Registration Dossier

Administrative data

Description of key information

The test item showed minimal reactivity towards peptides (OECD 442C, reference 7.4.1 -1) and does not activate keratinocytes (OECD 442D, reference 7.4.1 -2). An in silico assessment of the test item did not indicate a skin sensitisation potential (reference 7.4.1 -3). Therefore the test item is not considered to be a skin sensitiser.

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:
25 April 2017 - 12 July 2017
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))
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)
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system)

Synthetic peptides used:
- cysteine peptide with an amino acid sequence of Ac-RFAACAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 111016HS-MHeW0117
- lysine peptide with an amino acid sequence of Ac-RFAAKAA, JPT Peptide Technologies GmbH; > 95%; Lot. No.: 220114HSDWW0117

Controls used:
- Positive control: Cinnamic aldehyde 100 mM in acetonitrile
- Co-elution control: test item or positive control without cysteine or lysine peptide
- Reference controls: cysteine or lysine peptide in acetonitrile with and without test item; 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.

Test substance preparation:
- The test substance was prepared as a 100 mM preparation in acetonitrile.

Peptide stock solution preparation:
- 19.33 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (37.535 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
- 19.85 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 (37.083 mL) to reach a concentration of 0.667 mM.

Experimental procedure:
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.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and
phase separation prior and after HPLC analysis, if a precipitate or phase separation was observed
after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at
low speed (100 -400x g) to force precipitates to the bottom of the vial.
After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides via HPLC.


HPLC conditions:
Peptide depletion was monitored by HPLC coupled with an UV detector at A = 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 (80.1% ( v/v) trifluoroacetic acid in water) and 50% phase B (0.085% ( v/v) trifluoroacetic acid in acetonitrile) 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.

Calculation and data evaluation:
The concentration of the cysteine and lysine peptide was determined in each sample form absorbance at A = 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.
PPD = (1- (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in Refrence Control C)) * 100
Sensitising potential of the test item is predicted from the mean cysteine and lysine PPD value. The test item is considered positive to be a skin sensitiser in accordance with UN GHS "Category 1", if the mean depletion of both peptides exceeds the threshold of the respective prediction model. Negative depletion is considered as "0" when calculating the mean. Sensitizing potential might not be predictable if the test item was incubated using a concentration differently from 100 mM.
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.
By using the prediction model 2 (cysteine 1:10 prediction model) the threshold of 13.89% peptide depletion was used to support the discrimination between skin sensitisers and non-sensitisers.

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 64.37%.
Key result
Parameter:
other: combined peptide depletion [%]
Run / experiment:
mean depletion of both peptide depletions
Value:
2.82
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
0.92
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
4.73
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Precipitation and phase seperation was observed for the lysine sampls and the positive control samples. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 3:  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

1325.0602

0.1445

71.28

71.95

0.69

0.95

1261.7949

0.1375

72.65

1295.4968

0.1412

71.92

Test Item

4577.8272

0.5026

0.21

0.92

0.78

85.09

4507.0835

0.4948

1.75

4551.3691

0.4997

0.79

Table 4:  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

1766.7614

0.2083

56.65

56.79

1.06

1.87

1715.1394

0.2021

57.91

1801.3400

0.2123

55.80

Test Item

4033.5288

0.4766

0.00*

4.73

5.04

106.67

3857.3479

0.4557

4.15

3620.4099

0.4277

10.03

* value set to zero due to negative depletion

Table 5:  Categorisation of the Test Item

Predicition 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

2.82

Minimal Reactivity

no sensitizer

0.92

Minimal Reactivity

no sensitizer

Positive Control

64.37

High Reactivity

sensitizer

71.95

Moderate Reactivity

sensitizer

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the peptides. The test item might be considered as “non-sensitiser”.
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.
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.

In the present study the test item was given into water, based on the results of the pre-experiments. The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 131.17 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.

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 the samples of the test item. A slight precipitation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis.

