Registration Dossier

Administrative data

Description of key information

OECD 442C: skin sensitizer

OECD 442D: non-sensitizer to skin

OECD 442 E: skin sensitizer

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-09-05 to 2018-09-12
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 66.92%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
3.28
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
100
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

15.3680

0.5340

16.0900

0.5340

STD2

7.7670

0.2670

8.0180

0.2670

STD3

3.8360

0.1335

4.0080

0.1335

STD4

1.8710

0.0667

1.9940

0.0667

STD5

0.9040

0.0334

0.9980

0.0334

STD6

0.4260

0.0167

0.5000

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.2840

0.1493

72.34

72.48

0.20

0.27

4.2280

0.1473

72.70

4.2760

0.1490

72.39

Test Item

14.7380

0.5110

4.85

3.28

1.92

58.61

15.3130

0.5308

1.13

14.8890

0.5162

3.87

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

5.8160

0.1934

61.85

61.36

0.58

0.95

5.8670

0.1950

61.51

5.9890

0.1991

60.71

Test Item

0.0000

0.0003

100.00

100.00

0.00

0.00

0.0000

0.0003

100.00

0.0000

0.0003

100.00

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

51.64

High Reactivity

positive

3.28

Minimal Reactivity

negative

Positive Control

66.92

High Reactivity

positive

72.48

Moderate Reactivity

positive

Interpretation of results:
other: The data generated with this test should be considered in the context of integrated approached such as IATA.
Conclusions:
In this study under the given conditions the test item showed high reactivity towards both peptides. The test item is considered as “sensitiser”.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Executive summary:

In the present study the test material was dissolved in acetonitrile, based on the results of the pre-experiments.

Based on a molecular weight of 632.78 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.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item precipitation was observed when diluted with the lysine peptide solution.After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.

Precipitation of the test item with both peptide peaks was observed. Therefore, the given peak areas and corresponding peptide values can only be considered as an estimation of the peptide depletion.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was6.38% (51.64%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 1 the test item can be considered as 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 66.92%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-09-14 to 2018-11-05
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 (6.02 (experiment 1) and 2.35 (experiment 2)).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
1.04
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 2000 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
58.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
1.38
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 2000 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
171.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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
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

93.7

100.0

96.8

4.5

8.00

97.8

93.9

95.8

2.8

16.00

102.6

103.1

102.8

0.4

32.00

103.8

105.8

104.8

1.4

64.00

103.8

83.1

93.5

14.6

Test Item

0.98

84.9

76.8

80.9

5.7

1.95

79.9

83.0

81.5

2.3

3.91

83.4

83.4

83.4

0.0

7.81

77.2

78.1

77.7

0.6

15.63

74.6

81.7

78.2

5.0

31.25

68.5

75.7

72.1

5.1

62.50

72.3

69.6

71.0

1.9

125.00

73.9

72.1

73.0

1.2

250.00

61.9

80.6

71.2

13.2

500.00

69.7

83.6

76.7

9.8

1000.00

75.3

80.4

77.9

3.6

2000.00

58.6

171.8

115.2

80.1

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

1.03

1.07

1.09

1.07

0.03

 

8.00

1.29

1.31

1.39

1.33

0.06

 

16.00

1.54

1.76

1.65

1.65

0.11

*

32.00

2.51

2.09

2.48

2.36

0.23

*

64.00

6.93

4.60

6.53

6.02

1.25

*

Test Item

0.98

0.80

0.93

0.81

0.85

0.07

 

1.95

0.88

1.17

0.90

0.98

0.16

 

3.91

0.76

0.86

0.70

0.77

0.08

 

7.81

0.82

1.16

0.76

0.91

0.21

 

15.63

0.78

0.77

0.77

0.77

0.01

 

31.25

0.79

0.87

0.80

0.82

0.04

 

62.50

0.82

0.79

0.73

0.78

0.05

 

125.00

0.80

0.84

0.79

0.81

0.03

 

250.00

0.79

0.86

0.73

0.79

0.07

 

500.00

0.91

1.00

0.89

0.94

0.06

 

1000.00

0.89

0.83

0.83

0.85

0.04

 

2000.00

1.31

0.93

0.89

1.04

0.23

 

* = 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

1.13

0.99

1.07

1.07

0.07

 

8.00

1.02

1.34

1.38

1.25

0.20

 

16.00

1.37

1.26

1.69

1.44

0.23

 

32.00

1.53

1.88

2.01

1.81

0.25

*

64.00

2.28

2.25

2.52

2.35

0.15

*

Test Item

0.98

0.73

1.16

0.78

0.89

0.24

 

1.95

0.86

1.07

1.14

1.02

0.15

 

3.91

0.70

0.98

1.00

0.89

0.17

 

7.81

0.79

0.87

0.80

0.82

0.04

 

15.63

0.72

0.78

0.83

0.78

0.06

 

31.25

0.82

0.88

1.00

0.90

0.09

 

62.50

0.82

0.78

0.83

0.81

0.02

 

125.00

0.87

0.85

0.89

0.87

0.02

 

250.00

0.99

1.02

0.98

1.00

0.02

 

