Registration Dossier

Administrative data

Description of key information

OECD 442 C: Due to the observed precipitation in the cysteine peptide experiment a prediction cannot be made

OECD 442 D: positive

OECD 442 E: 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:
key study
Study period:
2017-12-01 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:
direct peptide binding assay
Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 62.40%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
5.58
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0.21
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

5221.6792

0.5340

4254.6284

0.5340

STD2

2648.5273

0.2670

2161.7087

0.2670

STD3

1327.5009

0.1335

1051.8925

0.1335

STD4

670.7400

0.0667

525.2503

0.0667

STD5

334.8562

0.0334

260.5259

0.0334

STD6

166.3505

0.0167

130.8325

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1609.4324

0.1633

68.11

68.17

0.05

0.08

1604.4860

0.1628

68.21

1605.4248

0.1629

68.19

Test Item

4722.0444

0.4814

4.97

5.58

0.54

9.77

4682.2471

0.4774

5.77

4670.3384

0.4761

6.01

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

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

4024.7078

0.5039

0.13

0.21

0.15

72.44

4025.2893

0.5039

0.12

4014.3962

0.5026

0.39

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

-

-

-

5.58

Minimal Reactivity

negative

Positive Control

62.40

High Reactivity

positive

68.17

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 in the cysteine peptide experiment 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:

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.

In the present study the test item was dissolved in N.N-dimethylformamide (DMF) based on the results of the pre-experiments. Based on a molecular weight of 324.82 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 the100 mM solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). 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 (droplets) was observed for the co-elution control of the positive control. The sample was not centrifuged prior to the HPLC analysis.

A minor co-elution of the test item with the lysine peptide peak was observed. The peak area determined in the co-elution controls corresponds to 0.0104 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (5.58%). 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.

Since precipitation was observed for the lysine and cysteine peptide samples containing also test item after the incubation period, no final prediction can be made.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-11-14 to 2018-02-23
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 (7.59(experiment 1); 3.43 (experiment 2)).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
6.41
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 31.25 µM
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
3.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 31.25 µM
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: EC1.5 [µM]
Run / experiment:
2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
29.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
40.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

87.6

83.5

85.6

2.8

8.00

92.1

87.0

89.6

3.6

16.00

100.1

96.3

98.2

2.7

32.00

106.4

100.6

103.5

4.1

64.00

112.2

111.7

111.9

0.4

Test Item

0.98

133.1

114.0

123.5

13.5

1.95

126.1

119.8

122.9

4.4

3.91

97.6

108.9

103.2

8.0

7.81

50.8

63.0

56.9

8.7

15.63

42.7

49.1

45.9

4.5

31.25

29.4

40.6

35.0

7.9

62.50

29.1

37.5

33.3

5.9

125.00

27.1

40.0

33.5

9.1

250.00

29.4

45.8

37.6

11.6

500.00

35.7

47.4

41.5

8.2

1000.00

39.3

57.0

48.2

12.6

2000.00

59.0

64.8

61.9

4.1

 

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

1.24

1.44

1.13

1.27

0.16

 

8.00

1.21

1.45

1.24

1.30

0.13

 

16.00

1.49

1.44

1.46

1.46

0.02

 

32.00

2.30

1.82

2.32

2.15

0.28

*

64.00

5.99

8.93

7.86

7.59

1.49

*

Test Item

0.98

1.96

2.54

1.85

2.12

0.37

*

1.95

3.31

2.50

2.06

2.62

0.64

*

3.91

3.58

3.81

3.48

3.63

0.17

*

7.81

5.86

5.45

4.93

5.41

0.46

*

15.63

3.64

3.82

3.02

3.49

0.42

*

31.25

7.10

6.91

5.23

6.41

1.03

*

62.50

6.17

5.77

5.03

5.66

0.58

*

125.00

4.87

5.34

4.79

5.00

0.30

*

250.00

4.09

5.31

4.29

4.56

0.66

*

500.00

4.21

4.13

3.96

4.10

0.13

*

1000.00

3.30

4.34

3.28

3.64

0.60

*

2000.00

2.29

3.47

2.34

2.70

0.67

*

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

Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.05

1.07

1.12

1.08

0.04

 

