Registration Dossier

Administrative data

Description of key information

In an in chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, no peptide depletion of cysteine or lysine was observed. The test item is therefore considered as non-sensitiser (2017).

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 therefore considered as non-sensitiser (2017).

In an in vitro skin sensitisation assay (h-CLAT) according to OECD Guideline 442E, the test induced the expression of surface marker (CD54, CD86). Thus the test substance is considered as sensitiser (2017).

In conclusion, the test item is considered to be non-sensitising according to the IATA, since the results on the first two key events of skin sensitisation (direct peptide reactivity and keratinocyte activation) were negative.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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.
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.
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
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

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

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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2017-05-15 to 2017-06-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
2016-07
Deviations:
yes
Remarks:
The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
Replacement of animal testing.
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
Reasons for the Choice of THP-1 Cells:
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Due to a technical error, 1884 μg/mL instead of the calculated 1601 μg/mL was the highest tested test item concentration in the first h-CLAT run. n addition, due to strong cytotoxicity (cell viability < 50%) observed in the four highest tested test item concentrations of the first h-CLAT run, the concentrations for the second run were adjusted. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different stainings (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITClabelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures. The cells with the different antibodies or the IgG1 were gently mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added. Before using the flow cytometer, the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNAbound 7-AAD fluorescence signal.
Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

Controls for XTT and h-CLAT:
Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously.
Medium Control and Solvent Control: Culture medium
Positive Control: DNCB 2,4-dinitrochlorobenzene, CAS No.: 97-00-7 final concentration: 2 and 3 μg/mL, Purity ≥ 99 %
Solvent Control: DMSO Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2 %, Purity ≥ 99 %
Solvent Control for the Positive Control: (h-CLAT) DMSO (Dimethyl sulfoxide, CAS No. 67-68-5 in culture medium, final concentration 0.2 %, Purity ≥ 99 %
Key result
Parameter:
other: RFI (%)
Run / experiment:
CD86
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI (%)
Run / experiment:
CD54
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Table 1: Results of the first h-CLAT run

 

Concentration

(μg/mL)

RFI (%)

CD 54 Antibody

RFI (%)

CD 86 Antibody

Cell Viability

(%)

Medium

Control

-

100.0

100.0

100.0

DMSO

Control

-

100.0

100.0

100.0

Positive

Control

(DNCB)

2.0

282.1*

497.5*

72.1

3.0

370.1*

511.3*

69.8

Test Substance

526

153.1

108.7

91.1

631

206.1*

140.7

90.3

757

365.3*

268.0*

68.9

908

457.1*

820.0*

64.3

1090

531.6*

4038.7*

8.4

1308

823.5*

5779.3*

5.8

1570

626.5*

5292.0*

5.3

1884

658.2*

6648.0*

5.3

Shaded test groups: cell viability below 50 %, are excluded from the evaluation

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 150 % and CD54 200 %).

Table 2: Results of the second h-CLAT run

 

Concentration

(μg/mL)

RFI (%)

CD 54 Antibody

RFI (%)

CD 86 Antibody

Cell Viability

(%)

Medium

Control

-

100.0

100.0

100.0

DMSO

Control

-

100.0

100.0

100.0

Positive

Control

(DNCB)

2.0

284.3*

574.8

71.5

3.0

407.0*

712.6

67.2

Test Substance

310

114.5

125.7

93.6

372

128.2

122.8

93.1

446

135.5

121.8

89.7

535

128.2

80.2

87.9

642

151.8

127.7

84.4

771

239.1*

171.3*

81.3

925

282.7*

213.9*

77.8

1110

308.2*

219.8*

67.7

* RFI value of CD86 or CD54 exceeded the positive criteria (CD86 150 % and CD5 4200 %).

