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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: inconclusive due to precipitation of the test material

OECD 442 D: non-sensitizer

OECD 442 E: non-sensitizer

Test item showed minimal reactivity towards the cysteine peptide in the OECD Guideline 442C. Due to the observed precipitation the prediction model does not apply and a prediction cannot be made. However, the test item does not activate keratinocytes (OECD 442D) and did not upregulate the expression of the cell surface markers (OECD 442E). Therefore, it can be concluded that the test item is not sensitizing to skin.

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:
2017-10-19 to 2017-11-25
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))
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
Version / remarks:
2013
Deviations:
no
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 64.04%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0
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
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

4523.9644

0.5340

4455.4287

0.5340

STD2

2255.1311

0.2670

2248.6604

0.2670

STD3

1081.2599

0.1335

1103.8511

0.1335

STD4

538.3588

0.0667

555.1791

0.0667

STD5

264.6839

0.0334

278.7628

0.0334

STD6

131.3584

0.0167

141.0930

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

1320.4471

0.1577

70.95

71.10

0.18

0.25

1315.4908

0.1571

71.06

1304.7782

0.1559

71.29

Test Item

4595.2119

0.5431

0.00

0.00

0.00

n/a

4572.8872

0.5405

0.00

4551.4190

0.5379

0.00

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

1768.9691

0.2117

57.97

56.98

1.07

1.87

1804.7944

0.2160

57.12

1858.1759

0.2224

55.85

Test Item

4243.7490

0.5080

0.00

0.00

0.00

n/a

4209.2564

0.5038

0.00

4209.8970

0.5039

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

0.00

Minimal Reactivity

no sensitiser

0.00

Minimal Reactivity

no sensitizer

Positive Control

64.04

High Reactivity

sensitizer

71.10

Moderate Reactivity

sensitizer

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 was dissolved in acetonitrile, based on the results of the pre-experiments

Based on a molecular weight of 338 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). Phase separation was observed for the samples of the test item. Samples of the positive control were centrifuged prior to the HPLC analysis.

For the 100 mM stock 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 samples of the positive control (excluding the co-elution control of the positive control). Phase separation was also observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria of the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

Due to the observed phase separation after the incubation period in the lysine peptide samples, prediction model 2 based on the cysteine peptide depletion only should be considered. However, precipitation was observed as well in the cysteine experiment.

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was  6.38% (0%).

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 64.04%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-15 to 2018-04-30
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)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
Version / remarks:
2015
Deviations:
no
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 (4.26 (experiment 1); 4.34 (experiment 2)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 15.63 µM
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 15.63 µM
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
90.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at 15.63 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
81.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at 15.63 µM
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

102.8

101.3

102.1

1.1

8.00

113.1

100.5

106.8

8.9

16.00

113.5

98.8

106.2

10.4

32.00

125.3

101.8

113.5

16.6

64.00

131.2

96.8

114.0

24.3

Test Item

0.98

102.9

97.6

100.3

3.7

1.95

97.2

102.0

99.6

3.4

3.91

96.9

99.6

98.3

1.9

7.81

94.9

94.4

94.6

0.4

15.63

90.7

81.4

86.1

6.5

31.25

22.9

15.9

19.4

5.0

62.50

0.0

0.0

0.0

0.0

125.00

0.0

-0.1

-0.1

0.1

250.00

0.1

-0.1

0.0

0.1

500.00

0.2

0.0

0.1

0.1

1000.00

0.5

0.0

0.2

0.3

2000.00

0.5

0.1

0.3

0.3

Induction of Luciferase Activity Experiment 1 (Batch D017011497)

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

1.13

1.14

1.12

1.13

0.01

 

8.00

1.20

1.12

1.07

1.13

0.07

 

16.00

1.33

1.61

1.23

1.39

0.19

 

32.00

2.10

1.76

1.72

1.86

0.21

*

64.00

4.82

4.21

3.76

4.26

0.53

*

Test Item

(0.98

1.10

1.06

[2.46]

1.54

0.80)

 

1.95

1.16

0.99

0.95

1.03

0.11

 

3.91

1.07

1.17

0.99

1.08

0.09

 

7.81

1.22

1.06

1.15

1.14

0.08

 

15.63

1.81

1.34

1.34

1.50

0.27

 

31.25

0.70

1.38

1.30

1.13

0.37

 

62.50

0.00

0.68

0.24

0.31

0.34

 

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

[outlier] according to statistical tests of Grubbs, Nalimov and Dixon

( ) concentration will not be used for evaluation

Induction of Luciferase Activity Experiment 2 (D017144397)

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

1.14

1.06

0.99

1.07

0.07

 

