Registration Dossier

Administrative data

Description of key information

Based on the results of an in chemico/in vitro test strategy the test item showed minimal peptide reactivity (DPRA test, OECD TG 442C) and does not activate keratinocytes ( ARE-Nrf2 luciferase test, OECD TG 422D). Therefore, the substance is not predicted to be a skin sensitizer (reference 7.4.1-1 and -2).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2019-01-14 to 2019-01-18
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:
February 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
January 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.
Key result
Parameter:
other: mean peptide depletion of both peptides [%]
Value:
0.12
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS: All acceptance criteria are fulfiled
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Table 1: Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Conc. [nM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.7360

4.8660

4.6910

0.1551

0.1593

0.1537

69.56

68.73

69.85

69.38

0.58

0.84

Test item

15.5650

15.5010

15.5250

0.5050

0.5029

0.5037

0.00

0.39

0.23

0.21

0.19

94.37

  

Table 2: Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.4950

4.6220

4.7210

0.1644

0.1690

0.1726

67.20

66.27

65.55

66.34

0.83

1.25

Test Item

13.7810

13.7280

13.6890

0.5029

0.5010

0.4996

0.00

0.00

0.10

0.03

0.06

173.21

 

Table 3: Categorization of the Test Item

Prediction Model

Prediction Model 1

Prediction model 2 8Cysteine Peptide 7 Test Item Ratio: 1:10)

Test substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

0.12

Minimal Reactivity

Negative

0.21

Minimal Reactivity

Negative

Positive Control

67.86

High Reactivity

Positive

69.38

Moderate Reactivity

Positive

 

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no-co-elution was observed, prediction model 1 based on the combination of cysteine and lysine depletion should be considered.

Interpretation of results:
other: no peptide binding
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:
The test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay according to OECD guideline 442C. The test item is considered as "non-sensitiser".
Executive summary:

In this study the test item was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 73.1 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.
All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.
No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C acetonitrile).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.12%). Based on the prediction model 1 the test item can be considered as non-sensitiser.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.
In conclusion the test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay and the test item is considered as "non-sensitiser".

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-12-19 to 2019-02-25
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:
June 2018
Deviations:
no
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
Version / remarks:
March 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
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.

Cell line:
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 12 in experiment 1; P 02 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.

Test Item Exposure Medium
Dulbecco's Modified Eagle Medium (GlutaMAX™) (Gibco Life Science, Cat. No.: 21885-025, Lot No.: 2007923) with 1.0 g/L D-glucose and 1 mM Na-Pyruvate. The medium was supplemented with 1% fetal bovine calf serum (Biochrom, Cat. No.: S 0615, Lot No.: 0298G).

Dose Groups
1. Negative Control: 1% (v/v) DMSO in test item exposure medium
2. Positive Control: CA: 4 uM, 8 uM, 16 uM; 32 uM; 64 uM
3. Test Item: 12 concentrations of the test item

Each concentration step of the test item and the positive control was assessed in three replicates in every independent run. The negative control was assessed using six replicates per 96-well plate in every independent run.

Preparation of the Test Item
All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO, CAS No.: 67-68-5, purity >99%; AppliChem; Lot No.: 0001336139). A stock solution of 200 mM was prepared by pre-weighing the test material into a suitable tube.
Vortex mixing was used to aid solubilisation.
Based on the stock solution a set of twelve master solutions in 100% solvent was prepared. The stock solution of the test item was diluted eleven times using a constant dilution factor of 1:2. Then the 100x concentrated master solutions were further diluted 1:25 in cell culture medium resulting in a 4% share of the solvent.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Experimental Procedure
A cell suspension of 8 x 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 x 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel, cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 mL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity
After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1 sec. before assessing the luciferase activity for 2 sec. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 over the weekend. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 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 consisted of three replicates for every concentration step of the test item and the positive control. In case of discordant results a third independent run is performed.
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student's t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
The lowest concentration with >1.5 fold luciferase activity induction was the value determining the EC15 value. It was checked in each case whether this value was below the IC30 value, indicating that there was less than 30% reduction on cellular viability at the EC15 determining concentration.

Prediction Model:
A KeratinoSens™ prediction is considered positive of the following conditions will be 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 an 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. A negative result for test items with a log Kow >7 has to be interpreted with care due to the applicability.

Acceptance Criteria:
The test meets acceptance criteria of:
- 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 postive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the postive control is within 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.
Positive control results:
See table 4 in the section "Any other information on results incl. tables"
Key result
Parameter:
other: Induction of Luciferase Activity (Imax)
Run / experiment:
1
Value:
1.15
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: see details in secton 'Any other information on results incl. tables'
Key result
Parameter:
other: Induction of Luciferase Activity (Imax)
Run / experiment:
2
Value:
1.17
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: see details in secton 'Any other information on results incl. tables'

Table 1: 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

8.00

16.00

32.00

64.00

95.8

106.7

94.1

78.5

74.8

88.1

97.1

94.3

95.0

94.4

92.0

101.9

94.2

86.8

84.6

5.4

6.8

0.2

11.7

13.8

Test item

0.98

1.95

3.91

7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

2000.00

90.0

101.8

97.1

94.6

94.1

86.1

99.6

98.2

90.9

91.3

82.9

70.6

100.9

100.0

95.1

95.5

88.0

88.8

93.1

87.5

89.0

87.9

86.6

92.3

95.5

100.9

96.1

95.0

91.0

87.5

96.4

92.9

90.0

89.6

84.8

81.5

7.7

1.3

1.4

0.7

4.3

1.9

4.6

7.5

1.4

2.4

2.7

15.4

  

