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Diss Factsheets

Administrative data

Description of key information

OECD442D: In vitro Skin Sensitisation; ARE-Nrf2 Luciferase Test Method. 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.

No significant luciferase induction >1.5 was found in the tested concentration range.

OECD442E: In vitro Skin Sensitisation Test; Human Cell Line Activation Test (h-CLAT). This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test item which formed a stable suspension in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) was previously determined by three XTT tests. The method has been developed for the evaluation of the skin sensitisation potential by measuring phenotypic changes, such as CD86 and CD54 expression on dendritic cells. The human leukemia cell line THP-1 is used as surrogate for human myeloid dendritic cells, since these cells show also enhanced CD86 and/or CD54 expression when treated with sensitisers.

The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any tested test item concentration in both runs.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 February 2018 to 22 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 442E: hCLAT human Cell Line Activation Test
Version / remarks:
(2017)
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The Human Cell Line Activation Test (h-CLAT) was conducted as an alternative to in vivo skin sensitisation tests. The selected in vitro skin sensitisation test was considered scientifically valid for the evaluation of skin sensitisation hazard of chemicals, based on the ECVAM. Information from this test should be used in combination with other information within a weight-of-evidence approach and not as a stand-alone test method.
Details on the study design:
DETAILS ON TEST SYSTEM
- Species: Human monocytic leukaemia cell line
- Method(s): XTT test and h-CLAT test
- Expiration date: Not specified
- Date of initiation of testing: 09 October 2019

TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment/exposure: The assays were incubated at 37°C
Incubation duration: 24 ± 0.5 hours

CONTROL SAMPLES
Controls: A culture medium with no additives was used as the blank and vehicle control; DMSO and DNCB was used as a positive control.

AMOUNT/CONCENTRATION APPLIED
Test items concentrations: 17, 21, 25, 30, 36, 43, 52 and 62 μg/mL
Positive control concentrations: The concentration for the XTT test were 2.0 and 3.0 μg/mL
Replicates: 7 replicates were used for each test item concentration

Positive control results:
The actual RFI values for CD86 were 519.2 %, 674.2%, 725.3% and 652.8% and 230.8 %, 482.7%, 296.9% and 342.0% for CD54. The RFI values of both CD86 and CD54 antibodies fulfilled the positive control criteria (CD86 ≥ 150% and CD54 ≥ 200%).
Key result
Run / experiment:
other: 1/ Experiment 1: CD 54 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
95.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/ Experiment 1: CD 86 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
98.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 1: CD 54 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
109.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 1: CD86 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
96.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 1: CD 54 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
117.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 1: CD 86 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
87.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 1: CD 54 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
87
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 1: CD 86 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
64.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 1: CD 54 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
93.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 1: CD 86 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
68.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 1: CD 54 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
63
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 1: CD 86 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
64.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 1: CD 54 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
90.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 1: CD 86 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
81.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 1: CD 54 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
79.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 1: CD 86 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
75.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/ Experiment 2: CD 54 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
119.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1/ Experiment 2: CD 86 (17 μg/mL)
Parameter:
other: RFI (%)
Value:
84.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 2: CD 54 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
111.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2/ Experiment 2: CD 86 (21 μg/mL)
Parameter:
other: RFI (%)
Value:
105.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 2: CD 54 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
101.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 3/ Experiment 2: CD 86 (25 μg/mL)
Parameter:
other: RFI (%)
Value:
101.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 2: CD 54 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
91.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 4/ Experiment 2: CD 86 (30 μg/mL)
Parameter:
other: RFI (%)
Value:
70.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 2: CD 54 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
96
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 5/ Experiment 2: CD 86 (36 μg/mL)
Parameter:
other: RFI (%)
Value:
84.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 2: CD 54 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
100.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 6/ Experiment 2: CD 86 (43 μg/mL)
Parameter:
other: RFI (%)
Value:
77
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 2: CD 54 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
98.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 7/ Experiment 2: CD 86 (52 μg/mL)
Parameter:
other: RFI (%)
Value:
80.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 2: CD 54 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
105.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 8/ Experiment 2: CD 86 (62 μg/mL)
Parameter:
other: RFI (%)
Value:
94.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
- Acceptance criteria met for negative control: The negative control was more than 90 % in comparison to the medium control (100 %).
- Acceptance criteria met for positive control: In the positive control DNCB, the RFI values were 230.8%, 482.7%, 296.9% and 342.0% for CD54 and 519.2%, 674.2%, 725.3% and 652.8% for CD86. All values were within the historical laboratory data (CD54 ≥ 200% and CD86 ≥ 150%). The cell viability was also more than 50 % (73.3%, 70.1% 74% and 73.2%). Therefore, the acceptance criteria for the positive control was fulfilled.
- Acceptance criteria met for variability between replicate measurements: Not specified
- Range of historical values if different from the ones specified in the test guideline: No applicable


