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Diss Factsheets

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.

For the test material the in vitro weight of evidence approach was used to gain sufficient information on the skin sensitisation potential of this compound. In general evidence is accumulated from the chemical reactivity of the molecule, and from responses obtained in in vitro assays that address key events of the biological mechanism associated with sensitisation. The chemical structure and the metabolism was investigated by a QSAR analysis showing no reactive groups or elements in the structure or in the metabolites that could lead to skin sensitisation. In vitro assays performed for three key events have been performed. Two of these three key events, namely the initiating event of protein binding and the activation of dendridic cells assessed by expression of specific cell surface markers, provide no evidence for skin sensitisation. The Keratinosens assay showed evidence for inflammatory response and indicated changes in gene expression associated with specific cell signaling pathways. There are two clear negative results in the battery of on total four assessments and only one positive signal. Overall the absence of structural alerts in the parent molecule and in the metabolites generated after biotransformation, and the negative outcome in the in vitro assays provided along with the evidence for absence of protein binding potential provide sufficient arguments to conclude that there is no potential for skin sensitisation associated with the test material.

An additional in vivo assay was performed to fulfil registration requirements in China. This assay was negative confirming the results from the initial weight of evidence analysis.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-12-11 to 2018-01-26
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 68.75%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
0.86
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:
0.2
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

Cystein Peptide

Lysine Peptide

Peak Area at 220 nm

Peptide Concentration [mM]

Peak Area at 220 nm

Peptide Concentration [mM]

STD1

17.2695

0.5340

14.7569

0.5340

STD2

8.6330

0.2670

7.4271

0.2670

STD3

4.3121

0.1335

3.6929

0.1335

STD4

2.1439

0.0667

1.8123

0.0667

STD5

1.0337

0.0334

0.9009

0.0334

STD6

0.5111

0.0167

0.4518

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 Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.0850

0.1268

75.22

74.85

0.50

0.67

4.1130

0.1276

75.05

4.2401

0.1316

74.28

Test Item

16.3481

0.5054

0.00

0.86

0.75

86.60

15.9824

0.4941

1.30

15.9835

0.4942

1.29

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

5.3694

0.1943

61.31

62.65

1.16

1.85

5.0902

0.1842

63.32

5.0901

0.1842

63.32

Test Item

13.9418

0.5039

0.00

0.20

0.18

91.93

13.8787

0.5016

0.24

13.8616

0.5010

0.36

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 / Item 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

0.53

Minimal Reactivity

-

0.86

Minimal Reactivity

-

Positive Control

68.75

High Reactivity

sensitiser

74.85

Moderate Reactivity

sensitiser

Interpretation of results:
study cannot be used for classification
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. Due to the observed phase separation 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 isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 222.42 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of thetest item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item (including the co-elution control). Samples were not 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 test item and the positive control (including the co-elution controls). Samples were not centrifuged prior to the HPLC analysis.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was  6.38% (0.53%). Since a phase separation (droplet on top of the sample-peptide solution) was observed, 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 68.75%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-01-24 to 2018-04-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on the study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.97 (experiment 1); 3.67 (experiment 2)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
3.43
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 62.50 µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
110.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
17.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
2.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 250 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
79.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Value:
40.83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
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

94.3

98.3

96.3

2.9

8.00

97.1

101.9

99.5

3.4

16.00

96.6

100.9

98.7

3.0

32.00

94.3

103.6

98.9

6.6

64.00

93.7

88.9

91.3

3.4

Test Item

0.98

100.0

86.4

93.2

9.6

1.95

99.4

98.5

98.9

0.6

3.91

99.3

94.4

96.9

3.4

7.81

105.7

102.6

104.2

2.2

15.63

109.8

104.0

106.9

4.1

31.25

115.3

109.4

112.4

4.2

62.50

110.4

122.5

116.5

8.6

125.00

41.0

134.9

87.9

66.4

250.00

0.2

79.1

39.6

55.8

500.00

0.1

0.0

0.1

0.1

1000.00

0.4

0.0

0.2

0.2

2000.00

0.3

0.0

0.2

0.2

 

