Registration Dossier

Administrative data

Description of key information

Skin sensitisation is considered as an allergic response following skin contact. The sensitisation potential can be assessed by numerous assays including in vivo studies and in vitro

experiments.

The in vitro assays performed show that due to the physicochemical properties of the compound, namely logP and limited solubility, the in vitro approach is technically challenging. Two of the three assays performed are inconclusive thus a conclusion based on in vitro data is not possible.

However, QSAR analysis of the chemical structure provides additional information. This analysis resulted in two structural alerts for skin sensitisation. The subsequent EC3 prediction based on information of close structural analogues reveals a moderate potential for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to the QMRF and QPRF files provided under the section attached justification.
Qualifier:
according to guideline
Guideline:
other: ECHA Guidance on IR/CSA R.6 QSARs and grouping of chemicals
Principles of method if other than guideline:
Estimates the skin sensitising properties of chemicals using structural alert relationships.
GLP compliance:
no
Specific details on test material used for the study:
see QPRF
Parameter:
other: Structural Alert for skin sensitisaton
Value:
2
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Two alerts for skin sensitisation have been identified. See QPRF for details.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Using Derek and Meteor Nexus , the skin sensitising potential assessment resulted in two alerts for skin sensitisation. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate. Due to the prediction result, the compound should be regarded as moderate skin sensitiser.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-06-25 to 2018-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.38%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0.52
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

15.0360

0.5340

14.1920

0.5340

STD2

7.5920

0.2670

7.1540

0.2670

STD3

3.7630

0.1335

3.5770

0.1335

STD4

1.8500

0.0667

1.7790

0.0667

STD5

0.8890

0.0334

0.8770

0.0334

STD6

0.4300

0.0167

0.4310

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

4.1310

0.1469

70.73

71.32

0.53

0.74

4.0270

0.1433

71.47

3.9870

0.1418

71.75

Test Item

14.4290

0.5114

0.00

0.52

0.89

172.02

14.1140

0.5002

0.01

13.8970

0.4925

1.54

 

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

5.3010

0.1990

60.30

59.44

0.84

1.41

5.4220

0.2036

59.39

5.5240

0.2074

58.63

Test Item

13.4640

0.5057

0.00

0.00

0.00

-

13.3870

0.5028

0.00

13.4230

0.5042

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

0.26

Minimal Reactivity

-

0.52

Minimal Reactivity

-

Positive Control

65.38

High Reactivity

sensitiser

71.32

Moderate Reactivity

sensitiser

Interpretation of results:
other: The result should be considered in the context of IATA.
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. 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 492 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.

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

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was  6.38% (0.26%).Since precipitation wasobserved, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline,no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM.Therefore, no prediction can be made.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 65.38%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-07-23 to 2018-09-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on the study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (6.98 (experiment 1); 3.11 (experiment 2).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.56
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 2000 µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
125.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
1 867.82
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: clearly above threshold of 1000 µM
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.44
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 2000 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
117.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
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
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

80.8

102.0

91.4

15.0

8.00

83.6

101.1

92.3

12.3

16.00

84.6

107.0

95.8

15.9

32.00

84.8

109.9

97.3

17.7

64.00

83.4

111.1

97.3

19.6

Test Item

0.98

122.0

127.7

124.9

4.0

1.95

132.1

118.6

125.4

9.6

3.91

116.0

103.8

109.9

8.6

7.81

75.1

100.4

87.7

17.9

15.63

114.4

106.8

110.6

5.4

31.25

84.5

95.3

89.9

7.6

62.50

30.2

86.2

58.2

39.6

125.00

78.2

85.1

81.6

4.9

250.00

29.0

87.7

58.3

41.5

500.00

71.9

91.7

81.8

14.0

1000.00

91.0

107.1

99.1

11.4

2000.00

125.4

117.6

121.5

5.5

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

1.22

1.07

1.13

1.14

0.08

 

8.00

1.26

1.49

1.28

1.34

0.12

 

16.00

1.72

1.96

1.73

1.80

0.14

*

32.00

2.50

2.81

2.72

2.68

0.16

*

64.00

6.59

7.67

6.67

6.98

0.60

*

Test Item

0.98

1.42

1.40

1.31

1.38

0.06

 

1.95

1.24

1.09

1.16

1.16

0.07

 

3.91

1.53

1.12

1.19

1.28

0.22

 

7.81

1.18

1.42

1.14

1.24

0.15

 

15.63

1.10

1.11

1.08

1.10

0.01

 

31.25

1.11

1.01

0.88

1.00

0.12

 

62.50

1.07

0.86

0.88

0.94

0.12

 