Table 1- Results from h-CLAT experiment 1

 

Concentration (µg/mL)

RFI (%) CD 54 Antibody

RFI (%) CD 86Antibody

Cell Viability

(%)

Medium Control

-

100.0

100.0

100.0

DMSO control

-

100.0

100.0

100.0

Positive control (DNCB)

2.0

230.8

519.2

73.3

3.0

482.7

674.2

70.1

 

 

 

Test item

17

95.7

98.6

93.7

21

109.8

96.8

94.1

25

117.4

87.5

94.4

30

87.0

64.8

92.3

36

93.5

68.7

90.3

43

63.0

64.8

95.6

52

90.2

81.5

92.0

62

79.3

75.4

86.3

 

Table 2- Results from h-CLAT experiment 2

 

Concentration (µg/mL)

RFI (%) CD 54 Antibody

RFI (%) CD 86Antibody

Cell Viability

(%)

Medium Control

-

100.0

100.0

100.0

DMSO control

-

100.0

100.0

100.0

Positive control (DNCB)

2.0

296.9

725.3

74.0

3.0

342.0

652.8

73.2

 

 

 

Test item

17

119.4

84.8

98.1

21

111.3

105.8

99.5

25

101.6

101.6

97.2

30

91.1

70.2

92.6

36

96.0

84.1

92.0

43

100.8

77.0

86.6

52

98.4

80.9

85.1

62

105.6

94.2

81.7

Interpretation of results:
study cannot be used for classification
Conclusions:
The test item with a log Pow of 3.02 and 3.32, did not cause THP-1 cell activation up to the highest tested concentration (62 μg/mL) under test conditions. Therefore, the test item is considered negative for the third key event of skin sensitisation adverse outcome pathway (AOP).
Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 442E: In Vitro Skin Sensitisation: Human cell line activation test (h-CLAT) and is compliant with GLP.

The skin sensitising potential of the test item was determined by the third key event of the skin sensitisation Adverse Outcome Pathway (AOP). The test item concentrations of 17, 21, 25, 30, 36, 43, 52 and 62 μg/mL were used in the definitive study. Two independent runs were conducted for the study.

The negative control was more than 90 % in comparison to the medium control (100 %). In the positive control DNCB, the RFI values were 230.8%, 482.7%, 296.9% and 342.0% for CD54 and 519.2%, 674.2%, 725.3% and 652.8% for CD86. All values were within the historical laboratory data (CD54 ≥ 200% and CD86 ≥ 150%). The cell viability was also more than 50 % (73.3%, 70.1% 74% and 73.2%). Therefore, all validity criteria were met. The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry.

The test item with a log Pow of 3.02 and 3.32, did not cause THP-1 cell activation up to the highest tested concentration (62 μg/mL) under test conditions. Therefore, the test item is considered negative for the third key event of skin sensitisation adverse outcome pathway (AOP).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 March 2018 to 12 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
(2015)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
other: KeratinoSens (Luciferase test method)
Justification for non-LLNA method:
The Are-Nrf2 Luciferase test method was conducted as an alternative to in vivo skin sensitisation tests. The selected in vitro skin sensitisation test was considered scientifically valid for the evaluation of skin sensitisation hazard of chemicals, based on the ECVAM. Information from this test should be used in combination of other information within a weight-of-evidence approach and not as a stand-alone test method.
Details on the study design:
DETAILS ON TEST SYSTEM
- Model used: KerantinoSensTM assay
- Species: Human keratinocytes (HaCaT)
- Expiration date: Not specified
- Date of initiation of testing: 05 March 2018

TEMPERATURE USED FOR TEST SYSTEM
Temperature used during treatment/exposure: The KerantinoSensTM assay were incubated at 37°C
Incubation duration: 48 hours

CONTROL SAMPLES
Controls: A blank with no seeded cells was used; DMSO was used as a negative control and cinnamic aldehyde was used as a positive control.

