Registration Dossier

Administrative data

Description of key information

Skin sensitisation

Based on the results of an in chemico/in vitro test strategy the test item is peptide reactive (DPRA test, OECD TG 442C) and does activate keratinocytes ( ARE-Nrf2 luciferase test, OECD TG 422D). Therefore, the substance is 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:
2017-11-29 to 2018-01-20
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 04, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system):
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C.

Preparation of the cysteine or lysine-containing peptides:
18.87 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (36.69 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
20.56 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (39.09 mL) to reach a concentration of 0.667 mM.
All peptides used for this study were stored at -80 °C and protected from light. Peptides were thawed only immediately prior to use.

Preparation of the test item:
The test item was freshly prepared immediately prior to use. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared.

Positive control, reference controls and co-elution control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides.
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.

Incubation of the test item with the cysteine and lysine peptide:
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.

Preparation of the HPLC standard calibration curve:
A standard calibration curve was generated for both, the cysteine and the lysine peptide. Peptide standards were prepared in a solution of 20% acetonitrile : 80% buffer (v/v) using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide (dilution buffer (DB)). A serial dilution of the peptide stock solution (0.667 mM) using the respective DB was performed.

HPLC Preparation and Analysis:
Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected. HPLC analysis for the cysteine and lysine peptide was performed concurrently (if two HPLC systems were available) or on separate days. If analysis was conducted on separate days all test chemical solutions were freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample started 22 to 26 hours after the test chemical was mixed with the peptide solution. The HPLC run sequence was set up in order to keep the HPLC analysis time less than 30 hours.

Data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves derived from the standard solutions.
By using the prediction model 1 (cysteine 1:10 / lysine 1:50 prediction model) the threshold of 6.38% average peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers. Application of the prediction model for assigning a test item to a reactivity class (i.e. low, moderate or high reactivity) may perhaps prove useful to inform potency assessment within the framework of an IATA. In the framework of an IATA the test substance may be considered as non-sensitiser to skin in accordance with UN GHS “No Category”, if the mean depletion of both peptides is below 6.38%.

Acceptance Criteria:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.
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 65.38%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
98.62
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
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. Precipitation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.
For the 100 mM stock solution of the test item 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. Precipitation was observed for the samples of the test item (including the respective co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the co-elution control of the positive control sample. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

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

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

17.8700

0.5340

4254.6284

0.5340

STD2

9.0120

0.2670

2161.7087

0.2670

STD3

4.5180

0.1335

1051.8925

0.1335

STD4

2.2520

0.0667

525.2503

0.0667

STD5

1.1010

0.0334

260.5259

0.0334

STD6

0.5300

0.0167

130.8325

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine 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

4.4470

0.1325

74.33

74.15

0.18

0.25

4.4770

0.1334

74.15

4.5100

0.1344

73.96

Test Item

0.3270

0.0096

98.07

98.62

1.20

1.22

0.3740

0.0110

97.80

0.0000

-0.0002

100.00

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

1675.9452

0.2100

57.93

56.62

1.13

2.00

1753.5808

0.2197

55.98

1754.5223

0.2198

55.95

Test Item

4064.3474

0.5088

0.00

0.00

0.00

--

4043.3840

0.5062

0.00

4041.7593

0.5060

0.00

A minor/major peak in the co-elution control of the test item was observed at the retention time of the lysine peptide (peak area = 78.755). Therefore, co-elution of test item and peptide occurred and the given peak areas do not properly reflect the amount of lysine peptide present in the test item samples. The values can only be considered as an estimation of the peptide depletion and will not be used for evaluation. Since co-elution with the lysine peptide was observed, prediction model 2 should be considered.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

Reactivity Class

DPRA Prediction

0.00% PPD 13.89%

No or minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2
(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

-

-

-

98.62

High Reactivity

positive

Positive Control

65.38

High Reactivity

positive

74.15

Moderate Reactivity

positive

Interpretation of results:
other: peptide depletion positive
Remarks:
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Conclusions:
In this study under the given conditions the test item showed high reactivity towards the cysteine peptide. Even though a precipitate was observed a positive result was obtained.
Executive summary:

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.

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. Precipitation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item, 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. Precipitation was observed for the samples of the test item (including the respective co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the co-elution control of the positive control sample.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Co-elution of the test item with the lysine peptide was observed. The peak area determined in the co-elution controls corresponds to

0.0102 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was > 13.89% (98.62%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 2 the test item can be considered as 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 65.38%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-11-14 to 2018-03-20
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:
February 04, 2015
Deviations:
no
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
Details on study design:
Details on study design:
The test described in the OECD Guideline 442D is proposed to address the second key event in the Adverse Outcome Pathway (AOP) underlying skin sensitisation. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens™ cell line by measuring the induction of an ARE dependent gene product, the luciferase gene.

