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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:
key study
Study period:
05 May 2017 - 28 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
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)
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

Details on study design:
Skin sensitisation (In vitro test system)
Cell line used: KeratinoSensTM (Givaudan, Switzerland)

Technical material and conditions:
- Maintenance Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS + 1 % Geneticin (final concentration: 500 µg/mL)
- Assay Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 10 % FBS
- Test Item Exposure Medium: D-MEM with 1.0 g/L D-glucose and Na-Pyruvate + 1 % FBS
- Luciferace reagent: Luciferase Assay Substrate (Promega, Cat. No.: E1501)
- Assay Buffer: Luciferase Assay Buffer (Promega, Cat. No.: E1501)
- Lysisbuffer: Luciferase Cell Culture Lysis (Promega Cat. No.: E1531)
- MTT Solution: MTT stock solution: 5 mg/mL MTT in DPBS
- SDS solution: 10% (wlv) SDS in dist. water
- DPBS solution: DPBS solution (without Ca2+/Mg2+)

Controls used:
- Vehicle control: DMSO: 1% (vlv) in test item exposure medium
- Positive control: Cinnamic aldehyde in DMSO, 4 µM, 8 µM, 16 µM; 32 µM; 64 µM
- Blank control: vehicle control without cells

Test procedure:
Each concentration step of the test item (twelve concentrations) and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per plate in every independent run. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a plate reader. Cell viability was determined by a MTT assay. The test substance was incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 10% SDS solution the plate was incubated at 37 °C ± 1 °C and 5% CO2 overnight. The OD was measured at A = 600 nm.

Data Analysis:
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consists of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run should be performed.

Acceptance criteria:
- 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.

Evaluation of results:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

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 (3.26 (experiment 1); 6.31 (experiment 2); 5.70 (experiment 3)). Therefore, the positive control fullfilled the validity criteria of the test.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
1.24
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
1.02
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: Imax
Run / experiment:
3
Value:
1.15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
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 OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

Table1: Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Experiment 3

Mean

SD

Solvent Control

-

100.0

100.0

100.0

100.0

0.0

Positive Control

4.00

92.5

94.3

102.4

96.4

5.2

8.00

99.0

102.2

105.5

102.2

3.2

16.00

93.9

102.4

112.2

102.8

9.2

32.00

91.4

105.8

106.2

101.1

8.4

64.00

85.0

106.7

121.1

104.3

18.2

Test Item

0.98

87.6

104.4

110.4

100.8

11.9

1.95

129.2

98.3

103.6

110.3

16.5

3.91

122.4

101.9

96.6

107.0

13.6

7.81

114.1

93.4

99.1

102.2

10.7

15.63

100.3

94.6

98.5

97.8

2.9

31.25

87.0

86.4

94.7

89.4

4.7

62.50

57.6

92.0

101.5

83.7

23.1

125.00

28.2

100.4

95.2

74.6

40.3

250.00

52.5

97.7

92.8

81.0

24.8

500.00

42.3

108.3

95.1

81.9

34.9

1000.00

35.1

102.7

94.7

77.5

36.9

2000.00

31.0

102.8

108.2

80.7

43.1

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

1.07

1.21

1.03

1.10

0.10

 

8.00

1.20

1.31

1.50

1.34

0.15

 

16.00

1.31

1.34

1.39

1.35

0.04

 

32.00

1.43

1.98

2.32

1.91

0.45

*

64.00

2.71

4.17

2.90

3.26

0.79

*

Test Item

0.98

0.70

1.39

0.97

1.02

0.35

 

1.95

1.11

1.07

0.95

1.04

0.09

 

3.91

0.85

1.19

0.85

0.96

0.20

 

7.81

0.99

1.08

0.84

0.97

0.12

 

15.63

1.25

1.32

1.15

1.24

0.08

 

31.25

1.57

1.01

0.87

1.15

0.37

 

62.50

0.97

0.90

0.78

0.88

0.10

 

125.00

1.07

1.03

1.03

1.04

0.02

 

250.00

1.01

0.92

0.76

0.90

0.13

 

500.00

0.84

1.18

1.10

1.04

0.18

 

1000.00

0.87

0.96

0.91

0.91

0.05

 

2000.00

0.75

1.11

1.15

1.00

0.22

 

* = 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

1.19

1.24

1.06

1.16

0.09

 

8.00

1.16

1.12

1.32

1.20

0.11

 

16.00

1.37

1.55

1.59

1.50

0.12

*

32.00

2.02

2.27

2.16

2.15

0.13

*

64.00

6.51

5.74

6.68

6.31

0.50

*

Test Item

0.98

0.83

1.11

1.13

1.02

0.16

 