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

1.16

1.21

1.05

1.14

0.08

 

8.00

1.19

1.25

1.13

1.19

0.06

 

16.00

1.68

1.45

1.38

1.50

0.16

*

32.00

1.80

1.81

2.03

1.88

0.13

*

64.00

5.22

4.72

4.96

4.97

0.25

*

Test Item

0.98

1.26

1.03

1.03

1.11

0.13

 

1.95

0.98

1.23

0.97

1.06

0.15

 

3.91

1.22

1.21

1.18

1.21

0.02

 

7.81

1.27

1.22

1.11

1.20

0.08

 

15.63

1.69

1.47

1.20

1.45

0.24

 

31.25

1.85

1.96

1.95

1.92

0.06

*

62.50

3.40

3.50

3.38

3.43

0.07

*

125.00

2.14

3.25

2.55

2.65

0.56

*

250.00

0.06

0.04

0.04

0.05

0.02

 

500.00

0.21

0.05

0.13

0.13

0.08

 

1000.00

0.03

0.01

0.01

0.02

0.01

 

2000.00

0.02

0.02

0.05

0.03

0.01

 

* = 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

1.03

1.11

0.93

1.02

0.09

 

8.00

1.14

1.08

0.94

1.05

0.10

 

16.00

1.40

1.41

1.24

1.35

0.10

 

32.00

1.66

1.68

1.54

1.62

0.07

*

64.00

3.05

4.47

3.48

3.67

0.73

*

Test Item

0.98

0.98

1.25

1.06

1.10

0.14

 

1.95

1.12

1.21

1.17

1.16

0.04

 

3.91

1.21

1.47

1.20

1.29

0.15

 

7.81

1.07

1.28

0.92

1.09

0.18

 

15.63

1.28

1.32

1.10

1.23

0.12

 

31.25

1.23

1.35

1.13

1.24

0.11

 

62.50

2.11

2.33

1.85

2.10

0.24

*

125.00

2.18

2.70

2.06

2.31

0.34

*

250.00

2.56

2.77

2.77

2.70

0.12

*

500.00

0.01

0.01

0.00

0.01

0.01

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.02

0.00

0.01

0.01

 

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

Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.14

1.02

1.08

0.08

 

8.00

1.19

1.05

1.12

0.10

 

16.00

1.50

1.35

1.43

0.11

 

32.00

1.88

1.62

1.75

0.18

*

64.00

4.97

3.67

4.32

0.92

*

Test Item

0.98

1.11

1.10

1.10

0.01

 

1.95

1.06

1.16

1.11

0.07

 

3.91

1.21

1.29

1.25

0.06

 

7.81

1.20

1.09

1.15

0.08

 

15.63

1.45

1.23

1.34

0.15

 

31.25

1.92

1.24

1.58

0.49

 

62.50

3.43

2.10

2.76

0.94

 

125.00

2.65

2.31

2.48

0.24

*

250.00

0.05

2.70

1.37

1.88

 

500.00

0.13

0.01

0.07

0.08

 

1000.00

0.02

0.00

0.01

0.01

 

2000.00

0.03

0.01

0.02

0.01

 

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

17.20

40.83

29.01

16.71

Imax

3.43

2.70

3.06

0.51

IC30[µM]

98.88

278.67

188.77

127.13

IC50[µM]

116.87

341.90

229.38

159.12

 

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

13.0

pass

11.7

pass

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

≥ 1

3.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

15.91

pass

24.77

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.97

pass

3.67

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.3

3.3

41

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

≥ 1

2.3

0.6

41

EC1.5 PC

7 < x < 34 µM

20.4

6.7

41

Induction PC at 64 µM

2.00 < x < 8.00

3.3

1.1

41

Interpretation of results:
other: should be considered in the context of integrated approached such as IATA
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Under the condition of this study the test item is therefore 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 DMSO. Based on a molecular weight of 222.41 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.

