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Description of key information

Key study: OECD Guideine 442C. GLP study. The test item showed mean depletion of 0.43% for Lysine and 0.04% for Cysteine, i.e. an overall average of 0.23% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Key study: OECD Guideline 442D. GLP study. The luciferase induction (Imax) was lower than 1.5 and no EC1.5 was determined. Thus, under the retained experimental conditions test item may be classified as not skin sensitizer.

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:
key study
Study period:
18 December 2018 - 21 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Details on the study design:
Test system:
Cysteine peptide: Supplier RS synthesis, LLC (Ac RFAACAA-COOH, Batch no. 170622, purity of 94.40%)
Lysiine peptide: Supplier RS synthesis, LLC (Ac-RFAAKAA-COOH, Batch no. 170705, purity of 92.93%)

Controls:
Positive control: 100 mM cinnamaldehyde (CAS N°: 104-55-2), purity ≥ 95% (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
Co-elution control: 100 mM test item in the appropriate buffer (cf § 7.5) (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
4 references, made with 0.667 mM peptides solutions in acetonitrile or in the test item solvent, are included in each set of analysis: (i.e. 0.500 mM as final concentration in the reaction mixtures):
-Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy
-Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time
-Reference control C: prepared with the positive control solvent.in order to check its influence on the peptide stability and with acetonitrile
-Reference control C’: prepared with purified water, the test item solvent, in order to check its influence on the peptide stability

Study course:
The test item was prepared at 100 mM in purified water and the positive control was prepared at 100 mM in acetonitrile. They were incubated in excess with the peptides at 1:10 and 1:50 ratio for cysteine and lysine peptides respectively. Each sample was tested 3 times from 3 independent solutions. The vials were capped and mixed carefully avoiding bubbling, then placed in the dark, in HPLC system sampler at 25°C ± 2.5°C. HPLC analysis started 24 hours ± 2 hours after addition of peptides.

Immediately upon addition of the test item solution to the peptide solution, just prior the beginning of the HPLC analysis and at the end of the analysis, samples vials were checked. Solution was cloudy right after preparation and at the end of analysis but no precipitation neither phase shift was observed prior the beginning and at the end of the analysis which shows the stability of the solution

The HPLC column was installed and equilibrated at 30°C with 50% of phase A and 50% of phase B, for at least 20 minutes before use. After the equilibration phase, 7 µl of each sample are automatically injected and the HPLC analysis was performed with a flow of 0.40 ml per minute at 0, 13.5, 14 and 19 minutes. The column was re-equilibrated to initial conditions (90% Phase A and 10% of phase B) at least 4 minutes between each injection.

Sequence of analysis:
The analysis was programmed according to the following principles:
-The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
-The reference controls C were placed at the beginning of each repetition.
-TThe standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.

Lysine and cysteine analysis were conducted on separate day and test item was freshly prepared for both assays on each day. The analysis was timed to assure that the injection of the first sample starts 22 to 26 hours after the test item was mixed with the peptide solution. The HPLC analysis time was less than 30 hours.


Positive control results:
See below.
Key result
Run / experiment:
other: 1
Parameter:
other: Depletion in Lysine peptide %
Value:
1.28
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Depletion in Lysine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Depletion in Lysine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: Depletion in Cysteine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Depletion in Cysteine peptide %
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 3
Parameter:
other: Depletion in Cysteine peptide %
Value:
0.11
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Mean depletion in Lysine peptide % was 0.43.
Mean depletion in Cysteine peptide % was 0.04.
Mean depletion % was 0.23.