8.00

1.17

1.29

1.25

1.24

0.07

 

16.00

1.31

1.35

1.68

1.45

0.20

 

32.00

1.93

1.85

1.69

1.83

0.12

*

64.00

3.67

2.88

3.74

3.43

0.48

*

Test Item

0.98

1.47

1.74

1.54

1.58

0.14

*

1.95

2.16

2.65

2.55

2.45

0.26

*

3.91

2.42

2.74

2.39

2.52

0.20

*

7.81

2.80

3.41

2.73

2.98

0.37

*

15.63

2.61

3.35

3.25

3.07

0.40

*

31.25

3.09

3.58

3.03

3.23

0.30

*

62.50

2.85

3.33

3.20

3.13

0.25

*

125.00

3.07

3.13

2.92

3.04

0.11

*

250.00

3.14

3.07

2.94

3.05

0.10

*

500.00

3.09

2.73

2.77

2.86

0.20

*

1000.00

2.88

2.37

2.83

2.69

0.28

*

2000.00

2.39

2.21

2.43

2.34

0.12

*

* = 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

1.27

1.08

1.18

0.13

 

8.00

1.30

1.24

1.27

0.04

 

16.00

1.46

1.45

1.46

0.01

 

32.00

2.15

1.83

1.99

0.23

*

64.00

7.59

3.43

5.51

2.94

 

Test Item

0.98

2.12

1.58

1.85

0.38

 

1.95

2.62

2.45

2.54

0.12

*

3.91

3.63

2.52

3.07

0.78

 

7.81

5.41

2.98

4.20

1.72

 

15.63

3.49

3.07

3.28

0.30

*

31.25

6.41

3.23

4.82

2.25

 

62.50

5.66

3.13

4.39

1.79

 

125.00

5.00

3.04

4.02

1.39

 

250.00

4.56

3.05

3.81

1.07

 

500.00

4.10

2.86

3.48

0.87

 

1000.00

3.64

2.69

3.17

0.67

*

2000.00

2.70

2.34

2.52

0.25

*

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

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
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 as sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the presentstudy the test material was dissolved in THF.

Based on a molecular weight of 324.82 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culturemedium and a stable suspension was formed. The following concentration range was tested in the assay:

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

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 6.41 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 29.4%. A significant luciferase induction >1.5 was found in the whole tested concentration range. Therefore, no EC1.5value could be calculated. A cytotoxic effect was observed from 7.81 µM onwards.

In the second experiment, a max luciferase activity (Imax) induction of 3.23 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 40.6%. A significant luciferase induction >1.5 was found in the whole tested concentration range. Therefore, no EC1.5value could be calculated. A cytotoxic effect was observed from 7.81 µM onwards.

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:
key study
Study period:
2018-09-03 to 2018-10-09
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 09 October 2017
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 both experiments. The threshold of 150% for CD86 (310% experiment 1; 314% experiment 2) and 200% for CD54 (232% experiment 1; 256% experiment 2) were clearly exceeded.
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1
Value:
157
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 800 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1
Value:
78
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 223.27 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2
Value:
185
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 800 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2
Value:
73
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 223.27 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

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 Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

95.00

Solvent Control

THF

--

95.60

Test material

C8

6.25

93.80

C7

12.50

94.80

C6

25.00

94.40

C5

50.00

94.10

C4

100.00

93.30

C3

200.00

92.30

C2

400.00

94.10

C1

800.00

94.30

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

-

97.2

97.2

96.6

3303

1165

606

2697

559

91

73

545

192

Solvent Control 2 (THF)

0.20%

97.0

96.6

96.7

3221.0

1150.0

624.0

2597

526

100

100

516

184

Solvent Control 1 (DMSO)