Interpretation of results:
other: Under the condition of this study the test substance did induce the surface marker expression (CD54, CD86) and is threrefore considered as 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 skin sensitisation assay (h-CLAT) according to OECD Guideline 442E, the test induced the expression of surface marker (CD54, CD86). Thus the test substance is in this study considered as sensitiser.
Executive summary:

In an in vitro skin sensitisation assay (h-CLAT) according to OECD Guideline 442E, the dendritic cell activation potential (third key event of a skin sensitisation AOP) was assessed. The test substance was suspended in culture medium when administered to THP-1 cells for 24 ± 0.5 hours at concentretions of 526, 631, 757, 908, 1090, 1308, 1570 and 1884 μg/mL. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2500 μg/mL up to the highest tested concentration (5000 μg/mL). The mean CV75 value of both XTT tests was calculated as 1334.1 μg/mL. However, due to a technical error, 1884 μg/mL instead of 1601 μg/mL was the highest tested test item concentration in the first h-CLAT run. Due to strong cytotoxicity (cell viability < 50 %) observed in the first h-CLAT run, the concentrations for the second run were adjusted and the following concentrations of the test item (dissolved in culture medium) were tested: 310, 372, 446, 535, 642, 771, 925 and 1110 μg/mL. Positive (DNCB), negative (medium) and solvent (DMSO) controls were valid. The RFI of CD86 and CD54 was greater than 150 % and 200 %, respectively, in at least one concentration of both independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT study.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 2017-05-22 to 2017-05-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015-02-05
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
Replacement method for animals testing.
Details on study design:
Skin sensitisation (in chemico test system) - Details on study design:
Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).
Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.
Preparation of Stability Controls and Precision Controls
Stability controls (Reference Control B) and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile.
Preparation of Positive Control Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of PI 26448 and the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either PI 26448 or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.
Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Acetonitrile solutions of PI 26448 were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either PI 26448 or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.
Incubation
The appearance of the PI 26448 and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.
Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of PI 26448 and the associated positive controls was quantified by HPLC using UV.

Key result
Parameter:
other: Peptide Depletion (%)
Run / experiment:
Cysteine
Value:
-0.516
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Negative value count as zero.
Key result
Parameter:
other: Peptide Depletion
Run / experiment:
Lysine
Value:
0.046
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
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

All analytical acceptance criteria for each peptide run were met.

Table 2: Acceptance criteria

 

Peptide

Standard Linearity

Positive control depletion (%)

Reference controls

Test item

Acceptance criteria

Cysteine

r2>0.99

60.8-100
(SD <14.9 %)

0.45-0.55 mM (CV <14.9 %)

SD <14.9 %

Lysine

r2>0.99

40.2-69.0
(SD <11.6 %)

0.45-0.55 mM (CV <11.6 %)

SD<11.6 %

Achieved results

Cysteine

r2>0.999

70.0
(SD, 0.30 %, n=3)

0.502 mM (CV 0.88 %, n=6)

SD 0.61 % (n=3)

Lysine

r2>0.999

59.8
(SD, 0.99 %, n=3)

0.511 mM (CV 1.30 %, n=6)

SD 1.07 % (n=3)

CV      Coefficient of Variation

SD      Standard deviation

Table 3: Depletion of peptide in the presence of PI 26448

 

Mean peak area of reference control (µV.sec)

Mean peak area of PI 26448 (µV.sec)

Mean Depletion of PI 26448 (%)

Cysteine

857880 (n=6)

862310 (n=3)

-0.516

Lysine

782340 (n=6)

778740 (n=3)

0.460

 

Table 4: Depletion model  

Mean of Cysteine and Lysine% depletion

Reactivity Class

DPRA Prediction

0%≤ mean% depletion ≤6.38%

No or minimal reactivity

Negative

6.38%< mean% depletion ≤22.62%

Low reactivity

Positive

22.62%< mean% depletion ≤42.47%

Moderate reactivity

42.47%< mean% depletion ≤100%

High reactivity

No co-elution peaks were observed in either the Cysteine or Lysine assays.   

According to the Depletion model, the reactivity was classed as no to minimal and the DPRA prediction is negative and PI 26448 is therefore a potential non skin sensitizer.


 

Table 5: Overall Achieved Depletion Values

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

PI 26448

-0.5161

0.0460

0.00

No to minimal

Negative

1        Negative value count as zero.

Table 6: Individual Achieved Depletion Values, Cysteine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD (%)

Positive control

256166

112.78

70.1

70.03

0.99

256241

112.81

70.1

260636

114.75

69.6

PI 264484

865406

381.32

-0.877

-0.516

0.40

862903

373.58

-0.586

858630

376.43

-0.0870

Data generated under study BY21MQ

CV    Coefficient of Variation

1                Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2                Calculated against a mean Reference Control area of 857880 µV.sec (n=6)

3                Individual and mean concentration values are outside of the range set in the historic data (Annex 1). This has not impacted on the data reported as the mean depletion value is within acceptance criteria limits.