500.00

0.95

0.97

1.04

0.99

0.05

 

1000.00

0.96

0.98

1.03

0.99

0.03

 

2000.00

1.09

1.14

1.92

1.38

0.46

 

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

Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

Positive Control

4.00

1.07

1.07

1.07

0.00

8.00

1.33

1.25

1.29

0.06

16.00

1.65

1.44

1.55

0.15

32.00

2.36

1.81

2.08

0.39

64.00

6.02

2.35

4.18

2.59

Test Item

0.98

0.85

0.89

0.87

0.03

1.95

0.98

1.02

1.00

0.03

3.91

0.77

0.89

0.83

0.09

7.81

0.91

0.82

0.87

0.06

15.63

0.77

0.78

0.77

0.00

31.25

0.82

0.90

0.86

0.06

62.50

0.78

0.81

0.79

0.02

125.00

0.81

0.87

0.84

0.04

250.00

0.79

1.00

0.90

0.14

500.00

0.94

0.99

0.96

0.04

1000.00

0.85

0.99

0.92

0.10

2000.00

1.04

1.38

1.21

0.24

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

n.a.

Imax

1.04

1.38

1.21

0.24

IC30[µM]

591.52

n.a.

n.a.

n.a.

IC50[µM]

n.a.

n.a.

n.a.

n.a.

n.a.: not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control PC (1% DMSO)

< 20%

4.0

pass

13.7

pass

CV Solvent Control TI (1% THF)

<20%

9.0

pass

14.2

pass

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

≥ 1

3.0

pass

2.0

pass

EC1.5 PC

± 2 x SD of historical mean

12.28

pass

18.54

pass

Induction PC at 64 µM

2 .00 < x < 8.00

6.02

pass

2.35

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.6

3.5

96

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

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96

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 not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test material was dissolved in THF. Based on a molecular weight of 632.78 g/mol a stock solution of 200 mM was prepared.

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

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

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

After incubation, precipitations were observed microscopically in cell culture medium at a concentration range from 15.6 µM – 2000 µM in the first main experiment and at a concentration of 2000 µM in the second main experiment.

In the first experiment, a slight cytotoxicity in the concentrations 31.25 µM, 250 µM, 500 µM and 2000 µM was observed, the concentrations were excluded from evaluation. No significant luciferase induction > 1.5 was found in the remaining test concentrations. Therefore, no EC1.5 value could be calculated.

In the second experiment, only at 62.5 µM a slight cytotoxicity was observed, the concentration was excluded from evaluation. 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:
weight of evidence
Study period:
2018-12-10 to 2019-02-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on study design:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test
item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely
dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in
the human monocytic cell line THP-1. The expression of the cell surface markers compared to the
respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (296% experiment 1; 350% experiment 2; 408% experiment 3) and 200% for CD54 (299% experiment 1; 358% experiment 2; 409% experiment 3) were clearly exceeded.
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1
Value:
214
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 1000 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1
Value:
137
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 578.70 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2
Value:
107
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 482.25 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2
Value:
55
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 279.08 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
3
Value:
306
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 482.25 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
3
Value:
162
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 279.08 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 3

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

85.4

306

>150

84.7

220

>200

yes

pass

NiSO4

100 µg/mL

81.8

251

>150

80.9

363

>200

yes

pass

LA

1000 µg/mL

94.0

93

150

93.9

93

200

no

pass

Results of the Cell Batch Activation Test 4

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

84.5

342

>150

85.8

334

>200

yes

pass

NiSO4

100 µg/mL

81.7

280

>150

81.7

578

>200

yes

pass

LA

1000 µg/mL

95.3

90

150

95.6

123

200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

94.50

Solvent Control

THF

--

95.50

Test material

C8

7.81

95.20

C7

15.63

95.80

C6

31.25

94.60

C5

62.50

94.80

C4

125.00

95.20

C3

250.00

95.40

C2

500.00

94.80

C1

1000.00

95.00

Calculated CV75 [µg/mL]

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

NoSD

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

-

97.5

97.2

96.4

1230

761

518

712

243

90

93

237

147

Solvent Control 2 (THF)

0.20%

97.0

97.0

96.7

1115.0

743.0

514.0

601

229

100

100

217

145

Solvent Control 1 (DMSO)

0.20%

96.8

96.9

96.9

1289

760

500

789

260

100

100

258

152

DNCB

4.00

85.8

84.9

84.0

2958

1396

619

2339

777

296

299

478

226

 

Test material

1000

95.3

94.5

94.4

1853

854

566

1287

288

214

126

327

151

833.33

96.6

97.0

96.8

1452

843

542

910

301

151

131

268

156

694.44

96.6

96.6

95.9

1602

837

536

1066

301

177

131

299

156

578.70

97.4

97.5

97.1

1350

856

542

808

314

134

137

249

158

482.25

96.3

97.0

96.9

1446

868

558

888

310

148

135

259

156

401.88

97.2

97.0

97.0

1375

826

547

828

279

138

122

251

151

334.90

97.2

97.4

97.2

1344

801

537

807

264

134

115

250

149

279.08

96.4

96.8

96.6

1370

786

545

825

241

137

105

251

144

 