In the first experiment, a max luciferase activity (Imax) induction of 3.43 was determined at a test item concentration of 62.50 µM. The corresponding cell viability was 110.4%. The lowest tested concentration with a significant luciferase induction >1.5 (1.92) was found to be 31.25 µM. The corresponding cell viability was >70% (115.3%).The calculated EC1.5 was <1000 µM (17.20 µM).

In the second experiment, a max luciferase activity (Imax) induction of 2.70 was determined at a test item concentration of 250.00 µM. The corresponding cell viability was 79.1%. The lowest tested concentration with a significant luciferase induction >1.5 (2.10) was found to be 62.50 µM. The corresponding cell viability was >70% (122.5%).The calculated EC1.5 was <1000 µM (40.83 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-07-20 to 2018-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: “In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on the study design:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test
item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely
dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in
the human monocytic cell line THP-1. The expression of the cell surface markers compared to the
respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both
experiments. The threshold of 150% for CD86 (331% experiment 1; 407% experiment 2) and
200% for CD54 (543% experiment 1; 344% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
111
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 279.08 µg/mL
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
103
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 279.08 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
142
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 401.88 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
142
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 1000 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 22)

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

85.3

329

>150

85.1

434

>200

yes

pass

NiSO4"old"

100 µg/mL

88.6

294

>150

89.4

447

>200

yes

pass

NiSO4"new"

100 µg/mL

88.7

298

>150

88.7

456

>200

yes

pass

LA

1000 µg/mL

96.7

84

£150

96.6

107

£200

no

pass

Results of the Dose Finding Assay

Sample

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

94.40

Solvent Control

THF

--

95.40

Test item

C8

7.81

96.30

C7

15.63

96.00

C6

31.25

95.10

C5

62.50

95.00

C4

125.00

94.80

C3

250.00

94.80

C2

500.00

95.30

C1

1000.00

96.20

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 Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

96.2

96.5

96.0

1431

813

568

863

245

102

79

252

143

Solvent Control 2 (THF)

0.20%

94.6

96.0

95.2

1607.0

898.0

569.0

1038

329

100

100

282

158

Solvent Control 1 (DMSO)

0.20%

96.5

95.5

96.2

1422

883

572

850

311

100

100

249

154

DNCB

4.0

80.1

80.1

79.5

3477

2349

660

2817

1689

331

543

527

356

Test item

1000

96.9

96.5

96.8

1187

812

568

619

244

60

74

209

143

833.33

96.5

96.4

96.5

1422

815

550

872

265

84

81

259

148

694.44

96.6

96.5

96.6

1278

815

549

729

266

70

81

233

148

578.70

96.5

95.0

96.4

1535

838

543

992

295

96

90

283

154

482.25

96.2

96.9

97.0

1312

860

555

757

305

73

93

236

155

401.88

96.9

96.6

96.8

1557

860

550

1007

310

97

94

283

156

334.90

96.3

96.2

96.5

1506

895

569

937

326

90

99

265

157

279.08

96.8

96.7

96.1

1725

912

574

1151

338

111

103

301

159

CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

Corrected Mean Fluorescence Intensity

Relative Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

96.1

96.2

96.6

1992

1129

641

1351

488

100

104

311

176

Solvent Control 2 (THF)

0.20%

97.4

97.2

96.8

1967

1100

636

1331

464

100

100

309

173

Solvent Control 1 (DMSO)

