Registration Dossier

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:
2018-07-23 to 2018-08-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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:
Sodium toluene-4-sulphinate
EC Number:
212-538-5
EC Name:
Sodium toluene-4-sulphinate
Cas Number:
824-79-3
Molecular formula:
C7H8O2S.Na
IUPAC Name:
sodium toluene-4-sulphinate
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 (6.43 (experiment 1); 7.85 (experiment 2)).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.07
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:
86.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.17
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 125 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
96.3
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.

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

86.4

113.7

100.0

19.4

8.00

92.4

116.6

104.5

17.1

16.00

96.0

117.3

106.6

15.0

32.00

86.6

122.4

104.5

25.3

64.00

84.1

109.9

97.0

18.2

Test Item

0.98

100.9

102.6

101.8

1.2

1.95

97.9

103.6

100.8

4.0

3.91

91.5

109.4

100.5

12.6

7.81

91.2

102.9

97.1

8.3

15.63

88.3

100.3

94.3

8.5

31.25

88.5

96.5

92.5

5.7

62.50

86.2

104.1

95.2

12.7

125.00

80.7

96.3

88.5

11.0

250.00

81.8

102.1

92.0

14.3

500.00

85.9

98.4

92.1

8.8

1000.00

68.5

86.5

77.5

12.8

2000.00

63.9

84.9

74.4

14.8

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

1.62

1.18

1.36

1.39

0.22

 

8.00

1.27

1.20

1.14

1.20

0.07

 

16.00

1.49

1.63

1.53

1.55

0.08

*

32.00

2.20

1.84

2.24

2.09

0.22

*

64.00

7.00

6.59

5.69

6.43

0.67

*

Test Item

0.98

1.08

1.02

1.01

1.04

0.04

 

1.95

0.99

0.94

1.04

0.99

0.05

 

3.91

1.12

1.07

1.03

1.07

0.04

 

7.81

1.01

1.01

1.11

1.04

0.06

 

15.63

1.04

0.92

1.12

1.03

0.10

 

31.25

0.94

0.97

0.95

0.96

0.02

 

62.50

1.16

0.94

1.12

1.07

0.12

 

125.00

1.05

0.86

1.02

0.97

0.10

 

250.00

0.87

1.08

0.92

0.95

0.11

 

500.00

0.82

0.82

0.83

0.82

0.01

 

1000.00

0.82

0.78

0.67

0.76

0.08

 

2000.00

0.60

0.73

0.61

0.65

0.07

 

* = 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

1.19

1.24

1.47

1.30

0.15

 

8.00

1.26

1.30

1.56

1.37

0.16

 

16.00

1.52

1.51

1.90

1.65

0.22

*

32.00

2.38

2.29

2.99

2.55

0.38

*

64.00

6.77

6.61

10.16

7.85

2.00

*

Test Item

0.98

0.94

0.89

1.19

1.01

0.16

 

1.95

0.98

0.87

1.04

0.97

0.09

 

3.91

0.88

1.01

1.15

1.01

0.13

 

7.81

1.18

0.89

1.03

1.03

0.14

 

15.63

0.98

1.01

1.09

1.03

0.05

 

31.25

0.87

0.89

1.11

0.96

0.13

 

62.50

0.94

1.07

0.99

1.00

0.07

 

125.00

1.07

0.95

1.48

1.17

0.28

 

250.00

0.85

0.77

1.18

0.93

0.22

 

500.00

0.82

0.71

1.16

0.90

0.24

 

1000.00

0.77

0.67

0.95

0.80

0.14

 

2000.00

0.60

0.53

0.84

0.66

0.16

 

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

Induction of Luciferase Activity – Overall Induction

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

1.39

1.30

1.34

0.06

 

8.00

1.20

1.37

1.29

0.12

 

16.00

1.55

1.65

1.60

0.07

*

32.00

2.09

2.55

2.32

0.33

*

64.00

6.43

7.85

7.14

1.00

*

Test Item

0.98

1.04

1.01

1.02

0.02

 

1.95

0.99

0.97

0.98

0.02

 

3.91

1.07

1.01

1.04

0.04

 

7.81

1.04

1.03

1.04

0.01

 

15.63

1.03

1.03

1.03

0.00

 

31.25

0.96

0.96

0.96

0.00

 

62.50

1.07

1.00

1.04

0.05

 

125.00

0.97

1.17

1.07

0.14

 

250.00

0.95

0.93

0.94

0.01

 

500.00

0.82

0.90

0.86

0.05

 

1000.00

0.76

0.80

0.78

0.03

 

2000.00

0.65

0.66

0.65

0.01

 

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5

n.a.

n.a.

n.a.

n.a.

Imax

1.07

1.17

1.12

0.07

IC30

955.91

n.a.

n.a.

n.a.

IC50

n.a.

n.a.

n.a.

n.a.

n.a. = not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

9.4

pass

16.4

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

14.84

pass

11.73

pass

Induction PC at 64 µM

2.00 < x < 8.00

6.43

pass

7.85

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
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.


Executive summary:

In the present study the test material was dissolved in DMSO. Based on a molecular weight of 214.23 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, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

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.