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. Precipitation and phase separation was observed for the samples of the positive 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 precipitations and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitizing 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.

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% (2.82%). Based on the prediction model 1 (Cysteine 1:10/ Lysine 1:50 Prediction Model) 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 64.37%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 May 2017 - 28 July 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system)
Cell line used: KeratinoSensTM (Givaudan, Switzerland)

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

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

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

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

Acceptance criteria:
- The luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations.
- The average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8.
- The EC1.5 value of the positive control is within two standard deviations of the historical mean.
- The average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Evaluation of results:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.26 (experiment 1); 6.31 (experiment 2); 5.70 (experiment 3)). Therefore, the positive control fullfilled the validity criteria of the test.
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
1.24
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
1.02
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Parameter:
other: Imax
Run / experiment:
3
Value:
1.15
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table1: Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Experiment 3

Mean

SD

Solvent Control

-

100.0

100.0

100.0

100.0

0.0

Positive Control

4.00

92.5

94.3

102.4

96.4

5.2

8.00

99.0

102.2

105.5

102.2

3.2

16.00

93.9

102.4

112.2

102.8

9.2

32.00

91.4

105.8

106.2

101.1

8.4

64.00

85.0

106.7

121.1

104.3

18.2

Test Item

0.98

87.6

104.4

110.4

100.8

11.9

1.95

129.2

98.3

103.6

110.3

16.5

3.91

122.4

101.9

96.6

107.0

13.6

7.81

114.1

93.4

99.1

102.2

10.7

15.63

100.3

94.6

98.5

97.8

2.9

31.25

87.0

86.4

94.7

89.4

4.7

62.50

57.6

92.0

101.5

83.7

23.1

125.00

28.2

100.4

95.2

74.6

40.3

250.00

52.5

97.7

92.8

81.0

24.8

500.00

42.3

108.3

95.1

81.9

34.9

1000.00

35.1

102.7

94.7

77.5

36.9

2000.00

31.0

102.8

108.2

80.7

43.1

Table2: Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.07

1.21

1.03

1.10

0.10

 

8.00

1.20

1.31

1.50

1.34

0.15

 

16.00

1.31

1.34

1.39

1.35

0.04

 

32.00

1.43

1.98

2.32

1.91

0.45

*

64.00

2.71

4.17

2.90

3.26

0.79

*

Test Item

0.98

0.70

1.39

0.97

1.02

0.35

 

1.95

1.11

1.07

0.95

1.04

0.09

 

3.91

0.85

1.19

0.85

0.96

0.20

 

7.81

0.99

1.08

0.84

0.97

0.12

 

15.63

1.25

1.32

1.15

1.24

0.08

 

31.25

1.57

1.01

0.87

1.15

0.37

 

62.50

0.97

0.90

0.78

0.88

0.10

 

125.00

1.07

1.03

1.03

1.04

0.02

 

250.00

1.01

0.92

0.76

0.90

0.13

 

500.00

0.84

1.18

1.10

1.04

0.18

 

1000.00

0.87

0.96

0.91

0.91

0.05

 

2000.00

0.75

1.11

1.15

1.00

0.22

 

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

Table3: Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.19

1.24

1.06

1.16

0.09

 

8.00

1.16

1.12

1.32

1.20

0.11

 

16.00

1.37

1.55

1.59

1.50

0.12

*

32.00

2.02

2.27

2.16

2.15

0.13

*

64.00

6.51

5.74

6.68

6.31

0.50

*

Test Item

0.98

0.83

1.11

1.13

1.02

0.16

 

1.95

0.81

0.92

0.82

0.85

0.06

 

3.91

0.80

0.86

0.99

0.88

0.10

 

7.81

1.05

0.79

0.89

0.91

0.13

 

15.63

0.87

0.77

0.86

0.84

0.05

 

31.25

0.97

1.05

1.05

1.02

0.05

 

62.50

0.90

0.80

0.97

0.89

0.09

 

125.00

0.93

0.87

0.87

0.89

0.03

 