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

-

96.1

96.5

96.9

1548

914

513

1035

401

133

172

302

178

Solvent Control 2 (THF)

0.20%

96.9

95.7

96.8

1570

1043

519

1051

524

100

100

303

201

Solvent Control 1 (DMSO)

0.20%

96.7

96.9

96.7

1297

751

518

779

233

100

100

250

145

DNCB

4.0

76.9

84.0

83.0

3392

1502

667

2725

835

350

358

509

225

 

Test material

1000.00

96.20

95.50

96.10

1592

825

584

1008

241

96

46

273

141

833.33

95.90

95.80

95.60

1604

831

577

1027

254

98

48

278

144

694.44

96.00

96.40

96.10

1557

806

561

996

245

95

47

278

144

578.70

96.30

96.50

95.80

1492

818

572

920

246

88

47

261

143

482.25

96.70

96.30

95.90

1668

777

542

1126

235

107

45

308

143

401.88

96.90

94.90

93.50

1560

762

558

1002

204

95

39

280

137

334.90

96.40

96.20

96.40

1472

863

592

880

271

84

52

249

146

279.08

96.90

96.90

96.60

1704

882

593

1111

289

106

55

287

149

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

-

97.3

97.1

96.7

1019

832

586

433

246

101

101

174

142

Solvent Control 2 (THF)

0.20%

96.9

95.9

95.3

951

868

570

381

298

100

100

167

152

Solvent Control 1 (DMSO)

0.20%

97.5

97.3

97.4

963

779

535

428

244

100

100

180

146

DNCB

4.0

86.4

88.2

87.4

2328

1577

580

1748

997

408

409

401

272

 

Test material

1000.0

95.8

95.6

95.7

969

1190

712

257

478

67

160

136

167

833.33

96.3

96.3

95.9

1534

1006

589

945

417

248

140

260

171

694.44

96.2

95.8

96.0

1731

1014

706

1025

308

269

103

245

144

578.70

95.1

94.7

95.5

1829

1149

695

1134

454

298

152

263

165

482.25

96.2

96.1

96.3

1787

1070

623

1164

447

306

150

287

172

401.88

96.4

96.2

96.3

1693

985

611

1082

374

284

126

277

161

334.90

96.3

96.3

96.2

1750

1244

810

940

434

247

146

216

154

279.08

96.9

96.7

96.3

1667

1100

616

1051

484

276

162

271

179

Acceptance Criteria

Acceptance Criterion

range

Experiment 1

pass/fail

Experiment 2

pass/fail

Experiment 3

pass/fail

cell viability medium and solvent controls [%]

>90

96.4

-

97.5

pass

95.7

-

96.9

pass

95.3

-

97.5

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

8

pass

RFI of positive control of CD86

≥150

296

pass

350

pass

408

pass

RFI of positive control of CD54

≥200

299

pass

358

pass

409

pass

RFI of DMSO solvent control of CD86

<150

111

pass

75

pass

99

pass

RFI of DMSO solvent control of CD54

<200

107

pass

58

pass

99

pass

RFI of THF solvent control of CD86

<150

84

pass

102

pass

88

pass

RFI of THF solvent control of CD54

<200

94

pass

131

pass

121

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

237

pass

302

pass

174

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

258

pass

250

pass

180

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

217

pass

303

pass

167

pass

MFI ratio CD54/IgG1 for medium control [%]

>105

147

pass

178

pass

142

pass

MFI ratio CD54/IgG1 for DMSO control [%]

>105

152

pass

145

pass

146

pass

MFI ratio CD54/IgG1 for THF control [%]

>105

145

pass

201

pass

152

pass

Historical Data

Criterion

mean

SD

N

Viability Solvent Control [%]

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 (THF) of CD86

115.0

15.1

112

RFI of solvent control (THF) of CD54

118.8

25.5

112

MFICD86 / MFIIgG1 for medium control [%]

202.4

50.0

112

MFICD86 / MFIIgG1 for THF control [%]

221.6

58.5

112

MFICD54 / MFIIgG1 for medium control [%]

141.0

24.7

112

MFICD54 / MFIIgG1 for THF control [%]

147.7

25.6

112

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.

The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

In the present study the test material was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 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.

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

1000; 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In all experiments, dose finding experiment and main experiment, precipitation of the test item was observed for all the concentration steps when mixing the test item stock solutions with cell culture medium.

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

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 95.3% (CD86), 94.5% (CD54) and 94.4% (isotype IgG1 control) in the first experiment, 96.20% (CD86), 95.50% (CD54) and 96.10% (isotype IgG1 control) in the second experiment and to 95.8% (CD86), 95.6% (CD54) and 95.7% (isotype IgG1 control) in the third experiment.

The expression of the cell surface marker CD86 was upregulated to 214% in the first experiment and to 306% in the third experiment. The upregulation above the threshold of 150% was observed at concentrations of 1000, 833.33 and 694.44 µg/mL in the first experiment and at concentrations of 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, and 279.08 µg/mL in the third experiment. In contrast, the expression of 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 there is sufficient information for classification for skin sensitisation class 1 (H317) according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.