125.00

0.78

0.89

0.86

0.84

0.06

 

250.00

0.72

1.11

0.75

0.86

0.22

 

500.00

0.78

1.13

0.89

0.93

0.18

 

1000.00

1.03

1.33

1.00

1.12

0.18

 

2000.00

1.40

1.93

1.34

1.56

0.32

*

* = 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

1.32

1.19

1.14

1.22

0.09

 

8.00

1.35

1.27

1.30

1.31

0.04

 

16.00

1.50

1.29

1.47

1.42

0.11

 

32.00

2.00

1.78

2.00

1.93

0.13

*

64.00

3.66

2.55

3.11

3.11

0.55

*

Test Item

0.98

1.23

1.47

1.58

1.43

0.18

 

1.95

1.07

1.17

1.17

1.14

0.06

 

3.91

0.96

1.07

1.08

1.03

0.07

 

7.81

1.09

1.27

1.03

1.13

0.12

 

15.63

0.94

1.12

0.95

1.01

0.10

 

31.25

0.94

1.09

0.89

0.98

0.10

 

62.50

0.79

0.85

0.76

0.80

0.05

 

125.00

0.91

0.91

0.83

0.88

0.05

 

250.00

0.92

0.94

0.84

0.90

0.06

 

500.00

0.95

1.10

0.90

0.98

0.11

 

1000.00

1.03

1.26

0.94

1.08

0.16

 

2000.00

1.28

1.70

1.34

1.44

0.23

 

* = 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

1.14

1.22

1.18

0.06

8.00

1.34

1.31

1.32

0.03

16.00

1.80

1.42

1.61

0.27

32.00

2.68

1.93

2.30

0.53

64.00

6.98

3.11

5.04

2.74

Test Item

0.98

1.38

1.43

1.40

0.03

1.95

1.16

1.14

1.15

0.02

3.91

1.28

1.03

1.16

0.18

7.81

1.24

1.13

1.19

0.08

15.63

1.10

1.01

1.05

0.07

31.25

1.00

0.98

0.99

0.02

62.50

0.94

0.80

0.87

0.10

125.00

0.84

0.88

0.86

0.03

250.00

0.86

0.90

0.88

0.03

500.00

0.93

0.98

0.96

0.04

1000.00

1.12

1.08

1.10

0.03

2000.00

1.56

1.44

1.50

0.08

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

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control PC (1% DMSO)

< 20%

12.7

pass

9.4

pass

CV Solvent Control TI (1% THF)

<20%

14.9

pass

18.6

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

10.74

pass

18.50

pass

Induction PC at 64 µM

2.00 < x < 8.00

6.98

pass

3.11

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 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 492 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 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 1.56 was determined at a test item concentration of 2000 µM.

The corresponding cell viability was 125.4%. No further significant luciferase induction > 1.5 was found in the tested concentration range.The calculated EC1.5 was > 1000 µM (1867.82 µM).

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:
weight of evidence
Study period:
2018-10-02 to 2018-11-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
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 (323% experiment 1; 365% experiment 2) and
200% for CD54 (446% experiment 1; 387% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
114
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 625 µg/mL
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
102
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 900 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
119
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 900 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
104
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 251.17 µg/mL
Other effects / acceptance of results:
Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Results of the Cell Batch Activation Test (Batch 1)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

87.5

373

>150

88.1

358

>200

yes

pass

NiSO4

100 µg/mL

83.5

295

>150

82.1

603

>200

yes

pass

LA

1000 µg/mL

95.7

81

≤150

95.8

100

≤200

no

pass

Results of the Cell Batch Activation Test (Batch 2)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

83.4

364

>150

82.3

419

>200

yes

pass

NiSO4

100 µg/mL

79.1

351

>150

77.9

688

>200

yes

pass

LA

1000 µg/mL

96.2

77

≤150

95.9

105

≤200

no

pass

 

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

96.80

Solvent Control

THF

--

96.20

test material

C8

7.03

96.40

C7

14.06

96.20

C6

28.13

96.90

C5

56.25

96.80

C4

112.50

96.80

C3

225.00

96.10

C2

450.00

96.50

C1

900.00

94.90

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

-

96.4

95.8

95.7

1402

882

533

869

349

79

78

263

165

Solvent Control 2 (THF)

0.20%

95.2

95.2

95.2

1555.0

942.0

552.0

1003

390

100

100

282

171

Solvent Control 1 (DMSO)

