81.4

80.6

4482

3075

704

3778

2371

373

375

637

437

Test item

144.07

20.5

20.9

20.7

1419

1427

710

709

717

70

113

200

201

120.06

64.9

65.2

66.6

2312

2447

812

1500

1635

148

259

285

301

100.05

78.5

78.9

78.8

2399

1890

799

1600

1091

158

173

300

237

83.37

79.6

81.2

81.0

2373

1815

767

1606

1048

158

166

309

237

69.48

86.5

86.0

86.1

2192

1722

811

1381

911

136

144

270

212

57.90

88.0

88.3

87.6

2113

1684

852

1261

832

124

132

248

198

48.25

89.3

90.0

88.9

2060

1610

835

1225

775

121

123

247

193

40.21

91.6

91.1

91.4

1984

1647

907

1077

740

106

117

219

182

 

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

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

95.0

95.3

95.0

2630

1252

784

1846

468

70

93

335

160

Solvent Control

0.20%

95.0

94.8

94.9

3417

1275

774

2643

501

100

100

441

165

DNCB

4.0

83.0

83.9

82.7

7420

2393

714

6706

1679

254

335

1039

335

Test item

144.1

12.0

12.3

12.0

9305

6023

2211

7094

3812

268

761

421

272

120.06

30.2

31.2

29.6

2927

1586

640

2287

946

87

189

457

248

100.05

61.2

62.4

62.6

3158

1959

668

2490

1291

94

258

473

293

83.37

74.7

76.0

75.9

3371

1616

753

2618

863

99

172

448

215

69.48

83.8

83.4

82.8

3340

1476

764

2576

712

97

142

437

193

57.90

86.2

86.8

86.0

2952

1429

780

2172

649

82

130

378

183

48.25

87.4

87.6

87.8

2918

1374

873

2045

501

77

100

334

157

40.21

89.0

89.7

88.9

3056

1332

803

2253

529

85

106

381

166

 

Interpretation of results:
other: The result 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 in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1. However, due to the severe cytotoxic effects at the high test item concentrations it cannot be excluded, that the upregulation of the cell surface marker was caused by the cell stress.
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 item was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 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.

A CV75 of 120.05 ± 17.31 µg/mL was derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

144.07, 120.05, 100.05, 83.37, 69.48, 57.90, 48.25 and 40.21 µg/mL

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.

Severe cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 11.4% (CD86), 10.1% (CD54) and 11.6% (isotype IgG1 control) in the first experiment, to 20.5% (CD86), 20.9% (CD54) and 20.7% (isotype IgG1 control) in the second experiment and 12.0% (CD86), 12.3% (CD54) and 12.0% (isotype IgG1 control) in the second experiment. Severe cytotoxic effects (cell viability < 50%) was observed at the highest two concentrations in experiment 1 and 3 and at the highest concentration only in experiment 2.

In experiment 1 the expression of the cell surface marker CD86 and CD54 was not upregulated above the respective threshold values (150% for CD86 and 200% for CD54).

In experiment 2 the expression of the cell surface marker CD86 was upregulated to 158%. The upregulation above the threshold of 150% was observed starting from a concentration of 83.37 µg/mL, whereas the two highest concentrations showed no upregulation above the threshold. The expression of the cell surface marker CD54 was upregulated to 259%. The upregulation above the threshold of 200% was observed at a concentration of 120.06 µg/mL.

To verify the results a third experiment was performed.

In experiment 3 the expression of the cell surface marker CD54 was upregulated to 258%. The upregulation above the threshold of 200% was observed at a concentration of 100.05 µg/mL. In contrast, the expression of cell surface marker CD86 was not upregulated above the threshold of 150%. Furthermore, for both cell surface marker an upregulation above the respective threshold values was observed at the highest concentration of 144.10 µg/mL, respectively. However, at this concentration the cell viability was < 50%.

Since the cell surface marker clearly exceeded the threshold in two independent experiments the test item considered to be a skin sensitiser. However, due to the severe cytotoxic effects at the high test item concentrations it cannot be excluded, that the upregulation of the cell surface marker was caused by the cell stress.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (259% experiment 1; 373% experiment 2; 254% experiment 3) and 200% for CD54 (244% experiment 1; 375% experiment 2; 335% experiment 3) were clearly exceeded.

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 information there is sufficient evidence that the substance needs to classified as skin sensitiser class 1 according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.