OTHER EFFECTS:
- Visible damage on test system: No coelution of the test item with the peptides has been highlighted.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
Reference A: Lysine peptide concentration (mM) = 0.509, Cysteine peptide concentration (mM) = 0.502
Reference C (positive control diluent, acetonitrile): Lysine peptide concentration (mM) = 0.503, Cysteine peptide concentration (mM) = 0.498
Reference C' (test item diluent, purified water): Lysine peptide concentration (mM) = 0.480, Cysteine peptide concentration (mM) = 0.487
Validty criteria = 0.500 ± 0.050

Reference B/C: Lysine peptide CV% = 0.51, Cysteine peptide CV% = 1.76
Validity criteria < 15%

- Acceptance criteria met for positive control:
Mean depletion in Lysine peptide % was 51.17 ± 1.29 (validity criteria 40.2-69.4)
Mean depletion in Cysteine peptide % was 68.83 ± 0.55 (validity criteria 60.8-100)

Test item results:

 

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

 

 

Repetition 1

1.28

0

 

 

Repetition 2

0

0

 

Mean Depletion %

Repetition 3

0

0.11

 

Mean

0.43

0.04

 

0.23

SD

(Standard Deviation)

0.74

0.06

 

 

SD Validity criteria

< 11.6%

< 14.9%

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed mean depletion of 0.43% for Lysine and 0.04% for Cysteine, i.e. an overall average of 0.23% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA.

Executive summary:

An in-chemico skin sensitization test was performed according to the OECD Guideline 442 (GLP study). The principle is based on chemical reactivity of the test item with proteins. The interaction between the molecule and lysine or cysteine rich peptides was detected with a high performance liquid chromatography (HPLC). The remaining concentration of peptides was measured after 24 hours of incubation with the test item at 25°C. It was measured with the UV detector of the HPLC system, after gradient elution, at 220 nm. The depletion rates of lysine and cysteine peptides were then used to distinguish the skin sensitizer and non-sensitizer. The test item showed mean depletion of 0.43% for Lysine and 0.04% for Cysteine, i.e. an overall average of 0.23% reflecting no or a minimal reactivity and thus a negative prediction for the DPRA. All the validity criteria were fullfilled.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2018 - 26 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Details on the study design:
Test series (two independent repetitions):
Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM (3 replicates)
Negative control (treatment culture medium, 1% DMSO and 1% non-heat inactivated foetal calf serum): 6 wells of solvent control (1% DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate (3 replicates).
Positive control (cinnamaldehyde): 5 concentrations of cinnamaldehyde on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2 (3 replicates).

Test system:
Source of cells (KeratinoSens™): Givaudan.
Cells are cultured in maintenance medium at 37°C, 5% CO2. Cells are exempt of mycoplasma. Cells should be 80-90% confluent, and care is taken to ensure that cells are never grown to full confluence: for a start on Monday, cells are trypsinized and seeded according to a ratio 1/6 and, for a start on Tuesday, according to a ratio 1/12. Cells were used at passage 19 in repetition 1 and passage 21 in repetition 2.

Test protocol:
Cells seeding (first day)
After removal the culture medium from the culture flask, the cell layer was rinsed with PBS, 0.05% EDTA, Ca2 + and Mg² + free to remove all traces of serum. The solution of trypsin-EDTA was added and left for a few minutes at 37°C, 5% CO2 until the detachment of cells. The action of trypsin was stopped by addition of maintenance medium. Cell concentration was determined on Malassez cell. Cells suspension was adjusted to a density of 8.104 cells/ml in seeding medium. 125 µl of the cell suspension at 8.104 cells/ml (i.e. 104 cells per well) were distributed in three white cell culture plates (96 wells) for the induction measurement and two transparent cell culture plates (96 wells) to assess the cytotoxicity. In order to ensure a good homogeneity of seeding, cells suspension was regularly mixed all along the seeding.

Contact between the cells and the test and reference items (second day):
In the 5 seeded plates, the medium was aspirated and replaced with 150 µl of treatment medium. Then the 4 X plate was replicated 5 times: 50 µl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37°C, 5% CO2).

Luciferase activity (day 4)
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate (luciferine + ATP + lysing agent) were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

Cell viability assessment with MTT method (day 4)
After 48 hours, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then, 225 µl of staining solution diluted at 0.6 mg/ml in treatment medium (from the 5 mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37°C, 5% CO2). After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbances were measured at 540 nm

Positive control results:
The gene induction is statistically significant above the threshold of 1.5 in more than one dose,
The EC1.5 is between IDEA Lab historical data: mean EC1.5 value ± 2 SD.
The average induction, in each repetition, for cinnamaldehyde at 64 µM is between 2 and 8.
There is clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
Key result
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
1.33
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
1.04
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In both repetition, Imax is lower than 1.5, no EC1.5 is determined.