AMOUNT/CONCENTRATION APPLIED
Test items concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM
Positive control concentrations: 4 µM, 8 µM, 16 µM; 32 µM; 64 µM

Positive control results:
Cinnamic aldehyde showed significant luciferase activity induction that was above the acceptable criteron threshold of >1.5 (2.0 in experiment 1/ 3.0 in experiment 2).
Key result
Run / experiment:
other: 1
Parameter:
other: Max luciferase activity induction (Imax)
Value:
1.32
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 2
Parameter:
other: (Imax) max luciferase activity induction
Value:
1.4
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: Imax) max luciferase activity induction
Value:
1.36
Vehicle controls validity:
not valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS: Cell viability (Cytotoxicity) was also examined for both experiments. The cell viability was 12.0 % at the test item concentration 125 µM in experiment 1. The cell viability was 90.5 % at the test item concentration 7.81 µM in experiment 2.
- Visible damage on test system: None

DEMONSTRATION OF TECHNICAL PROFICIENCY: Not applicable

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control from the study had an average coefficient of variation luminescence reading that was <20 % (Experiment 1: 14.1 %, Experiment 2: 11.3 %).
- Acceptance criteria met for positive control: The positive control from the study had a mean luciferase activity induction that was >1.5 (Experiment 1: 2.0, Experiment 2: 3.0).
- Acceptance criteria met for variability between replicate measurements: The standard deviation for the negative and positive controls were within two standard deviations of the laboratory historical mean data for the EC1.5 values

Table 1: 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

95.9

104.8

100.3

6.3

8.00

100.8

98.3

99.6

1.7

16.00

98.4

107.3

102.8

6.3

32.00

94.0

104.8

99.4

7.7

64.00

92.4

105.5

99.0

9.3

Test Item

0.98

97.7

101.9

99.8

3.0

1.95

99.7

97.6

98.7

1.5

3.91

110.8

92.2

101.5

13.2

7.81

114.2

90.5

102.4

16.8

15.63

111.6

101.8

106.7

6.9

31.25

113.7

99.0

106.4

10.4

62.50

120.5

98.3

109.4

15.7

125.00

12.0

111.0

61.5

70.0

250.00

0.0

107.7

53.9

76.1

500.00

0.1

114.0

57.1

80.6

1000.00

0.1

0.2

0.2

0.1

2000.00

123.0

0.2

61.6

86.9

 

Table 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

1.22

1.52

1.41

1.38

0.15

 

8.00

0.96

1.31

1.29

1.19

0.20

 

16.00

1.08

1.59

1.23

1.30

0.26

 

32.00

2.32

2.97

2.42

2.57

0.35

*

64.00

3.28

7.07

3.59

4.64

2.11

*

Test Item

0.98

1.00

1.29

0.93

1.07

0.19

 

1.95

1.19

1.19

0.99

1.13

0.11

 

3.91

0.99

1.40

0.84

1.08

0.29

 

7.81

0.81

0.81

0.86

0.82

0.03

 

15.63

0.89

0.81

0.90

0.87

0.05

 

31.25

0.94

1.04

0.87

0.95

0.08

 

62.50

1.03

0.94

0.88

0.95

0.08

 

125.00

1.20

1.70

1.08

1.32

0.33

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.01

0.00

0.00

0.00

 

1000.00

1.15

0.55

0.00

0.57

0.58

 

2000.00

1.04

0.80

0.31

0.72

0.37

 

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

 

Table 3: 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

1.23

1.11

1.24

1.19

0.07

 

8.00

1.25

1.26

1.93

1.48

0.39

 

16.00

1.60

1.50

1.48

1.53

0.07

*

32.00

2.18

2.03

2.22

2.14

0.10

*

64.00

3.70

2.24

3.94

3.29

0.92

*

Test Item

0.98

1.28

1.19

1.08

1.18

0.10

 

1.95

0.89

0.98

1.07

0.98

0.09

 

3.91

1.37

1.27

1.36

1.33

0.06

 

7.81

1.59

1.27

1.36

1.40

0.17

 