8.00

1.20

1.39

1.54

1.37

0.17

 

16.00

1.28

1.59

1.57

1.48

0.18

 

32.00

2.36

2.74

2.58

2.56

0.19

*

64.00

4.04

5.02

3.97

4.34

0.58

*

Test Item

0.98

0.99

1.06

1.08

1.04

0.05

 

1.95

0.95

1.02

0.95

0.98

0.04

 

3.91

0.97

0.97

1.02

0.99

0.03

 

7.81

1.04

1.05

1.13

1.07

0.05

 

15.63

1.19

1.04

1.11

1.11

0.08

 

31.25

0.70

0.85

0.82

0.79

0.08

 

62.50

0.55

0.77

0.71

0.68

0.11

 

125.00

0.00

0.35

0.14

0.16

0.18

 

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

 

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

1.13

1.07

1.10

0.05

 

8.00

1.13

1.37

1.25

0.17

 

16.00

1.39

1.48

1.44

0.07

 

32.00

1.86

2.56

2.21

0.50

 

64.00

4.26

4.34

4.30

0.06

*

Test Item

(0.98

1.54

1.04

1.29

0.35)

 

1.95

1.03

0.98

1.00

0.04

 

3.91

1.08

0.99

1.03

0.06

 

7.81

1.14

1.07

1.11

0.05

 

15.63

1.50

1.11

1.30

0.27

 

31.25

1.13

0.79

0.96

0.24

 

62.50

0.31

0.68

0.49

0.26

 

125.00

0.00

0.16

0.08

0.11

 

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

( ) concentration will not be used for evaluation

 

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

n.a.

Imax

1.50

1.11

1.30

0.27

IC30[µM]

20.39

18.35

19.37

1.44

IC50[µM]

25.00

23.12

24.06

1.33

n.a.: not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

7.8

pass

7.0

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

19.77

pass

16.27

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.26

pass

4.34

pass

 

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.3

3.3

41

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

≥ 1

2.3

0.6

41

EC1.5 PC

7 < x < 34 µM

20.4

6.7

41

Induction PC at 64 µM

2.00 < x < 8.00

3.3

1.1

41

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

In the present study the test material was dissolved in DMSO. Based on a molecular weight of 338 g/mol a stock solution of 200 mM was prepared.The main experiments were performed using two different batches (experiment 1: D017011497; experiment 2: D017144397) because of the expiry date of the first batch of the test item.

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, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second 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:
2018-04-30 to 2018-07-17
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
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
449
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 51.06 µg/mL
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
678
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 42.55 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
282
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 51.06 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
337
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 51.06 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 20)

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

89.2

266

>150

88.4

206

>200

yes

pass

NiSO4

100 µg/mL

82.3

220

>150

80.6

283

>200

yes

pass

LA

1000 µg/mL

96.2

79

150

96.9

101

200

no

pass

Results of the Cell Batch Activation Test (Batch 21)

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

85.5

251

>150

84.7

287

>200

yes

pass

NiSO4

100 µg/mL

74.9

249

>150

75.5

518

>200

yes

pass

LA

1000 µg/mL

94.8

67

150

95.0

108

200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Experiment 2

Experiment 3

Concentration applied [µg/mL]

Cell Viability [%]

Concentration applied [µg/mL]

Cell Viability [%]

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

94.30

--

93.90

--

95.20

Solvent Control

DMSO

--

95.00

--

92.90

--

95.30

Test item

C8

7.81

94.40

7.81

91.30

7.81

94.60

C7

15.63

93.00

15.63

89.20

15.63

93.90

C6

31.25

86.80

31.25

79.50

31.25

72.90

C5

62.50

79.20

62.50

29.10

62.50

4.50

C4

125.00

11.80

125.00

7.90

125.00

5.40

C3

250.00

7.30

250.00

4.30

250.00

5.90

C2

500.00

6.50

500.00

6.60

500.00

7.90

C1

1000.00

14.10

1000.00

9.70

1000.00

11.90

Calculated CV75 [µg/mL]

65.26

33.25

29.16

Mean CV75 [µg/mL]

42.55

SD CV 75 [µg/mL]

