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Description of key information

In chemico skin sensitisation:

Weight of evidence: OECD 442C. GLP study. The test item showed mean depletion of 0.30% for Lysine and 0.53% for Cysteine, i.e. an overall average of 0.42%, reflecting no or minimal reactivity, therefore a negative prediction of DPRA.

In vitro skin sensitisation:

Weight of evidence: OECD 442D. GLP study. The test item showed a Imax of 1.23 and a IC50 and a IC30 higher than 2000 µM in KeratinoSens™. Therefore, under this conditions the test item may be classified as not skin sensitizer.

Based on the available data, a negative prediction of DPRA and a EC50>2000 μM in the OECD 442D test, the 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:
weight of evidence
Study period:
January 16, 2017 - January 18, 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
Qualifier:
according to guideline
Guideline:
other: Protocol no.154 ECVAM DB ALM
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Details on the study design:
- Positive control: 100 mM cinnamaldehyde (CAS 104-55-2, Sigma Aldrich batch no. MKBT8955V) (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 appropiate buffer
- 4 references made with 0.500 mM (final concentration in the reaction mixtures) peptides solutions in acetonitrile or in the test item solvent were included in the analysis:
1) Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy,
2) 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.
3) Reference control C: prepared with acetonitrile, the positive control solvent, in order to check its influence on the peptide stability.
4) Reference control C': prepared with water, the test item solvent, in order to check its influence on the peptide stability.
- Test system: Cysteine peptide (RS Synthesis, LLC; ref. Ac RFAACAA-COOH; batch no. 160113; purity 95%) and Lysine peptide (RS Synthesis, LLC; ref. Ac RFAACAA-COOH; batch no. 160113; purity 91.2%).

Analytical method:
- Waters e2695 HPLC (Waters 2489 UV detector), cortecs column C18 2.7 µm, dimensions 2.1 x 100 mm
The HPLC column was installed and equilibrated at 30ºC with 50 % of phase A and 50% of phase B, for at least 20 minutes before use. The gradient was performed at least one time before to use the column.
- Conditions:
Mobile phase A: trifluoroacetic acid at 0.1% (v/v) in water; flow rate: 0.21 mL/min; injection volume: 7 μL; wavelength: 220 nm; sequence: 0, 10, 11, 13, 13.5 and 20 minutes.
Mobile phase B: trifluoroacetic acid at 0.085% (v/v) in acetonitrile; flow rate: 0.21 mL/min; injection volume: 7 μL; wavelength: 220 nm; sequence: 0, 10, 11, 13, 13.5 and 20 minutes.
- Method validation parameters: Linearity (R2 ≥ 0.999586) meet the criterion of approval (R2 ≥ 0.99).
A complete sequence was performed for each sample.
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 the analysis:
The analysis was programmed according to the following principles:
- The reference controls B were placed at the beginning and the end of the analysis (3 repetitions).
- The reference controls C were placed at the beginning of each repetition.
- The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.

Interpretation of the results:
- 0.00 % ≤ mean of cysteine and lysine % depletion ≤ 6.38 % = No or minimal reactivity = Negative DPRA Prediction
- 6.38 % ≤ mean of cysteine and lysine % depletion ≤ 22.62 % = Low reactivity = Positive DPRA Prediction
- 22.62 % ≤ mean of cysteine and lysine % depletion ≤ 42.47 % = Moderate reactivity = Positive DPRA Prediction
- 42.47 % ≤ mean of cysteine and lysine % depletion ≤ 100 % = High reactivity = Positive DPRA Prediction

Positive control results:
The depletion mean of cinnamaldehyde was 51.08 for Lysine and 76.99 for Cystine.
Run / experiment:
mean
Parameter:
other: % Lysine peptide depletion
Value:
0.3
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
mean
Parameter:
other: % Cystine peptide depletion
Value:
0.68
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: % depletion
Value:
0.42
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the expected DPRA prediction for the 10 proficiency substances was obtained. The resulted cysteine and lysine depletion values fall within the respective reference range for 9 out of the 10 proficiency substances for each peptide (8 out of 10 expected in the OECD guide line).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for reference control: The mean concentration of the peptides for the Reference control A and for the Reference control C met the concentration validity criteria mM: 0.500 ± 0.05 mM.
Reference control B/C the CV of the 9 controls B and C were less than 15% (%CV lysine= 1.03, %CV cystine= 1.60)
reference control C the mean
- Acceptance criteria met for positive control: yes, the depletion mean was 51.08 for Lysine (validity criteria between 40.2% and 69.4%) and 76.99 for Cystine (validity criteria between 60.8% and 100%)
- Acceptance criteria met for variability between replicate measurements: the SD of measurements were 0.28 for Lysine (SD valitidy criteria <11.6%) and 0.68 for Cysteine (SD validity criteria >14.9%)