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 2 in experiment 1; P 3 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2. For test item exposure, cells were cultured in medium for test item exposure.

Preparation of the test and control items:
All test item solutions were freshly prepared immediately prior to use. The test item was dissolved in dimethyl sulfoxide (DMSO). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial. A stable suspension was formed when diluted 1:100 in cell culture medium. 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.
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.
Cinnamic aldehyde (CA) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 μM – 64 μM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Dose Groups
Negative Control: 1% (v/v) DMSO in test item exposure medium
Positive Control: CA: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM
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 plate in every independent run.

Experimental Procedure:
A cell suspension of 8 × 10E4 cells/mL in assay medium was prepared. 125 μL of the cell suspension corresponding to 1 × 10E4 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 μL 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 (Gibco Life Science; Lot No.: 1909266). 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 1.000 ms before assessing the luciferase activity for 2.000 ms. 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 overnight or over the weekend. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Data evaluation:
The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item was calculated.
The EC1.5 was calculated by linear extrapolation, and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.
The IC50 and IC30 were calculated by linear extrapolation according and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
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 EC1.5 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 EC1.5 determining concentration.

Prediction Model
The test item is considered positive in accordance with UN GHS “Category 1” 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

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
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.35 (experiment 1); 4.70 (experiment 2)).
Key result
Parameter:
other: luciferase activity
Run / experiment:
Experiment 1 / 15.63 µM
Value:
5.03
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
Experiment 1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: luciferase activity
Run / experiment:
Experiment 2/ 3.91 µM
Value:
3.88
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
Experiment 2
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

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

87.2

87.0

87.1

0.1

8.00

77.0

66.4

71.7

7.5

16.00

78.6

76.3

77.4

1.6

32.00

61.8

59.3

60.6

1.8

64.00

56.8

48.8

52.8

5.7

Test Item

0.98

112.2

107.4

109.8

3.4

1.95

115.2

106.3

110.7

6.3

3.91

108.7

114.5

111.6

4.1

7.81

91.4

75.2

83.3

11.5

15.63

22.8

19.4

21.1

2.4

31.25

22.0

17.8

19.9

3.0

62.50

22.1

14.2

18.2

5.6

125.00

18.8

13.4

16.1

3.8

250.00

14.3

12.7

13.5

1.1

500.00

13.6

13.1

13.4

0.3

1000.00

12.7

12.0

12.4

0.5

2000.00

15.7

12.5

14.1

2.3

 

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

1.21

1.22

1.29

1.24

0.04

 

8.00

1.19

1.23

1.27

1.23

0.04

 

16.00

1.64

1.70

1.51

1.62

0.10

*

32.00

2.12

2.21

1.83

2.05

0.19

*

64.00

3.84

6.24

2.97

4.35

1.69

*

Test Item

0.98

2.64

3.47

3.10

3.07

0.42

*

1.95

3.39

4.01

3.89

3.77

0.33

*

3.91

3.86

4.25

4.49

4.20

0.32

*

7.81

4.17

5.04

5.23

4.81

0.57

*

15.63

4.41

5.13

5.55

5.03

0.57

*

31.25

4.56

5.20

5.33

5.03

0.41

*

62.50

4.00

4.76

4.57

4.44

0.39

*

125.00

3.55

3.53

3.41

3.50

0.07

*

250.00

2.28

2.77

2.55

2.53

0.25

*

500.00

2.77

3.68

3.23

3.23

0.45

*

1000.00

3.61

4.60

3.79

4.00

0.52

*

2000.00

2.95

3.96

3.12

3.34

0.54

*

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

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

1.16

1.18

1.10

1.15

0.04

 

8.00

1.16

1.10

1.09

1.12

0.04

 

16.00

1.65

1.69

1.49

1.61

0.11

*

32.00

2.23

1.88

1.93

2.01

0.19

*

64.00

4.84

4.51

4.75

4.70

0.17

*

Test Item

0.98

4.04

3.92

2.80

3.59

0.68

*

1.95

3.80

3.71

3.60

3.70

0.10

*

3.91

3.72

3.98

3.92

3.88

0.14

*

7.81

4.06

3.89

3.60

3.85

0.23

*

15.63

3.76

3.32

3.77

3.62

0.26

*

31.25

3.35

3.47

3.60

3.47

0.13

*

62.50

2.63

2.68

2.82

2.71

0.10

*

125.00

1.76

1.91

1.86

1.84

0.08

*

250.00

1.67

1.82

1.73

1.74

0.07

*

500.00

1.78

1.76

1.80

1.78

0.02

*

1000.00

2.12

2.24

2.32

2.23

0.10

*

2000.00

1.18

1.22

1.27

1.22

0.05

 

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

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

1.24

1.15

1.19

0.07

 

