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Description of key information

Skin sensitisation (in chemico): First key event (molecular initiating event): Weight of evidence. Test method according to the OECD Guideline 442C with GLP. The test item showed a mean depletion of lysine and cysteine peptides of 1.40%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Skin sensitisation (in vitro): Second key event (Activation of keratinocytes): Weight of evidence. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test substance may be classified as not skin sensitizer using the KeratinoSensTM test method.

Skin sensitisation (in vitro): Third key event (activation of dendritic cells): Data waiving (study scientifically not necessary). This test was not performed because the available method (OECD TG 442E) addressing this key event tends to produce false negatice results with substances with a Log Kow greater than 3.5, as it is the case.

Skin sensitisation (in vivo): Weight of evidence. Test method according to the OECD 442B Guideline with GLP. Under the experimental conditions the test item does not have to be classified as a skin sensitizer using the LLNA assay. The Stimulation Index (SI) calculated by individual approach were 1.31, 1.33 and 1.13 at concentrations of test item 25%, 50% and 100%, respectively.

Weight of evidence approach: As it was not possible to perform the in vitro/in chemico tests addressing all the three key events required in column 1 of REACH Annex VII, it was decided to conduct an in vivo LLNA assay according to column 2 of REACH Annex VII. Based on weight of evidence approach, the test substance does not need to be classified as 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:
28 May 2018 - 31 May 2018
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:
Details on study design:

TEST ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was water.

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, LLC; Ref. Ac RFAACAA-COOH; batch no 170622; purity 94.40%)
Lysine peptide (supplier RS Synthesis, LLC; Ref. Ac-RFAAKAA-COOH; batch no 170705; purity 92.93%)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: 100 mM test item in the appropriate buffer (i.e. 5 or 25 mM as final concentration in the reaction mixtures, respectively with cysteine and lysine).
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy.
Reference control C: prepared with acetonitrile, the positive control solvent in order to check its influence on the peptide stability.
Reference control C': prepared with water, the test item solvent, in order to check its influence on the peptide stability.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 50 µl was distributed in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution with water.
Positive control solution: 38.2 µl of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution with acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 12.7 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 25.4 ml of phosphate buffer (100 mM; pH 7.5 ± 0.05).
Lysine solution: 15 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 29 ml of ammonium acetate buffer (100 mM; pH 10.2 ± 0.05).

TEST SOLUTIONS
Samples were dissolved immediately before use (100 mM positive control solution was kept for the 2 runs).
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
All the replicates were prepared with the same peptide stock solutions.

1 ml of each solution was prepared according to the following quantities:
Cysteine test solution (0.5 mM Peptide, 5 mM Sample): 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of sample or solvent for Reference controls + 200 µl of acetonitrile.
Lysine test solution (0.5 mM Peptide, 25 mM Sample): 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of sample or solvent for Reference controls.

The vials were capped and mixed carefully avoiding bubbling, then placed in the 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, sample vials were checked.

Replicates: Each sample was tested 3 times from 3 independent solutions

HPLC ANALYSIS
-Apparatus: Waters e2695 HPLC; Waters 2489 UV detector; Xbridge Peptide BEH C18 column 3.5 µm ; dimensions 2.1 x 100 mm.
-Calibration curve: Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
-Equilibration of the column: at 30°C with 50% phase A (0.1% (v/v) trifluoroacetic acid in water) and 50% phase B (0.085% (v/v) trifluoroacetic acid in acetonitrile) for at least 20 minutes before running.
-Volume injected: 7 µl of each sample
-Flow rate: 0.40 ml/min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Duration of re-equilibration to the initial conditions between injections: at least 4 min.
-Analysis sequence:
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.
The 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.