Table 2: 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

1.28

1.17

1.25

1.23

0.06

 

8.00

1.35

1.37

1.32

1.35

0.03

 

16.00

1.66

1.44

1.63

1.58

0.12

*

32.00

1.93

2.24

2.07

2.08

0.16

*

64.00

3.35

3.82

3.18

3.45

0.33

*

Test Item

0.98

1.17

1.14

1.14

1.15

0.02

 

1.95

0.88

0.95

0.74

0.86

0.11

 

3.91

0.79

0.99

0.65

0.81

0.17

 

7.81

0.92

0.94

1.05

0.97

0.07

 

15.63

0.79

0.94

0.90

0.88

0.08

 

31.25

1.01

1.04

0.78

0.94

0.14

 

62.50

1.13

1.11

0.86

1.04

0.15

 

125.00

0.90

0.93

0.81

0.88

0.06

 

250.00

1.08

1.11

0.87

1.02

0.13

 

500.00

1.11

1.14

0.74

1.00

0.22

 

1000.00

1.24

1.01

0.93

1.06

0.16

 

2000.00

1.10

1.19

1.03

1.11

0.08

 

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

 

  Table 3: 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

1.21

1.26

1.13

1.20

0.06

 

8.00

1.35

1.38

1.20

1.31

0.10

 

16.00

1.46

1.62

1.43

1.50

0.10

*

32.00

2.50

1.95

1.93

2.13

0.32

*

64.00

4.20

4.36

4.66

4.41

0.23

*

Test Item

0.98

0.98

1.19

1.06

1.08

0.11

 

1.95

0.92

1.08

1.04

1.01

0.08

 

3.91

0.82

1.24

0.94

1.00

0.22

 

7.81

1.01

1.05

1.01

1.03

0.02

 

15.63

0.90

1.11

0.95

0.99

0.11

 

31.25

0.97

1.11

0.96

1.01

0.08

 

62.50

1.07

1.10

1.07

1.08

0.02

 

125.00

1.01

1.08

1.02

1.04

0.04

 

250.00

1.00

1.09

0.98

1.03

0.06

 

500.00

1.03

1.15

1.19

1.12

0.08

 

1000.00

1.00

1.08

0.98

1.02

0.05

 

2000.00

0.95

1.42

1.15

1.17

0.24

 

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

 

Table 4: 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

1.23

1.20

1.21

0.02

 

8.00

1.35

1.31

1.33

0.03

 

16.00

1.58

1.50

1.54

0.05

*

32.00

2.08

2.13

2.10

0.03

*

64.00

3.45

4.41

3.93

0.67

*

Test Item

0.98

1.15

1.08

1.11

0.05

 

1.95

0.86

1.01

0.94

0.11

 

3.91

0.81

1.00

0.90

0.13

 

7.81

0.97

1.03

1.00

0.04

 

15.63

0.88

0.99

0.93

0.08

 

31.25

0.94

1.01

0.98

0.05

 

62.50

1.04

1.08

1.06

0.03

 

125.00

0.88

1.04

0.96

0.11

 

250.00

1.02

1.03

1.02

0.01

 

500.00

1.00

1.12

1.06

0.09

 

1000.00

1.06

1.02

1.04

0.03

 

2000.00

1.11

1.17

1.14

0.05

 

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

Table 5: Additional Parameter

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

n.a.

Imax

1.15

1.17

1.16

0.01

IC30[µM]

n.a.

n.a.

n.a.

n.a.

IC50[µM]

n.a.

n.a.

n.a.

n.a.

 n.a.: not applicable

 

Table 6: Acceptance Criteria

Criterion

Range

Experiment 1

Pass/fail

Experiment 2

Pass/fail

CV Solvent Control

<20%

11.9

pass

5.1

pass

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

≥1

3.0

pass

3.0

pass

EC1.5 PC

± 2 x SD of historical mean

13.25

pass

15.84

pass

Induction PC at 64 µM

2.00 < x < 8.00

3.45

pass

4.41

pass

  

Table 7: 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

 

 

Interpretation of results:
other: no activation of keratinocytes
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:
The test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs according to OECD guideline 442D. Therefore, the test item can be considered as non-sensitiser.
Executive summary:

The test item was examined for sensitising properties in the ARE-Nrf2 luciferase test method according to OECD TG 442D. 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.
In the present study the test item was dissolved in DMSO. Based on a molecular weight of 73.1 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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. 

DPRA:

A study was conducted according to OECD TG 442C in order to evaluate the reactivity of the test item towards cysteine (Cys-) and lysine (Lys-) containing peptides. In this study the test item, was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 73.1 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.
All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.
For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged prior to the HPLC analysis.
Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant.
No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C acetonitrile).
The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.12%). Based on the prediction model 1 the test item can be considered as non-sensitiser.
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.86%.
In conclusion the test item showed minimal reactivity towards both peptides in this Direct Peptide Reactivity Assay and the test item is considered as "non-sensitiser".

ARE-Nrf2 luciferase test method

The test item was examined for sensitising properties in the ARE-Nrf2 luciferase test method according to OECD TG 442D. 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.
In the present study the test item was dissolved in DMSO. Based on a molecular weight of 73.1 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.


Conclusion:

Based on the results from the in chemico and in vitro studies and considering the Adverse Outcome Pathway (AOP) the test item is considered to ne not sensitising.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

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 available data on skin sensitisation, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the twelfth time in Regulation (EU) 2019/521.