8.00

1.23

1.12

1.17

0.08

 

16.00

1.62

1.61

1.61

0.01

*

32.00

2.05

2.01

2.03

0.03

*

64.00

4.35

4.70

4.52

0.25

*

Test Item

0.98

3.07

3.59

3.33

0.37

*

1.95

3.77

3.70

3.74

0.05

*

3.91

4.20

3.88

4.04

0.23

*

7.81

4.81

3.85

4.33

0.68

*

15.63

5.03

3.62

4.32

1.00

*

31.25

5.03

3.47

4.25

1.10

 

62.50

4.44

2.71

3.58

1.23

 

125.00

3.50

1.84

2.67

1.17

 

250.00

2.53

1.74

2.14

0.56

 

500.00

3.23

1.78

2.50

1.02

 

1000.00

4.00

2.23

3.11

1.25

 

2000.00

3.34

1.22

2.28

1.50

 

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a.

-

-

Imax

5.03

3.88

4.45

0.82

IC30[µM]

10.25

8.54

9.39

1.21

IC50[µM]

12.53

11.34

11.93

0.84

IC70 [µM]

14.81

14.15

14.48

0.47

n.a.: not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment2

pass/fail

CV Solvent Control

< 20%

8.6

pass

9.6

pass

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

≥ 1

3.0

pass

3.0

pass

EC1.5 PC

7 < x < 34 µM

13.60

pass

14.23

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.35

pass

4.70

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.3

3.3

41

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

≥ 1

2.3

0.6

41

EC1.5 PC

7 < x < 34 µM

20.4

6.7

41

Induction PC at 64 µM

2.00 < x < 8.00

3.3

1.1

41

Interpretation of results:
other: activation of keratinocytes
Remarks:
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
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 as sensitiser.
Executive summary:

In a study according OECD TG 442D the test item was dissolved in DMSO. Based on a molecular weight of 306.38 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 and a stable suspension was formed. 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 5.03 was determined at a test item concentration of 15.63 µM and 31.25 µM. The corresponding cell viability was 22.8% and 22.0%. The lowest tested concentration with a significant luciferase induction >1.5 (2.53) was found to be 250.00 µM. The corresponding cell viability was <70% (14.3%). The calculation of the EC1.5 value was not possible because each induction value was above the threshold.

In the second experiment, a max luciferase activity (Imax) induction of 3.88 was determined at a test item concentration of 3.91 µM. The corresponding cell viability was 114.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.74) was found to be 250.00 µM. The corresponding cell viability was <70% (12.7%). The calculation of the EC1.5 value was not possible because each induction value was above the threshold.

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 sensitizer. The controls confirmed the validity of the study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

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

DPRA test

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.

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. Precipitation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item, 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. Precipitation was observed for the samples of the test item (including the respective co-elution control). Samples were centrifuged prior to the HPLC analysis. Phase separation was observed for the co-elution control of the positive control sample.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Co-elution of the test item with the lysine peptide was observed. The peak area determined in the co-elution controls corresponds to

0.0102 mM of lysine peptide. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C.

The 100 mM stock solution of the test item showed high reactivity towards the synthetic peptides. The mean depletion of the cysteine peptide was > 13.89% (98.62%). Even though a precipitate was observed a positive result can still be used. Based on the prediction model 2 the test item can be considered as 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 65.38%.

KeratinoSens test

In a study according OECD TG 442D the test item was dissolved in DMSO. Based on a molecular weight of 306.38 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 and a stable suspension was formed. 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 5.03 was determined at a test item concentration of 15.63 µM and 31.25 µM. The corresponding cell viability was 22.8% and 22.0%. The lowest tested concentration with a significant luciferase induction >1.5 (2.53) was found to be 250.00 µM. The corresponding cell viability was <70% (14.3%). The calculation of the EC1.5 value was not possible because each induction value was above the threshold.

In the second experiment, a max luciferase activity (Imax) induction of 3.88 was determined at a test item concentration of 3.91 µM. The corresponding cell viability was 114.5%. The lowest tested concentration with a significant luciferase induction >1.5 (1.74) was found to be 250.00 µM. The corresponding cell viability was <70% (12.7%). The calculation of the EC1.5 value was not possible because each induction value was above the threshold.

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 sensitizer.

The controls confirmed the validity of the study.

Conclusion

Based on the results of an in chemico/in vitro test strategy the test item is peptide reactive (DPRA test, OECD TG 442C) and does activate keratinocytes ( ARE-Nrf2 luciferase test, OECD TG 422D). Therefore, the substance is predicted to be a skin sensitizer.

Justification for classification or non-classification

Classification, Labeling, 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 the test item is classified as skin sensitising Cat.1 (H317) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fourteenth time in Regulation (EU) No 2020/217.