0.20%

95.5

95.9

95.5

1635

987

538

1097

449

100

100

304

183

DNCB

4.00

80.1

80.6

78.8

4099

2559

556

3543

2003

323

446

737

460

 

test material

900

94.6

95.5

95.6

1632

975

577

1055

398

105

102

283

169

750

94.6

94.5

94.3

1722

975

596

1126

379

112

97

289

164

625

94.0

94.1

94.9

1740

951

595

1145

356

114

91

292

160

520.83

95.3

94.8

94.8

1660

921

579

1081

342

108

88

287

159

434.03

94.6

95.0

94.7

1719

963

600

1119

363

112

93

287

161

361.69

94.3

95.5

94.5

1680

957

598

1082

359

108

92

281

160

301.41

94.9

95.2

94.9

1728

977

584

1144

393

114

101

296

167

251.17

95.2

95.0

94.9

1632

955

600

1032

355

103

91

272

159

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

-

94.6

94.1

94.1

2259

1199

592

1667

607

107

88

382

203

Solvent Control 2 (THF)

0.20%

93.8

94.0

93.8

2053

1259

589

1464

670

100

100

349

214

Solvent Control 1 (DMSO)

0.20%

94.5

94.6

93.8

2124

1258

569

1555

689

100

100

373

221

DNCB

4.0

80.9

80.6

79.7

6271

3252

589

5682

2663

365

387

1065

552

 

test material

900

93.70

93.70

93.50

2380

1272

632

1748

640

119

96

377

201

750

93.40

94.00

94.20

2322

1218

634

1688

584

115

87

366

192

625

93.50

94.00

93.70

2324

1249

631

1693

618

116

92

368

198

520.83

94.70

94.80

94.40

2083

1238

623

1460

615

100

92

334

199

434.03

93.90

93.10

94.00

2335

1240

641

1694

599

116

89

364

193

361.69

92.80

93.10

93.00

2340

1253

643

1697

610

116

91

364

195

301.41

93.60

93.90

93.70

2367

1304

634

1733

670

118

100

373

206

251.17

94.00

94.20

93.60

2349

1329

632

1717

697

117

104

372

210

 

Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

95.2

-

96.4

pass

93.8

-

94.6

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

323

pass

365

pass

RFI of positive control of CD54

≥200

446

pass

387

pass

RFI of DMSO solvent control of CD86

<150

126

pass

93

pass

RFI of DMSO solvent control of CD54

<200

129

pass

114

pass

RFI of THF solvent control of CD86

<150

115

pass

88

pass

RFI of THF solvent control of CD54

<200

112

pass

110

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

263

pass

382

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

304

pass

373

pass

MFI ratio CD86/IgG1 for THF control [%]

>105

282

pass

349

pass

MFI ratio CD54/IgG1for medium control [%]

>105

165

pass

203

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

183

pass

221

pass

MFI ratio CD54/IgG1for THF control [%]

>105

171

pass

214

pass

Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

96.2

1.6

216

number of test doses with viability >50%

-

-

555

RFI of positive control of CD86

358.9

90.1

36

RFI of positive control of CD54

435.7

285.8

36

RFI of solvent control (THF) of CD86

102.9

17.4

36

RFI of solvent control (THF) of CD54

102.0

15.4

36

MFI ratio IgG1/CD86 for medium control [%]

285.6

124.3

36

MFI ratio IgG1/CD86 for THF control [%]

270.8

105.0

36

MFI ratio IgG1/CD54 for medium control [%]

308.6

137.1

36

MFI ratio IgG1/CD54 for THF control [%]

158.2

28.4

36

Interpretation of results:
other: The result should be considered in the context of IATA.
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 has to be considered as inconclusive.
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 at the highest soluble concentration of 450 mg/mL. For the dose finding assay stock solutions with a concentration range of 450 mg/mL to 3.52 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:

900; 750, 625, 520.83, 434.03, 361.69, 301.41, 251.17 µg/mL

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

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

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 94.6% (CD86), 95.5% (CD54) and 95.6% (isotype IgG1 control) in the first experiment and to 93.7% (CD86), 93.7% (CD54) and 93.5% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of the cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Because of a Log KOW value above 3.5 a negative result cannot be considered. Therefore, the test item has to be considered as inconclusive.

Endpoint:
skin sensitisation, other
Type of information:
other: Weight of Evidence Justification
Adequacy of study:
other information
Study period:
2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Key result
Parameter:
other:
Value:
1
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The structural alerts identified in the QSAR analysis and the moderate EC3 potential for skin sensitisation estimated from the data of structural analogues provide sufficient evi-dence for a classification in class 1B for skin sensitisation for the test item.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The structural alerts identified in the QSAR analysis and the moderate EC3 potential for skin sensitisation estimated from the data of structural analogues provide sufficient evidence for a classification in class 1B for skin sensitisation.