19.77

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

-

94.5

93.7

94.2

3327

1407

668

2659

739

97

102

498

211

Solvent Control

0.20%

94.5

93.2

93.9

3430

1410

686

2744

724

100

100

500

206

DNCB

4.00

79.5

79.3

79.8

8105

2427

619

7486

1808

273

250

1309

392

Test item

51.06

9.2

8.8

8.7

13774

5991

1461

12313

4530

449

626

943

410

42.55

9.1

9.4

9.0

12131

6367

1457

10674

4910

389

678

833

437

35.46

24.1

23.3

22.4

4265

2753

849

3416

1904

124

263

502

324

29.55

47.5

47.6

48.4

4093

1894

886

3207

1008

117

139

462

214

24.62

75.1

73.8

74.0

3763

1686

744

3019

942

110

130

506

227

20.52

84.2

83.7

84.1

3418

1612

692

2726

920

99

127

494

233

17.10

88.2

88.7

87.6

3337

1572

685

2652

887

97

123

487

229

14.25

90.6

90.8

90.6

3337

1502

693

2644

809

96

112

482

217

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

-

97.2

97.0

96.7

2751

1282

649

2102

633

100

100

424

198

Solvent Control

0.20%

97.1

97.0

97.0

2713

1251

618

2095

633

100

100

439

202

DNCB

4.0

84.2

83.7

83.2

7946

3000

651

7295

2349

348

371

1221

461

Test item

51.06

5.4

4.6

4.4

7306

3532

1398

5908

2134

282

337

523

253

42.55

30.3

30.1

29.5

3654

1796

704

2950

1092

141

173

519

255

35.46

53.4

54.6

53.8

3556

1590

689

2867

901

137

142

516

231

29.55

66.2

66.6

66.1

3678

1478

685

2993

793

143

125

537

216

24.62

77.1

76.5

77.0

3090

1408

694

2396

714

114

113

445

203

20.52

87.7

87.5

86.6

3205

1386

658

2547

728

122

115

487

211

17.10

90.3

90.6

90.1

3040

1294

640

2400

654

115

103

475

202

14.25

90.8

90.9

91.1

3078

1277

640

2438

637

116

101

481

200

 

Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

93.2

-

94.5

pass

96.7

-

97.2

pass

number of test dosed with viability >50% CD86

≥4

4

pass

6

pass

number of test dosed with viability >50% CD54

≥4

4

pass

6

pass

number of test dosed with viability >50% IgG1

≥4

4

pass

6

pass

RFI of positive control of CD86

≥150

273

pass

348

pass

RFI of positive control of CD54

≥200

250

pass

371

pass

RFI of solvent control of CD86

<150

103

pass

100

pass

RFI of solvent control of CD54

<200

98

pass

100

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

498

pass

424

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

500

pass

439

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

211

pass

198

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

206

pass

202

pass

Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

97.0

1.3

672

number of test doses with viability >50%

-

-

1786

RFI of positive control of CD86

401.0

146.8

112

RFI of positive control of CD54

576.6

312.0

112

RFI of solvent control of CD86

115.0

15.1

112

RFI of solvent control of CD54

118.8

25.5

112

MFI ratio IgG1/CD86 for medium control [%]

202.4

50.0

112

MFI ratio IgG1/CD86 for DMSO control [%]

221.6

58.5

112

MFI ratio IgG1/CD54 for medium control [%]

141.0

24.7

112

MFI ratio IgG1/CD54 for DMSO control [%]

147.7

25.6

112

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item is 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. 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. In the dose finding assay, precipitates and turbidity and oily droplets were observed for working solutions 1 – 4 (500 mg/mL – 62.5 mg/ml - 1000 µg/ml – 125 µg/mL applied concentration) when diluted 1:250 in cell culture medium.

A CV75 of 42.55 ± 19.77 µg/mL was derived in the dose finding assay.

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

51.06, 42.55, 35.46, 29.55, 24.63, 20.52, 17.10, 14.25 µg/mL

In the main experiment no precipitation or turbidity of the test item was observed for all tested concentration steps when diluted 1:250 in 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 9.2% (CD86), 8.8% (CD54) and 8.7% (isotype IgG1 control) in the first experiment and to 5.4% (CD86), 4.6% (CD54) and 4.4% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 449% and 389% in the first experiment and to 282% in the second

experiment. The upregulation above the threshold of 150% was observed at a concentration of 51.06 µg/mL and 42.55 µg/mL in the first experiment and at a concentration of 51.06 µg/mL in the second experiment.The expression of the cell surface marker CD54 was upregulated to 626%, 678% and 263% in the first experiment and to 337% in the second experiment. The upregulation above the threshold of 200% was observed at a concentration of 51.06 µg/mL, 42.55 µg/mL and 35.46 µg/mL in the first experiment and at a concentration of 51.06 µg/mL in the second experiment.

The induction of the expression of both cell surface markers occurred only at cytotoxic concentrations. Since there was no induction of the expression of the cell surface markers CD86 and CD54 in the other non-cytotoxic concentrations, the test item can be considered as non-sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.