DEVIATIONS FROM OECD GUIDELINE: No.
According to the guideline, the set-up parameters for HPLC analysis were adjusted to guarantee an appropriate elution and integration of the cysteine and lysine peptides. Proficiency substances recommended in the OECD guideline were performed in these conditions.
Positive control results:
The depletion mean rate was 68.05% for cysteine peptide and 55.13% for lysine peptide.
Key result
Run / experiment:
other: mean of 3 repetitions
Parameter:
other: %depletion in lysine peptide
Value:
0.39
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Mean of 3 repetitions
Parameter:
other: %depletion in cysteine peptide
Value:
2.42
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Mean of lysine and cysteine peptides
Parameter:
other: mean %depletion in peptides
Value:
1.4
Vehicle controls validity:
valid
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: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.496 mM for lysine and 0.516 mM for cysteine which are equal to 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B/C was 0.66% for lysine and 0.76% for cysteine which is lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.492 mM for lysine and 0.516 mM for cysteine which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the controls B/C was 0.66% for lysine and 0.76% for cysteine which is lower than 15%.
- Acceptance criteria met for reference control C': yes, the mean concentration of peptide was 0.471 mM for lysine and 0.508 mM for cysteine which are equal to 0.50± 0.05 mM.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 1.11% for cysteine and 2.37% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 68.05% for cysteine peptide and 55.13% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 0.41% for cysteine and 0.34% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.


Table 3: Positive control

Cinnamaldehyde

Depletion in Lysine Peptide %

Depletion in Cysteine Peptide %

Repetition 1

56.61

68.41

Repetition 2

56.39

68.93

Repetition 3

52.39

66.81

Mean Depletion

55.13

68.05

Depletion
Validity criteria

40.2 < Depletion < 69.4

60.8 < Depletion < 100

SD
(standard deviation)

2.37

1.11

SD Validity criteria

< 11.6 %

< 14.9 %

Table 4: Reference controls

 

Lysine Peptide

Cysteine Peptide

 

Concentration (mM)

Concentration (mM)

Concentration validity criteria (mM)

Reference A

0.496

0.516

0.500 ± 0.050

* Reference C

0.492

0.516

** Reference C’

0.471

0.508

 

 

 

 

 

CV %

CV %

CV validity criteria

Reference B/C

0.66

0.76

< 15 %

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

**Reference C': Test item diluent, i.e. = water

Table 5: Test item

 

Depletion in Lysine Peptide %

Depletion in Cysteine Peptide %

 

Repetition 1

0.63

2.02

Repetition 2

0.54

2.84

Mean Depletion %

Repetition 3

0

2.38

Mean depletion

0.39

2.42

1.40

SD
(Standard Deviation)

0.34

0.41

 

SD Validity criteria

< 11.6%

< 14.9%

No co-elution occurred of the test item neither with lysine nor with cysteine peptides

Interpretation of results:
other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.
Conclusions:
The test substance was found negative in the DPRA skin sensitisation test.

Executive summary:

A DPRA skin sensitization test was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item and Cinnamaldehyde (positive control) were prepared at 100 mM in water and acetonitrile as solvents, respectively. Reference controls A, B, C (acetonitrile) and C' (water) were prepared in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C ± 2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested 3 times from 3 independent solutions. All validity criteria were fulfilled. The test item showed mean depletion of 0.39% for lysine and 2.42% for cysteine, i.e. an overall average of 1.40%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 June 2018 - 06 July 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:
Details on study design:

REAGENTS AND MEDIA
-DMSO (Supplier ref.41640, Sigma Aldrich; Batch no SZBG2170H; purity 99.9%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 1943444)
-Non-heat inactivated foetal calf serum (Supplier ref. 10270098, Fisher Bioblock; Batch no 42Q3174K)
-Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin.
-Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum.
-Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum.
-Staining solution: 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS (phosphate buffered saline). Prepared extemporaneously and used within the day.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in maintenance medium.
-Passage number: cells were used at passage 20 in repetition 1 and passage 24 in repetition 2.

CONTROLS
-Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
-Negative (solvent) control: DMEM 1 g/l glucose, 1% DMSO, 1% non-heat inactivated foetal calf serum.

CELLS SEEDING.
-Culture plates: 5; 3 white cell culture plates (96 wells) for luminescence reading (induction measurement) + 2 transparent cell culture plates (96 wells) for absorbance reading (cytotoxicity).
-Cells suspension: 125 µl at 8x10e4 cells/ml in seeding medium were distributed in the culture plates.
-Cell density: 10e4 cells per well.
-Incubation: the seeded plates were incubated 24 hours ± 1 hour at 37ºC, 5% CO2

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Test item stock solution: The test item was diluted in DMSO at 200 mM.
Positive control stock solution: The positive control was prepared at 200 mM in DMSO then diluted to the final concentration of 6.4 mM.