4                Calculated against a mean Control C peak area of 854080 µV.sec (n=3)

Table 7: Individual Achieved Depletion Values, Lysine Peptide Depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion2(%)

Mean Depletion (%)

SD (%)

Positive control

305750

158.56

60.9

59.8

2.47

316448

163.91

59.6

320860

166.12

59.0

PI 264482

777456

394.53

0.624

0.460

0.25

777794

394.69

0.581

780982

396.29

0.174

Data generated under study BY21MQ

SD    Coefficient of Variation

1                Samples prepared at a concentration of 388 µg/mL (0.5 mM)

2        Calculated against a mean Reference Control area of 782340 µV.sec (n=6)

 

Interpretation of results:
other: Under the condition of this study the test substance did not induce peptide (cysteine or lysine) depletion 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 chemico direct peptide reactivity assay (DPRA) according to OECD Guideline 442C, no peptide depleition of cysteine or lysinewas observed. The test item is threrefore considered as non sensitiser.
Executive summary:

In an in chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C, the reactivity and sensitizing potential of 2-isobutyl-4-vinyl-1,3-dioxolane were determined. Solutions of the test item were successfully analysed by the validated DPRA analytical method in both cysteine and lysine containing synthetic peptides. Test item and controls were incubateted for at least 22 h at 25 °C prior to analysis. The concentration of cysteine and lysine was quantified by HPLC with UV detection. All acceptance criteria were met. Results of the positive control cinnamic aldehyde (100 mM) were valid. No cysteine or lysine depletion was observed. The overall result places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted to be a non-skin sensitizer according to the depletion model. 

Endpoint conclusion
Additional information:

The aim of the IATA is to replace in vivo tests with in vitro tests. However one in vitro method alone may be no sufficient to conclude on the absence of skin sensitisation potential of chemicals. Therefore, the integrated approach of the IATA concentrates on three key events of the skin senitising AOP (direct protein binding, keratinocyte activation and dendritic cell activation) for the assessment of skin senitising properties of chemicals.

The first key event in skin sensitisation initiation is the direct protein binding. In an In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C, the reactivity and sensitizing potential of2-isobutyl-4-vinyl-1,3-dioxolane were determined.Solutions of the test item were successfully analysed by the validated DPRA analytical method in both cysteine and lysine containing synthetic peptides. Test item and controls were incubateted for at least 22 h at 25 °C prior to analysis. The concentration of cysteine and lysine was quantified by HPLC with UV detection. All acceptance criteria were met. Results of the positive control cinnamic aldehyde (100 mM) were valid. No cysteine or lysine depletion was observed. The overall result places the test item in the reactivity class of “no or minimal reactivity” and therefore it is predicted to be a non-skin sensitizer according to the depletion model. 

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.

In an in vitro skin sensitisation assay (h-CLAT) according to OECD Guideline 442E (2017), the dendritic cell activation potential (third key event of a skin sensitisation AOP) of 2-isobutyl-4-vinyl-1,3-dioxolane was assessed. The test substance was suspended in culture medium when administered to THP-1 cells for 24 ± 0.5 hours at concentretions of 526, 631, 757, 908, 1090, 1308, 1570 and 1884 μg/mL. Cytotoxic effects were observed following incubation with the test item starting with the concentration of 1090 μg/mL up to the highest tested concentration. However, due to a technical error, 1884 μg/mL instead of 1601 μg/mL was the highest tested test item concentration in the first h-CLAT run. Due to strong cytotoxicity (cell viability < 50 %) observed in the first h-CLAT run, the concentrations for the second run were adjusted and the following concentrations of the test item (dissolved in culture medium) were tested: 310, 372, 446, 535, 642, 771, 925 and 1110 μg/mL. Positive (DNCB), negative (medium) and solvent (DMSO) controls were valid. The RFI of CD86 and CD54 was greater than 150 % and 200 %, respectively, in at least one concentration of both independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT study.

According to the results of the testing battery, the test item is not peptide reactive, does not activate keratinocytes, but activates dendritic cells. In accordance with the published evaluation scheme (Bauchet al., 2012; Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation No 1907/2006, the test item is judged not to be a skin sensitizer.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

Based on this data, the substance is not considered to be classified as skin sensitising under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.