15.63

0.98

1.04

1.12

1.05

0.07

 

31.25

0.96

1.07

1.01

1.01

0.05

 

62.50

1.10

1.10

1.03

1.08

0.04

 

125.00

0.93

1.22

1.14

1.10

0.15

 

250.00

1.12

1.37

1.12

1.20

0.15

 

500.00

1.32

1.35

1.40

1.36

0.04

 

1000.00

1.46

1.18

0.00

0.88

0.78

 

2000.00

0.00

0.04

0.00

0.01

0.02

 

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

 

Table 4: 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

1.38

1.19

1.29

0.14

 

8.00

1.19

1.48

1.33

0.21

 

16.00

1.30

1.53

1.41

0.16

 

32.00

2.57

2.14

2.36

0.30

*

64.00

4.64

3.29

3.97

0.95

*

Test Item

0.98

1.07

1.18

1.13

0.08

 

1.95

1.13

0.98

1.05

0.10

 

3.91

1.08

1.33

1.20

0.18

 

7.81

0.82

1.40

1.11

0.41

 

15.63

0.87

1.05

0.96

0.13

 

31.25

0.95

1.01

0.98

0.04

 

62.50

0.95

1.08

1.01

0.09

 

125.00

1.32

1.10

1.21

0.16

 

250.00

0.00

1.20

0.60

0.85

 

500.00

0.00

1.36

0.68

0.96

 

1000.00

0.57

0.88

0.72

0.22

 

2000.00

0.72

0.01

0.36

0.50

 

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

Table 5: Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a

-

-

Imax

1.32

1.40

1.36

0.06

IC30[µM]

n.a

693.49

-

-

IC50[µM]

n.a

781.36

-

-

n.a.:not applicable

Table 6: 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:
study cannot be used for classification
Conclusions:
In conclusion, the test item is considered negative for the second key event of skin sensitization Adverse Outcome Pathway (AOP).
The In vitro test method- OECD 442 D- ARE-Nrf2 Luciferase Test Method can only be used as a weight-of-evidence approach and may not be sufficient on its own to conclude on the skin sensitisation hazard potential.
Executive summary:

The study is assigned a reliability score of 1 (reliable without restrictions) as it followed OECD Guideline 442D: In VitroSkin Sensitisation: ARE-Nrf2 Luciferase Test Method and is compliant with GLP.

The skin sensitising potential of the test item was determined by the second molecular key event of the adverse outcome pathway. The test item concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM were used in the test assay.

The negative control from the study had an average coefficient of variation luminescence reading that was <20 % (Experiment 1: 14.1 %, Experiment 2: 11.3 %).The positive control from the study had a mean luciferase activity induction that was >1.5 (Experiment 1: 2.0, Experiment 2: 3.0). The standard deviation for the negative and positive controls were within two standard deviations of the laboratory historical mean data for theEC1.5values. Therefore, all validity criteria were met.

The test item under test conditions, is considered negative for the second key event of skin sensitization Adverse Outcome Pathway (AOP). The In vitro test method- OECD 442 D-ARE-Nrf2 Luciferase Test Method can only be used as a weight-of-evidence approach and may not be sufficient on its own to conclude on the skin sensitisation hazard potential.

 

 

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

OECD 422D:

The test item was dissolved in DMSO and a stock solution of 200 mM 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 1.32 was determined at a test item concentration of 125 µM. The corresponding cell viability was 12.0%.No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated.

In the second experiment, a max luciferase activity (Imax) induction of 1.40 was determined at a test item concentration of 7.81 µM. The corresponding cell viability was 90.5%.No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated.

OECD 422E:

The test item with a log Pow of 3.02 and 3.32 was tested in 2 independent runs. The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any tested test item concentration in both runs. No clear dose response could be observed for CD54 and CD86 in both runs. Therefore the h-CLAT prediction is considered negative for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In the in vitro luciferase test method, 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 nonsensitising in this assay covering the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).

In the in vitro h-CLAT assay, the test item with a log Pow of 3.02 and 3.32 did not activate THP-1 cells up to a concentration of 62 μg/mL under the test conditions of this study. Therefore the test item is considered negative for the third key event of the skin sensitisation AOP.

The data generated by both methods individually provide a Weight of Evidence in favour of classification as a non-sensitiser.