250.00

0.85

0.77

0.89

0.83

0.06

 

500.00

0.79

0.89

0.92

0.87

0.07

 

1000.00

0.87

0.88

0.57

0.77

0.17

 

2000.00

1.03

0.86

0.84

0.91

0.11

 

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

Table 4: Induction of Luciferase Activity Experiment 3

Experiment 3

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.17

1.33

1.21

1.24

0.08

 

8.00

1.35

1.34

1.08

1.25

0.15

 

16.00

1.46

1.55

1.27

1.43

0.14

 

32.00

2.11

1.98

1.64

1.91

0.24

*

64.00

6.96

5.89

4.26

5.70

1.36

*

Test Item

0.98

1.28

1.10

0.91

1.09

0.19

 

1.95

1.18

1.14

0.96

1.09

0.12

 

3.91

1.12

1.20

0.92

1.08

0.14

 

7.81

1.11

1.32

0.95

1.13

0.19

 

15.63

1.10

1.17

0.95

1.07

0.12

 

31.25

1.12

1.21

0.99

1.11

0.11

 

62.50

1.11

1.29

0.92

1.11

0.19

 

125.00

1.12

1.24

0.98

1.12

0.13

 

250.00

1.12

1.29

0.93

1.11

0.18

 

500.00

1.13

1.26

0.97

1.12

0.15

 

1000.00

1.08

1.26

0.93

1.09

0.16

 

2000.00

1.23

1.26

0.96

1.15

0.16

 

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

Table 5: Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Experiment 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.10

1.16

1.24

1.17

0.07

 

8.00

1.34

1.20

1.25

1.26

0.07

 

16.00

1.35

1.50

1.43

1.43

0.08

 

32.00

1.91

2.15

1.91

1.99

0.14

*

64.00

3.26

6.31

5.70

5.09

1.62

*

Test Item

0.98

1.02

1.02

1.09

1.05

0.04

 

1.95

1.04

0.85

1.09

1.00

0.13

 

3.91

0.96

0.88

1.08

0.98

0.10

 

7.81

0.97

0.91

1.13

1.00

0.11

 

15.63

1.24

0.84

1.07

1.05

0.20

 

31.25

1.15

1.02

1.11

1.09

0.07

 

62.50

0.88

0.89

1.11

0.96

0.13

 

125.00

1.04

0.89

1.12

1.02

0.11

 

250.00

0.90

0.83

1.11

0.95

0.14

 

500.00

1.04

0.87

1.12

1.01

0.13

 

1000.00

0.91

0.77

1.09

0.93

0.16

 

2000.00

1.00

0.91

1.15

1.02

0.12

 

Table 6: Additional Parameters

Parameter

Experiment 1

Experiment 2

Experiment 3

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

-

-

Imax

1.24

1.02

1.15

1.14

0.11

IC30[µM]

49.29

n.a.

n.a.

49.29

-

IC50[µM]

279.80

n.a.

n.a.

279.80

-

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

Table 7: Acceptance Criteria

Criterion

Range

Exp. 1

pass/fail

Exp. 2

pass/fail

Exp. 3

pass/fail

CV Solvent Control

< 20%

17.8

pass

8.4

pass

9.9

pass

No. of positive control

concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

3.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

20.35

pass

15.90

pass

18.43

pass

Induction PC at 64 µM

2.00 < x < 8.00

3.26

pass

6.31

pass

5.70

pass

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser. The data generated with this method may not be sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

In the present study the test item was dissolved in dist. water. Based on a molecular weight of 131.17 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed andluciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Within this first experiment a cytotoxic effect could be observed starting from 62.50 µM onwards. In the second experiment,no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Furthermore, no cytotoxic effect was observed within the second experiment. In the third experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Furthermore, no cytotoxic effect was observed within the third experiment. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser. The controls confirmed the validity of the study.