The depletion rate was calculated for each type of peptide according to the following formula:

Depletion % = [1- (Peptide peak area with the replicate injection/mean peptide peak area with the C base control)] x 100

Table 1. Reference control results.

 

Lysine Peptide

Cysteine Peptide

 

 

Concentration (nM)

Concentration (nM)

Concentration validity criteria (mM)

Reference A

0.508

0.500

0.500±0.050

* Reference C

0.510

0.476

* Reference C’

0.490

0.472

* Reference C: positive control diluant, i.e. acetonitrile

* Reference C’: test item diluent, i.e. water

 

Table 2. Coefficient of variation between reference controls B and C.

 

CV %

CV %

CV validity criteria

Reference B/C

1.03

1.60

< 15%

 

Table 3. Positive control results.

Cinnamaldehyde

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

Repetition 1

47.43

79.53

Repetition 1

54.48

76.39

Repetition 1

51.35

75.04

Mean

51.08

76.99

Depletion validity criteria

40.2 < Depletion > 69.4

60.8 < Depletion > 100

 

Table 4. Test item results.

 

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

Mean

Depletion %

Repetition 1

0.36

0.30

 

Repetition 1

0.55

0

 

Repetition 1

0

1.29

 

Mean

0.30

0.53

0.42

SD

0.28

0.68

 

SD Validity criteria

< 11.6%

< 14.9%

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed mean depletion of 0.30% for Lysine and 0.53% for Cysteine, i.e. an overall average of 0.42%, reflecting no or minimal reactivity, therefore a negative prediction of DPRA.
Executive summary:

To support the identification of the sensitization potential of the test item the method DPRA has been performed according to OECD 442C, following GLP. The method is based on the interaction between the test item and lysine or cysteine rich peptides detected with a high performance liquid chromatography (HPLC). Samples were dissolved immediately before use. Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively. The remaining concentration of peptides is measured after 24 hours of incubating with the test item at 25ºC. It is measured with the UV detector of the HPLC system, after gradient elution, at 220 nm. The test item showed mean depletion of 0.30% for Lysine and 0.53% for Cysteine, i.e. an overall average of 0.42%, reflecting no or minimal reactivity, therefore a negative prediction of DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 13, 2017 - March 3, 2017
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
Qualifier:
according to guideline
Guideline:
other: ECVAM DB-ALM protocol 155: KeratinoSens
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
Details on the study design:
- Cell line used: KeratinoSens™ (Givaudan) cultured in maintenance medium (DMEM 1 g/l glucosa, 9.1% non-heat inactivated doetal calf serum, 0.05% geneticin - stored at 5ºC ± 3°C) at 37ºC, 5% CO2. Cells were exempt of mycoplasma.
- Passage number and level of confluence of cells: cells were used at passage 15 in repetition 1, passage 17 in repetition 2 and passage 19 in repetition 3.
- Cell counting was performed using a Malassez cell (grid of 10 x 10 tiles and a total volume of 1 mm3), cells were counted on two lines (one vertical and one horizontal) and cells suspension was adjunted at 8E04 cells/ml in seeding medium (DMEM 1 g/l glucosa, 9.1% non-heat inactivated doetal calf serum - stored at 5ºC ± 3°C). 10E04 cells/ml were distributed in 3 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. The seeded plates were incubated for 24 hours ± 1 hour at 37°C, 5% CO2.
- Luminometer used: GloMax™ (Promega)
MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -Blue formazan (CAS # 57360-69-7) linearity range at 540 nm: 0 - 2.200 units of Absorbance
- Number of repititions and replicates: the study was composed of three independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.
- Test chemical concentrations:
Test item was diluted 100-fold (10X plate) in sterile water from a 200 mM stock solution and then diluted 25-fold in a new plate (4X plate). Finally, there was a further 4-fold dilution in the seeding plate (1X plate). Positive control was diluted 100-fold in DMSO (Sigma Aldrich Batch no. SZBG2170) from a 6.4 mM stock solution and then diluted 25-fold in a new plate in treatment medium and then further diluted 4-fold in the seeding plate.
Test item was tested at 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
Negative control: 6 wells of solvent control,1% DMSO in treatment medium (DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum - stored at 5°C ± 3°C), with cells and 1 well of solvent control without cells.
Positive control: 5 concentrations of Cinnamaldehyde (Sigma Aldrich Batch no. MKBT8955V) on each culture plate. The concentration varies from 4 to 64 µM according to a geometric progression of ratio 2.

