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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-02-10 to 2017-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on 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 (2.83 in experiment 1; 2.67 in experiment 2).

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
13.17
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
208.2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Value:
2.41
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
9.01
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
143.9
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Value:
2.56
Vehicle 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

Table1: 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

111.4

105.9

7.8

8.00

102.2

109.0

105.6

4.9

16.00

116.3

109.7

113.0

4.6

32.00

117.7

115.0

116.3

1.8

64.00

129.4

123.2

126.3

4.4

Test Item

0.98

143.4

109.1

126.2

24.3

1.95

153.7

113.5

133.6

28.4

3.91

202.9

139.1

171.0

45.1

7.81

206.8

136.7

171.7

49.6

15.63

203.6

141.3

172.5

44.1

31.25

208.2

137.5

172.8

50.0

62.50

205.9

140.7

173.3

46.1

125.00

217.7

143.9

180.8

52.2

250.00

208.3

143.4

175.9

45.9

500.00

210.1

144.3

177.2

46.6

1000.00

191.9

138.6

165.2

37.7

2000.00

198.0

129.2

163.6

48.7

 

Table2: 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

1.20

1.20

1.22

1.21

0.01

 

8.00

1.20

1.23

1.29

1.24

0.05

 

16.00

1.30

1.25

1.56

1.37

0.17

 

32.00

1.86

1.58

2.28

1.90

0.35

*

64.00

2.86

2.81

2.81

2.83

0.03

*

Test Item

0.98

1.16

1.14

1.12

1.14

0.02

 

1.95

1.20

0.98

1.22

1.14

0.14

 

3.91

2.38

2.73

2.99

2.70

0.31

*

7.81

11.91

8.86

11.80

10.86

1.73

*

15.63

13.08

10.89

12.28

12.08

1.11

*

31.25

14.54

11.62

13.36

13.17

1.47

*

62.50

12.97

11.71

13.08

12.58

0.76

*

125.00

14.70

10.24

13.51

12.82

2.31

*

250.00

11.87

9.37

11.35

10.86

1.32

*

500.00

10.34

9.06

11.72

10.37

1.33

*

1000.00

9.14

7.65

9.33

8.71

0.92

*

2000.00

4.97

5.52

7.25

5.91

1.19

*

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

Table3: 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

1.00

1.19

1.44

1.21

0.22

 

8.00

1.32

1.22

1.28

1.27

0.05

 

16.00

1.34

1.62

1.34

1.43

0.16

 

32.00

1.61

2.16

1.56

1.78

0.33

 

64.00

3.06

2.85

2.11

2.67

0.50

*

Test Item

0.98

1.07

1.06

1.16

1.10

0.05

 

1.95

1.24

1.12

1.16

1.17

0.06

 

3.91

1.91

2.74

2.01

2.22

0.45

*

7.81

7.92

8.46

7.20

7.86

0.63

*

15.63

8.14

8.75

7.57

8.15

0.59

*

31.25

8.72

9.63

8.39

8.92

0.64

*

62.50

9.36

9.19

8.14

8.90

0.66

*

125.00

9.41

9.58

8.05

9.01

0.84

*

250.00

8.17

7.68

6.62

7.49

0.79

*

500.00

8.26

6.90

6.52

7.23

0.92

*

1000.00

6.67

4.98

4.93

5.53

0.99

*

2000.00

4.98

3.57

5.50

4.68

1.00

*

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

Table4: 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

1.21

1.21

1.21

0.00

 

8.00

1.24

1.27

1.26

0.02

 

16.00

1.37

1.43

1.40

0.05

 

32.00

1.90

1.78

1.84

0.09

*

64.00

2.83

2.67

2.75

0.11

*

Test Item

0.98

1.14

1.10

1.12

0.03

 

1.95

1.14

1.17

1.15

0.03

 

3.91

2.70

2.22

2.46

0.34

*

7.81

10.86

7.86

9.36

2.12

*

15.63

12.08

8.15

10.12

2.78

*

31.25

13.17

8.92

11.05

3.01

*

62.50

12.58

8.90

10.74

2.61

*

125.00

12.82

9.01

10.92

2.69

*

250.00

10.86

7.49

9.18

2.38

*

500.00

10.37

7.23

8.80

2.23

*

1000.00

8.71

5.53

7.12

2.25

 

2000.00

5.91

4.68

5.30

0.87

*

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

Applicant's summary and conclusion

Interpretation of results:
other: The result must be considered in the context of an integrated approach.
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. Therefore, the test item can be considered to be a sensitiser in accordance with UN GHS category 1.
Executive summary:

In the present study the test material was dissolved in DMSO.

Based on a molecular weight of 395 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 13.17 was determined at a test item concentration of 31.25 µM. The corresponding cell viability was 208.2%. The lowest tested concentration with a significant luciferase induction >1.5 (2.70) was found to be 3.91 µM. The corresponding cell viability was >70% (202.9%). The calculated EC1.5was < 1000 µM (2.41 µM). Microscopically, signs of precipitates were observed at the 4 highest test item concentrations.This may be the reason for the decrease in luciferase activity at the highest test item concentrations.

In the second experiment, a max luciferase activity (Imax) induction of 9.01 was determined at a test item concentration of 125.0 µM. The corresponding cell viability was 143.9%. The lowest tested concentration with a significant luciferase induction >1.5 (2.22) was found to be 3.91 µM. The corresponding cell viability was >70% (139.1%). The calculated EC1.5was < 1000 µM (2.56 µM). Microscopically, signs of precipitates were observed at the 4 highest test item concentrations. This may be the reason for the decrease in luciferase activity at the highest test item concentrations.

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.