0.20%

97.4

96.9

96.9

1994

1110

640

1354

470

100

100

312

173

DNCB

4.0

85.0

84.6

85.5

6123

2232

615

5508

1617

407

344

996

363

Test item

1000.00

96.8

97.4

97.2

2279

1288

628

1651

660

124

142

363

205

833.33

97.0

97.1

97.1

2344

1120

621

1723

499

129

108

377

180

694.44

96.7

96.7

96.4

2430

1138

619

1811

519

136

112

393

184

578.70

97.0

97.4

97.3

2378

1184

633

1745

551

131

119

376

187

482.25

97.5

97.3

97.4

2052

1148

634

1418

514

107

111

324

181

401.88

97.6

97.0

97.0

2529

1159

642

1887

517

142

111

394

181

334.90

96.9

96.5

96.6

2206

1131

641

1565

490

118

106

344

176

279.08

96.3

96.7

96.7

2151

1080

625

1526

455

115

98

344

173

Interpretation of results:
other: 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 upregulate the expression of the cell surface markers in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

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

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 96.9% (CD86), 96.5% (CD54) and 96.8% (isotype IgG1 control) in the first experiment and to 96.8% (CD86), 97.4% (CD54) and 97.2% (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.

Therefore, the test item can be considered as non-sensitiser.

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:
0
Interpretation of results:
GHS criteria not met
Conclusions:
Using Derek and Meteor Nexus , the skin sensitising potential of the test item and of the metabolites therefor was estimated to be absent (Nothing to report). The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Mar 29, 2019 - Jun 11, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 10 weeks
- Weight at study initiation: Pre-test and Main test: 19.2 to 24.5 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 23 °C
- Humidity (%): 40 - 46 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6
Vehicle:
methyl ethyl ketone
Concentration:
1, 2, and 5% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Two test item concentrations were tested; a 5% and 10% concentration. The highest concentration was concentration that was considered to be suitable for animal welfare reasons
based on the known corrosive properties of the test item.

- Compound solubility: 5 and 10 % in MEK
- Irritation:yes (At a 10% test item concentration, variation in ear thickness during the observation period were more than 25% from Day 1 pre-dose values.)
- Lymph node proliferation response: -


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Standard statistical methods have been applied for data processing.
Positive control results:
Conc. SI
0%: 1.0
5% 1.3
10% 1.4
25% 5.2
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
Test Group: 1% in MEK
Key result
Parameter:
SI
Value:
1.4
Test group / Remarks:
Test Group: 2% in MEK
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
Test Group: 5% in MEK

Calculation of Stimulation Indices per Dose Group

Test item concentration
Group Calculation
Mean DPM per animal (2 lymph nodes)
SD
S.I.
MEK (Vehicle Control)
487
55
1.0
1 % Test Item in MEK
549
122
1.1
2 % Test Item in MEK 659
126
1.4
5 % Test Item in MEK 879
193
1.8


Interpretation of results:
GHS criteria not met
Conclusions:
The test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

Objective

The objective of this study was to evaluate whether the test material induces skin sensitization in mice after three epidermal exposures of the animals under the conditions

described in this report.

Study Design

The study was carried out based on the guidelines described in:

• OECD, Section 4, Health Effects, No.429 (2010).

• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".

• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.

Test item concentrations selected for the main study were based on the results of a pre-screen test. At a 10% test item concentration, variation in ear thickness during the observation

period were more than 25% from Day 1 pre-dose values. Therefore this concentration did not meet the selection criteria. At a 5% test item concentration, no signs of systemic toxicity

were noted and up to very slight irritation was observed. Therefore, this concentration was selected as highest concentration for the main study.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 1, 2 or 5% w/w on three consecutive days, by open application on

the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Methylethylketone (MEK)). Three days after the last exposure, all animals were injected

with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells,

radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated

for each group.

Results

The majority of auricular lymph nodes were considered normal in size, except for the nodes in three animals treated at 5% which were considered to be slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 1, 2 and 5% were 487, 549 and 879 DPM, respectively. The mean DPM/animal value for the

vehicle control group was 487 DPM. The SI values calculated for the test item concentrations 1, 2 and 5% were 1.1, 1.4 and 1.8, respectively.

Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 5%, the test material was considered not to be a skin sensitizer. It was established that the EC3

value (the estimated test item concentration that will give a SI =3) (if any) exceeds 5%.

Conclusion

The test item would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information there is no need to classified according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures.