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Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-12-07 to 2018-03-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
6-diazo-5,6-dihydro-5-oxonaphthalene-2-sulphonyl chloride
EC Number:
223-211-1
EC Name:
6-diazo-5,6-dihydro-5-oxonaphthalene-2-sulphonyl chloride
Cas Number:
3770-97-6
Molecular formula:
C10H5ClN2O3S
IUPAC Name:
6-(-lambda5-diazynylidene)-5-oxo-5,6-dihydronaphthalene-2-sulfonyl chloride
Test material form:
solid

In vitro test system

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.

Results and discussion

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (7.72 (experiment 1); 4.70 (experiment 2)).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
29.27
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:
3.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
526.22
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
16.99
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 1000 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
6.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Value:
294.94
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

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

The controls fullfilled the validity criteria of the test.

Any other information on results incl. tables

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

100.4

87.0

93.7

9.5

8.00

98.5

66.4

82.5

22.7

16.00

97.0

76.3

86.6

14.6

32.00

95.4

59.3

77.4

25.5

64.00

95.8

48.8

72.3

33.2

Test Item

0.98

94.1

107.1

100.6

9.2

1.95

94.0

101.5

97.8

5.3

3.91

90.9

101.1

96.0

7.2

7.81

89.8

100.8

95.3

7.8

15.63

88.4

100.0

94.2

8.3

31.25

90.4

93.8

92.1

2.4

62.50

79.7

85.6

82.7

4.2

125.00

53.5

63.2

58.3

6.9

250.00

40.8

47.8

44.3

5.0

500.00

37.2

53.4

45.3

11.5

1000.00

34.8

6.2

20.5

20.2

2000.00

3.8

2.9

3.4

0.7

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

1.28

1.23

1.23

1.25

0.03

 

8.00

1.44

1.35

1.29

1.36

0.07

 

16.00

1.82

1.66

1.50

1.66

0.16

*

32.00

2.52

2.75

1.68

2.32

0.57

*

64.00

7.62

(12.54#)

7.82

7.72

0.10

*

Test Item

0.98

1.03

1.04

1.07

1.05

0.02

 

1.95

0.99

1.01

0.92

0.97

0.05

 

3.91

1.08

1.06

0.95

1.03

0.07

 

7.81

1.16

1.27

1.14

1.19

0.07

 

15.63

1.00

1.08

1.01

1.03

0.04

 

31.25

1.03

1.07

0.93

1.01

0.07

 

62.50

1.04

1.16

0.95

1.05

0.10

 

125.00

1.09

0.95

0.88

0.97

0.11

 

250.00

1.03

1.15

0.80

0.99

0.18

 

500.00

1.30

1.51

1.29

1.37

0.13

 

1000.00

3.56

5.19

3.02

3.92

1.13

*

2000.00

38.70

33.84

15.27

29.27

12.37

*

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

#= outlier according to Grubbs, Nalimov and Dixon. Excluded from evaluation.

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

1.16

1.18

1.10

1.15

0.04

 

8.00

1.16

1.10

1.09

1.12

0.04

 

16.00

1.65

1.69

1.49

1.61

0.11

*

32.00

2.23

1.88

1.93

2.01

0.19

*

64.00

4.84

4.51

4.75

4.70

0.17

*

Test Item

0.98

1.17

1.20

1.00

1.13

0.11

 

1.95

0.97

1.03

0.88

0.96

0.08

 

3.91

0.97

0.91

0.99

0.96

0.04

 

7.81

0.87

0.92

0.82

0.87

0.05

 

15.63

0.88

0.98

1.07

0.98

0.09

 

31.25

0.87

0.81

0.81

0.83

0.04

 

62.50

0.93

0.95

0.78

0.89

0.09

 

125.00

1.09

1.06

0.85

1.00

0.13

 

250.00

1.27

1.04

0.96

1.09

0.16

 

500.00

6.05

2.42

1.64

3.37

2.35

 

1000.00

9.30

29.65

12.01

16.99

11.05

*

2000.00

0.01

0.01

1.52

0.51

0.87

 

* = 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

1.25

1.15

1.20

0.07

 

8.00

1.36

1.12

1.24

0.17

 

16.00

1.66

1.61

1.63

0.04

*

32.00

2.32

2.01

2.16

0.22

*

64.00

9.33

4.70

7.01

3.27

 

Test Item

0.98

1.05

1.13

1.09

0.06

 

1.95

0.97

0.96

0.97

0.01

 

3.91

1.03

0.96

0.99

0.05

 

7.81

1.19

0.87

1.03

0.22

 

15.63

1.03

0.98

1.00

0.04

 

31.25

1.01

0.83

0.92

0.13

 

62.50

1.05

0.89

0.97

0.12

 

125.00

0.97

1.00

0.99

0.02

 

250.00

0.99

1.09

1.04

0.07

 

500.00

1.37

3.37

2.37

1.42

 

1000.00

3.92

16.99

10.45

9.24

 

2000.00

29.27

0.51

14.89

20.33

 

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

526.22

294.94

410.58

163.54

Imax

29.27

16.99

23.13

8.69

IC30[µM]

85.61

106.04

95.82

14.45

IC50[µM]

159.32

231.00

195.16

50.69

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

13.1

pass

9.6

pass

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

≥ 1

3.0

pass

3.0

pass

EC1.5 PC

7 < x < 34 µM

11.72

pass

14.23

pass

Induction PC at 64 µM

2.00 < x < 8.00

7.72*

pass

4.70

pass

* = Mean out of replicate 1 and 3. 2 replicate is an outlier according to Grubbs, Nalimov and Dixon.

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

Applicant's summary and conclusion

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 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 DMSO.

Based on a molecular weight of 268.5 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 29.27 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 3.8%. The lowest tested concentration with a significant luciferase induction >1.5 (3.92) was found to be 1000 µM. The corresponding cell viability was <70% (34.8%).The calculated EC1.5 was <1000 µM (526.22)

In the second experiment, a max luciferase activity (Imax) induction of 16.99 was determined at a test item concentration of 1000 µM. The corresponding cell viability was 6.2%. The lowest tested concentration with a significant luciferase induction >1.5 (3.37) was found to be 500 µM. The corresponding cell viability was <70% (53.4%).The calculated EC1.5 was < 1000 µM (294.94).

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.

The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment one of the technical replicate exceeded the threshold with a fold induction of 12.54. On the one hand, the positive control clearly induces the luciferase induction in the KerationSens™ cell line, showing the capability of the test system to predict sensitisation. On the other hand, the results in this first experiment are clearly negative, indicating no oversensitivity of the test system. The single value of the positive control exceeding the upper threshold was therefore considered to be an outliner and not biological relevant. Furthermore all other controls confirmed the validity of the study