1.95

0.81

0.92

0.82

0.85

0.06

 

3.91

0.80

0.86

0.99

0.88

0.10

 

7.81

1.05

0.79

0.89

0.91

0.13

 

15.63

0.87

0.77

0.86

0.84

0.05

 

31.25

0.97

1.05

1.05

1.02

0.05

 

62.50

0.90

0.80

0.97

0.89

0.09

 

125.00

0.93

0.87

0.87

0.89

0.03

 

250.00

0.85

0.77

0.89

0.83

0.06

 

500.00

0.79

0.89

0.92

0.87

0.07

 

1000.00

0.87

0.88

0.57

0.77

0.17

 

2000.00

1.03

0.86

0.84

0.91

0.11

 

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

Table 4: Induction of Luciferase Activity Experiment 3

Experiment 3

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

1.17

1.33

1.21

1.24

0.08

 

8.00

1.35

1.34

1.08

1.25

0.15

 

16.00

1.46

1.55

1.27

1.43

0.14

 

32.00

2.11

1.98

1.64

1.91

0.24

*

64.00

6.96

5.89

4.26

5.70

1.36

*

Test Item

0.98

1.28

1.10

0.91

1.09

0.19

 

1.95

1.18

1.14

0.96

1.09

0.12

 

3.91

1.12

1.20

0.92

1.08

0.14

 

7.81

1.11

1.32

0.95

1.13

0.19

 

15.63

1.10

1.17

0.95

1.07

0.12

 

31.25

1.12

1.21

0.99

1.11

0.11

 

62.50

1.11

1.29

0.92

1.11

0.19

 

125.00

1.12

1.24

0.98

1.12

0.13

 

250.00

1.12

1.29

0.93

1.11

0.18

 

500.00

1.13

1.26

0.97

1.12

0.15

 

1000.00

1.08

1.26

0.93

1.09

0.16

 

2000.00

1.23

1.26

0.96

1.15

0.16

 

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

Table 5: Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Experiment 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.10

1.16

1.24

1.17

0.07

 

8.00

1.34

1.20

1.25

1.26

0.07

 

16.00

1.35

1.50

1.43

1.43

0.08

 

32.00

1.91

2.15

1.91

1.99

0.14

*

64.00

3.26

6.31

5.70

5.09

1.62

*

Test Item

0.98

1.02

1.02

1.09

1.05

0.04

 

1.95

1.04

0.85

1.09

1.00

0.13

 

3.91

0.96

0.88

1.08

0.98

0.10

 

7.81

0.97

0.91

1.13

1.00

0.11

 

15.63

1.24

0.84

1.07

1.05

0.20

 

31.25

1.15

1.02

1.11

1.09

0.07

 

62.50

0.88

0.89

1.11

0.96

0.13

 

125.00

1.04

0.89

1.12

1.02

0.11

 

250.00

0.90

0.83

1.11

0.95

0.14

 

500.00

1.04

0.87

1.12

1.01

0.13

 

1000.00

0.91

0.77

1.09

0.93

0.16

 

2000.00

1.00

0.91

1.15

1.02

0.12

 

Table 6: Additional Parameters

Parameter

Experiment 1

Experiment 2

Experiment 3

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

-

-

Imax

1.24

1.02

1.15

1.14

0.11

IC30[µM]

49.29

n.a.

n.a.

49.29

-

IC50[µM]

279.80

n.a.

n.a.

279.80

-

EC1.5: interpolated concentration for a 1.5 fold luciferase induction, Imax: maximal induction factor of luciferase activity compared to the solvent (negative) control measured at any test chemical concentration, IC30: concentration effecting a reduction of cellular viability by 30%, IC50: concentration effecting a reduction of cellular viability by 50%, n.a. = not applicable

Table 7: Acceptance Criteria

Criterion

Range

Exp. 1

pass/fail

Exp. 2

pass/fail

Exp. 3

pass/fail

CV Solvent Control

< 20%

17.8

pass

8.4

pass

9.9

pass

No. of positive control

concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

3.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

20.35

pass

15.90

pass

18.43

pass

Induction PC at 64 µM

2.00 < x < 8.00

3.26

pass

6.31

pass

5.70

pass

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. The data generated with this method may not be 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 item was dissolved in dist. water. Based on a molecular weight of 131.17 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 andluciferase 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 EC1.5value could be calculated. Within this first experiment a cytotoxic effect could be observed starting from 62.50 µM onwards. In the second experiment,no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Furthermore, no cytotoxic effect was observed within the second experiment. In the third experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Furthermore, no cytotoxic effect was observed within the third experiment. 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 controls confirmed the validity of the study.