- Description of evaluation and decision criteria used:
The test item is identified as potential skin sensitizer if the 4 following conditions are met in 2 of 2 or 2 of 3 repetitions. Otherwise the keratinosens™ prediction is cosidered as negative:
1) The Imax is strictly 1.5 fold higher of the basal luciferase activity* statistically significantly to the value obtained for the negative control (as determined by a two-tailed, unpaired Student's t-test on the raw RLU values). If the Imax is exactly equal to 1.5, the test item is rated as negative and no EC1.5 value is calculated.
2) The EC1.5 value is strictly below 1000 µM (or <200 µg/ml for the test item with no defined molecular weight)
3) At the lowest concentration with a gene induction above 1.5, the cell viability must be strictly above 70% (i.e EC1.5 < IC70).
4) There is an apparent overall dose-response for luciferase induction, which is similar between the repetitions.

- Description of study acceptance criteria used:
1) Positive control
- The gene induction must be statistically significant above the threshold of 1.5 in at least one dose.
- The EC1.5 value should be between IDEA Lab historical data: mean EC1.5 value ± 2 SD and the average induction, in each repetition, for cinmamaldehyde at 64 µM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
2) Negative control: for each repetition, the coefficient of variation of the solvent controls (3 x 6 wells) must be less than 20%. If for one repetition the validity criteria is not met, a third repetition should be considered.

Positive control results:
Imax = 3.59
IC1.5 = 20.47 (geometric mean)
Key result
Run / experiment:
mean
Parameter:
other: Imax
Value:
1.23
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Imax lower than 1.5, no EC1.5 is calculated
Key result
Run / experiment:
other: geometric mean
Parameter:
other: IC50
Value:
2 000
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: geometric mean
Parameter:
other: IC30
Value:
2 000
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: recommended substances for demonstrating technical proficiency with the KeratinoSens™ test method were tested to validate the method.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
Control solvent CV is higher than the expected value (because of higher RLU values on one of the three culture plates). However, induction are calculated plate by plate and the low CVs calculated for echa plate, similar between each other, allows us to validate the repetition.Therefore, Imax and EC1.5 fulfilled validity crieteria.
- Acceptance criteria met for positive control:
Repetition 1: Imax is slightly lower than the expected value (<2) but we can observe a clear dose-response with increasing luciferase activity inductions which allows to validate the test.
Repetition 1 and 3 EC1.5 is slightly higher than the historical data value. The EC1.5 is however in the OECD validation interval (7-30 µM).
- Acceptance criteria met for variability between replicate measurements: the CV% in one repetition was >20% and a third repetition was performed.
Repetition 2: Imax and EC1.5 fulfilled validity criteria and Control solvent CV is higher than the expected value (<20%) (because of higher RLU values on one of the three culture plates). This criteria is to demonstrate the homogeneity of the plates. If CV of each plate was higher than 20% it could lead to false results but CV being <<20% there is no risk since inductions are calculated plate by plate. The low CVs of each plate allow us to validate the repetition. Nevertheless, a third repetition was conducted.
- Range of historical values if different from the ones specified in the test guideline: Mean Imax = 5.29 ± 3.12; Mean EC1.5 = 12.59 ± 5.36, Mean IC70 > 64 µM; N= 117.

Table 1. Positive control results.

Cinnamaldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Imax

Rep 1

1.03

1.11

1.26

1.56

1.94

28.67

1.94

Rep 2

1.31

1.32

1.74

2.29

6.54

11.43

6.54

Rep 3

1.20

1.13

1.29

1.62

2.30

26.16

2.30

Mean

1.18

1.18

1.43

1.82

3.59

20.47*

3.59

* geometric mean

Table 2. Negative control results.

Control solvent

CV %

control solvent

Rep 1

10.89

Rep 2

24.77

Rep 3

16.17

 

Table 3. Coefficient of variation of the negative controls.

Control solvent

CV % control solvent

Plate 1

Plate 2

Plate 3

3 plates

Rep 1

7.7%

9.4%

5.4%

10.89%

Rep 2

6.1%

11.6%

6.2%

24.77%

Rep 3

2.7%

11.9%

10.8%

16.17%

 

Table 4. Test item results.

 

VIABILITY

INDUCTION

 

IC50 µM

IC70 µM

Imax

Linear EC1.5 µM

EC1.5 Lin/Log µM

Rep 1

> 2000

> 2000

1.18

-

-

Rep 2

> 2000

> 2000

1.05

-

-

Rep 3

> 2000

> 2000

1.46

-

-

Mean

-

-

1.23

-

-

Geometric mean

> 2000

> 2000

-

-

-

 

Imax are lower than 1.5, no EC1.5 is calculated.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item showed a Imax of 1.23 and a IC50 and IC30 higher than 2000 µM in KeratinoSens™. Therefore, under this conditions the test item may be classified as not skin sensitiser.
Executive summary:

The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, following GLP. A skin sensitiser is a substance that leads to an allergic response following skin contact. This allergic response includes inflammatory responses as well as gene expression associated with specific cell signalling pathways in the keratinocytes. One of the target genes is AKR1C2, and the test method KeratinoSens™ is based on the evaluation of the activation of this gene in transformed keratinocytes by monitoring the induction of the luciferase gene fused to AKR1C2. After 48 h of contact between the test item with KeratinoSens™ monolayer, the induction of the luciferase is quantified. A positive control and negative control were run in parallel, as well as a cytotoxicity assay. Three repetitions of the assay was performed on different days. The test item showed an Imax of 1.23 and a IC50 and a IC30 higher than 2000 µM in KeratinoSens™. Therefore, under these conditions the test item may be classified as not skin sensitiser.

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

In chemico skin sensitisation:

Weight of evidence: To support the identification of the sensitization potential of the test item the method DPRA has been performed according to OECD 442C, following GLP. The method is based on the interaction between the test item and lysine or cysteine rich peptides detected with a high performance liquid chromatography (HPLC). Samples were dissolved immediately before use. Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively. The remaining concentration of peptides is measured after 24 hours of incubating with the test item at 25ºC. It is measured with the UV detector of the HPLC system, after gradient elution, at 220 nm. The test item showed mean depletion of 0.30% for Lysine and 0.53% for Cysteine, i.e. an overall average of 0.42%, reflecting no or minimal reactivity, therefore a negative prediction of DPRA.

In vitro skin sensitisation:

Weight of evidence: The test method KeratinoSens™ has been performed as part of an integrated approach to support the identification of the sensitization potential of the test item according to OECD 442D, following GLP. A skin sensitizer is a substance that leads to an allergic response following skin contact. This allergic response includes inflammatory responses as well as gene expression associated with specific cell signalling pathways in the keratinocytes. One of the target genes is AKR1C2, and the test method KeratinoSens™ is based on the evaluation of the activation of this gene in transformed keratinocytes by monitoring the induction of the luciferase gene fused to AKR1C2. After 48 h of contact between the test item with KeratinoSens™ monolayer, the induction of the luciferase is quantified. A positive control and negative control were run in parallel, as well as a cytotoxicity assay. Three repetitions of the assay was performed on different days. The test item showed an Imax of 1.23 and a IC50 and a IC30 higher than 2000 µM in KeratinoSens™. Therefore, under these conditions the test item may be classified as not skin sensitizer.

Based on the available data, a negative prediction of DPRA and a EC50>2000 μM in the OECD 442D test, the test item may be classified as not skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information (a negative prediction of DPRA and a IC50>2000 μM in the OECD 442D test), the substance is not classified as skin sensitizer according to CLP Regulation (EC) no.1272/2008.