Endpoint:
skin sensitisation: in vitro
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
Please refer to the QMRF and QPRF files provided under the section attached justification.
Qualifier:
no guideline available
Principles of method if other than guideline:
Estimates the skin sensitising properties of chemicals using structural alert relationships.
GLP compliance:
no
Specific details on test material used for the study:
NCCCCCC(=O)O
Key result
Parameter:
other: alerts
Value:
0
Remarks on result:
no indication of skin sensitisation
Remarks:
QSAR predicted value. The substance is within the applicability domain of the model.
Interpretation of results:
other: Derek result: No alert were detected.
Conclusions:
Using Derek Nexus v5.0, no skin sensitising properties of the test item were determined. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.
Executive summary:

The skin sensitising properties were estimated using Derek Nexus v5.0. Skin sensitising properties were not detected based on the described QSAR method (Derek, 2017).

The adequacy of above prediction depends on the following conditions:

a) the (Q)SAR model is scientifically valid: the scientific validity is established according to the OECD principles for (Q)SAR validation;

b) the (Q)SAR model is applicable to the query chemical: a (Q)SAR is applicable if the query chemical falls within the defined applicability domain of the model;

c) the (Q)SAR result is reliable: a valid (Q)SAR that is applied to a chemical falling within its applicability domain provides a reliable result;

d) the (Q)SAR model is relevant for the regulatory purpose.

For assessment and justification of these 4 requirements the QMRF and QPRF files were developed and attached to this study record.

 

Description of the prediction Model

The prediction model was descripted using the harmonised template for summarising and reporting key information on (Q)SAR models. For more details please refer to the attached QSAR Model Reporting Format (QMRF) file. 

 

Assessment of estimation domain

The assessment of the estimation domain was documented in the QSAR Prediction Reporting Format file (QPRF). Please refer to the attached document for the details of the prediction and the assessment of the estimation domain.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. The protein reactivity of the test item was assessed in a Direct Peptide Reactivity Assay (DPRA). A Keratinocyte Activation Assay (LuSens) was performed to evaluate the keratinocyte activation potential. Furthermore, an in silico assessment using Derek Nexus v5.0 was carried out.

DPRA (reference 7.4.1 -1)

An in chemico direct peptide reactivity assay (DPRA) according to OECD 442C was performed. In the study the test item was given into water, based on the results of the pre-experiments. The test item was completely soluble and the resulting solution was used for further testing. Based on a molecular weight of 131.17 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.

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 the samples of the test item. A slight precipitation was observed for the samples of the positive control. Samples were not centrifuged prior to the HPLC analysis.

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. Precipitation and phase separation was observed for the samples of the positive 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 precipitations and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitizing 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.

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% (2.82%). Based on the prediction model 1 (Cysteine 1:10/ Lysine 1:50 Prediction Model) 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 64.37%.

LuSens (reference 7.4.1-2)

The keratinocyte activating potential of test substance was evaluated in a LuSens Assay (OECD 442D). The test item was dissolved in dist. water. Based on a molecular weight of 131.17 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM. Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed andluciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Within this first experiment a cytotoxic effect could be observed starting from 62.50 µM onwards. In the second experiment,no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated. Furthermore, no cytotoxic effect was observed within the second experiment. In the third experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Furthermore, no cytotoxic effect was observed within the third experiment. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser.The controls confirmed the validity of the study.

In silico assessment (reference 7.4.1-3)

For in silico assessment, Derek Nexus v5.0 was used (reference 7.4.1-3). No skin sensitising properties of the test item were estimated. The substance is within the applicability domain of the model. Thus the estimation can be regarded as accurate.

Conclusion

The test item showed minimal reactivity towards the peptides (DPRA, OECD 442C) (reference 7.4.1 -1) and does not activate keratinocytes (LuSens, OECD 442D) (reference 7.4.1 -2). In addition, an in silico assessment was performed also showing no indication of skin sensitisation (reference 7.4.1 -3). Therefore, the test item is considered not to have a skin sensitising potential (UN GHS: no category).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

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