0.20%

95.9

96.9

96.5

3525

1332

569

2956

763

100

100

620

234

DNCB

4.00

86.7

87.0

87.3

9787

2391

624

9163

1767

310

232

1568

383

Test material

800

93.7

93.3

93.2

4620

890

552

4068

338

157

64

837

161

666.67

93.5

93.2

93.3

4635

921

561

4074

360

157

68

826

164

555.56

94.9

94.4

95.2

3888

955

550

3338

405

129

77

707

174

462.96

95.0

95.2

95.4

3963

963

557

3406

406

131

77

711

173

385.80

95.3

95.1

91.3

3792

957

624

3168

333

122

63

608

153

321.50

93.2

93.5

93.0

4087

916

552

3535

364

136

69

740

166

267.92

95.3

95.2

95.8

3642

946

563

3079

383

119

73

647

168

223.27

95.7

95.6

95.5

3671

971

561

3110

410

120

78

654

173

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

-

94.4

94.7

93.8

4269

1460

585

3684

875

94

117

730

250

Solvent Control 2 (THF)

0.20%

95.1

94.8

95.2

3974

1398

605

3369

793

100

100

657

231

Solvent Control 1 (DMSO)

0.20%

94.1

94.2

93.9

4492

1336

585

3907

751

100

100

768

228

DNCB

4.0

80.4

79.5

79.2

12892

2541

622

12270

1919

314

256

2073

409

Test material

800

88.10

86.70

87.00

6865

1089

617

6248

472

185

60

1113

176

666.67

88.50

88.70

89.10

6594

1071

591

6003

480

178

61

1116

181

555.56

88.40

89.10

89.30

6589

1164

618

5971

546

177

69

1066

188

462.96

91.10

91.00

90.70

6055

1114

593

5462

521

162

66

1021

188

385.80

90.00

90.60

89.50

5808

1108

600

5208

508

155

64

968

185

321.50

91.20

91.00

91.80

5689

1023

583

5106

440

152

55

976

175

267.92

91.40

91.00

91.60

5494

1036

581

4913

455

146

57

946

178

223.27

91.70

91.20

91.30

5515

1189

613

4902

576

146

73

900

194

Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

95.9

-

97.2

pass

93.8

-

95.2

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

RFI of positive control of CD86

≥150

310

pass

314

pass

RFI of positive control of CD54

≥200

232

pass

256

pass

RFI of DMSO control of CD86

<150

110

pass

106

pass

RFI of DMSO control of CD54

<200

136

pass

86

pass

RFI of THF solvent control of CD86

<150

96

pass

91

pass

RFI of THF solvent control of CD54

<200

94

pass

91

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

545

pass

730

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

620

pass

768

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

516

pass

657

pass

MFI ratio CD54/IgG1for medium control [%]

>105

192

pass

250

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

234

pass

228

pass

MFI ratio CD54/IgG1for THF control [%]

>105

184

pass

231

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

MFICD86 / MFIIgG1for medium control [%]

285.6

124.3

36

MFICD86 / MFIIgG1for THF control [%]

270.8

105.0

36

MFICD54 / MFIIgG1for medium control [%]

308.6

137.1

36

MFICD54 / MFIIgG1for THF control [%]

158.2

28.4

36

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
In this study under the given conditions the test item did upregulate the 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 starting with the highest soluble concentrationfrom 400 mg/mL to 3.13 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:

800, 666.67, 555.56, 462.96, 385.80, 321.50, 267.92, 223.27 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 93.7% (CD86), 93.3% (CD54) and 93.2% (isotype IgG1 control) in the first experiment and 88.1% (CD86), 86.7% (CD54) and 87.0% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 157% (800 µg/mL and 666.67 µg/mL) in the first experiment and in almost all concentrations in the second experiment, except for the two lowest concentrations 267.92 µg/mL and 223.27 µg/mL (146%). 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 clearly 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

Based on the provided information, the test item is classified skin sensitisation cat. 1 and labelled with H317 according to the EU Regulation (EC) No 1272/2008 on Classification,Labelling and Packaging of Substances and Mixtures.