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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:
from 2017-04-25 to 2017-07-12
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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015-02-04
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Replacement method for animals testing.

Test material

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

In vitro test system

Details on study design:
Skin sensitisation (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:
Experiment 1: 32 µM, 1.91 (SD 0.45) fold induction; 64 µM, 3.26 (SD 0.79) fold Induction
Experiment 2: 32 µM, 1.76 (SD 0.21) fold induction; 64 µM, 2.81 (SD 0.64) fold Induction

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: max. Luciferase activity induction (Imax)
Run / experiment:
test substance concentration 2000 µM
Value:
1.24
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: Cell viability (%)
Run / experiment:
test substance concentration 2000 µM
Value:
86.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: EC 1.5
Run / experiment:
test substance concentration 2000 µM
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent 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

Induction of Luciferase Activity - Overall Induction:

 

Concentration [µM]

Fold Induction

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

Positive Control

4.00

1.10

1.10

1.10

0.00

8.00

1.15

1.34

1.24

0.13

16.00

1.39

1.35

1.37

0.03

32.00

1.61

1.91

1.76

0.21

64.00

2.36

3.26

2.81

0.64

Test Substance

0.98

1.06

0.91

0.99

0.11

1.95

1.01

1.10

1.06

0.07

3.91

0.97

1.04

1.00

0.05

7.81

1.03

1.08

1.06

0.04

15.63

1.03

0.80

0.91

0.17

31.25

1.06

0.91

0.98

0.11

62.50

0.93

0.87

0.90

0.05

125.00

1.14

0.88

1.01

0.19

250.00

1.11

0.98

1.05

0.10

500.00

1.10

0.97

1.03

0.09

1000.00

1.17

0.96

1.06

0.15

2000.00

1.24

1.07

1.15

0.12

Acceptance Criteria:

Criterion

Range

Experiment 1

Pass/Fail

Experiment2

Pass/Fail

CV Solvent Control

< 20 %

7.0

Pass

17.8

Pass

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

≥1

2.0

Pass

2.0

Pass

EC1.5 PC

7 < x < 34 µM

23.95

Pass

20.35

Pass

Induction PC at 64 µM

2.99 < x < 8.00

2.36

Pass

3.26

Pass

Applicant's summary and conclusion

Interpretation of results:
other: Under the condition of this study the test substance did not induce the luciferase activity and is threrefore considered as non sensitiser.
Remarks:
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.
Conclusions:
In an in vitro ARE-Nrf2 Luciferase assay (KeratinoSens™) according to OECD Guideline 442D, the test substance did not induce luciferase activity and no EC1.5 could be determined and is threrefore considered as non sensitiser.
Executive summary:

In the present study, the skin sensitising properties of 2-isobutyl-4-vinyl-1,3-dioxolane were determined in an in vitro ARE-Nrf2 Luciferase assay (KeratinoSens™) according to OECD Guideline 442D.

Based on a molecular weight of 156.22 g/mol a stock solution of 200 mM was prepared in water. Based on the stock solution a set of eleven 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 1.24 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 86.3 %. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Furthermore, no cytotoxic effect was observed. In the second experiment, a max luciferase activity (Imax) induction of 1.1 was determined at a test item concentration of 1.95 µM. The corresponding cell viability was 119.5 %. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Within the second experiment a cytotoxic effect was observed starting from 125 µM onward. 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 substance is therefore considered as non sensitiser.