OTHER EFFECTS:
- Visible damage on test system: Not stated.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Run 1: CV% = 17.4; Run 2: CV% = 5.8 (validity criteria CV < 20%)
- Acceptance criteria met for positive control:
Imax: Run 1 = 5.73; Run 2 = 3.68 (mean = 4.71) (validity criteria > 1.5)
Average induciton at 64 µM: Run 1 = 5.73; Run 2 = 3.68 (mean = 4.71) (validity criteria 2-8)
- Range of historical values if different from the ones specified in the test guideline:
The EC1.5 is between IDEA Lab historical data: mean EC1.5 value ± 2 SD

Test item:

VIABILITY

INDUCTION

IC30
 µM

IC50
 µM

Imax

Linear EC1.5
 µM

EC1.5 Lin/Log
 µM

Rep 1

153.98

181.70

1.33

-

-

Rep 2

143.60

173.90

1.04

-

-

Mean

 -

-

1.19

 -

 -

Geometric mean

148.70

177.76

 -

-

-

Positive control:

Cinnamaldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Imax

Rep 1

1.32

1.46

2.03

3.06

5.73

8.53

5.73

Rep 2

1.16

1.41

1.84

2.35

3.68

9.63

3.68

Mean

1.24

1.44

1.93

2.71

4.71

9.06*

4.71

Solvent control:

 Control solvent

CV %
 control solvent

Rep 1

17.4

Rep 2

5.8

Interpretation of results:
GHS criteria not met
Conclusions:
Under the retained experimental conditions test item may be classified as not skin sensitizer (Imax < 1.5).
Executive summary:

An in-vitro skin sensitization test was performed according to the OECD Guideline 442 D (GLP study). The test consists in evaluating the activation of AKR1C2 in transformed keratinocytes (KeratinoSens™), by monitoring the induction of the luciferase gene fused to AKR1C2. The luciferase produced by the cells complexes with luciferin which, in the presence of ATP, produces light measured in relative light units (RLU). After contact between the test tiem with a KeratinoSens™ monolayer, the induction of the luciferase was quantified. In parallel, the cytotoxicity was measured, in order to exclude a false positive generated by a skin irritation. The study was composed of two independent repetitions. For each repetition the test item and the reference items (positive control and solvent control) were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. In both repetition, the luciferase induction (Imax) was lower than 1.5 and no EC1.5 was determined. Thus, under the retained experimental conditions test item may be classified as not skin sensitizer. All validity criteria were fullfilled.

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

In chemico/in vitro studies assesing two key events of skin sensitisation were performed:

- Direct Peptide Reactivity Assay (DPRA) – B.59 / OECD TG 442C, provides information on the molecular initiation event of skin sensitization, i.e. protein binding of low molecular weight substances using synthetic heptapeptides containing cysteine and lysine amino acids. Based on the depletion of the synthetic heptapeptides by HPLC using UV detection (0.23%), the substance was determined not to be a skin sensitizer.

- ARE-Nrf2 Luciferase Test Method (KeratinoSensTM) – B.60 / OECD TG 442D, measures the induction of the luciferase gene as an indicator of the activity of the Keap1 -Nrf2 -ARE pathweay, which is considered to be a major regulator of cyto-protective responses to electrophile and oxidative stress by controlling the expression of detoxification, antioxidant and stress response enzymes and proteins. The substance was determined not to be a skin sensitizer since the luciferase induction (Imax) was lower than 1.5.

Regarding the third key event of skin sensitization, i.e. Human Cell Line Activation Test (h-CLAT) - OECD TG 442E, the study is not technically feasible since the substance has a log Pow >3.5 and this kind of substances tend to produce a higher rate of false negative results.

Based on a weight of evidence analysis and since the two feasible key events of the AOP were determined to be negative, the substance is concluded not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on available data (OECD 442C and OECD 442D negatives), the substance is not classified for skin sensitization according to the CLP Regulation (EC) no. 1272/2008.