1) Preparation of the 100 X plate: A 100-fold concentrated dilutions series was prepared in 96-well plate.
-Test item: The test item was placed in one of the rows B to F. 100 µl of DMSO were distributed from columns 1 to 11. 200 mM stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Positive control: 100 µl of DMSO were distributed in row G from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl of the column 11 in the column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Negative control: 100 µl of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.

2) Preparation of the 4 X dilution plate: The 100 X plate was diluted 25 fold in a new plate (4 X).

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
-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 (37ºC, 5% CO2).

REPLICATES: The study was composed of two 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.

LUCIFERASE ACTIVITY.
-Apparatus: Luminometer: GloMax™ (Promega)
-Validity of luminometer: validated according to the procedure described in Annex 3 of the OECD 442D guideline.
-Luciferase substrate: luciferine + ATP + lysing agent. Bright Glo™ Luciferase Assay System (Promega).
-Procedure: After 48 hours incubation, the medium was aspirated and each well was gently washed once with 200 µl of PBS. Then 100 µl of luciferase substrate 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.

CYTOTOXICITY ASSESSMENT.
-Apparatus: MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance.
-Procedure: After 48 hours incubation, 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 5mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37ºC, 5% CO2). After this contact time, the staining solution was eliminated and the cells were treated with 200 µl of 10% SDS (sodium dodecyl sulfate) a weekend in the dark (37°C, 5% CO2)(repetition 1) and one night in the dark (37ºC, 5% CO2) (repetition 2). After a 10 minutes homogenization, the absorbance was measured at 540 nm.

DEVIATIONS FROM OECD GUIDELINE: No
Positive control results:
Repetition 1: The maximal average induction of luciferase activity (Imax) was 3.80 at a concentration of 64 µM. The mean value EC1.5 was 11.52 µM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 2.90 at a concentration of 64 µM. The mean value EC1.5 was 10.48 µM.
Key result
Run / experiment:
other: Repetition 1
Parameter:
other: Imax
Value:
1.34
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Repetition 2
Parameter:
other: Imax
Value:
1.17
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Repetition 1
Parameter:
other: IC70 (µM)
Remarks:
(concentration at which cell viability is 70%)
Value:
82.86
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Run / experiment:
other: Repetition 2
Parameter:
other: IC70 (µM)
Remarks:
(concentration at which cell viability is 70%)
Value:
82.56
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: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442D were performed, obtaining values that fall within the respective reference range for 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 9.8% and for repetition 2 was 11.3% which are less than 20%.
- Acceptance criteria met for positive control: Yes, the luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The average induction values at 64 µM were 3.80 and 2.90 in each repetition which are between 2 and 8. The EC1.5 values were 11.52 and 10.48 µM for each repetition which are within 2 SD of the historical mean of the testing facility and between 7 µM and 30 µM based on the OECD validation dataset.




Table 1: Controls

Cinnamaldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Imax

Rep 1

1.19

1.28

1.78

2.35

3.80

11.52

3.80

Rep 2

1.19

1.38

1.78

2.26

2.90

10.48

2.90

Mean

1.19

1.33

1.78

2.31

3.35

10.99*

3.35

*geometric mean

Control solvent

CV %
 control solvent

Rep 1

9.8

Rep 2

11.3

Table 2: Test item

VIABILITY

INDUCTION

ID-18/04213

IC70
 µM

Imax

Linear EC1.5
 µM

EC1.5 Lin/Log
 µM

Rep 1

82.86

1.34

-

-

Rep 2

82.56

1.17

-

-

Mean

 -

1.25

-

-

Geometric mean

82.71

 -

-

-

Imax is less than 1.5, the EC1.5 is not determined.

Table 3: Test item mean viability percentage

Concentration µM

0,98

1,95

3,91

7,81

15,6

31,3

63

125

250

500

1000

2000

Rep 1

96,60

105,87

107,64

110,77

108,68

108,89

100,56

6,77

0,21

-0,52

0,52

0,42

Rep 2

99,77

108,15

112,83

113,25

108,58

109,22

103,48

-0,85

0,53

-0,74

-0,11

0,64

Viability

98,2

107,0

110,2

112,0

108,6

109,1

102,0

3,0

0,4

-0,6

0,2

0,5

Table 4: Test item mean induction

Concentration

µM

0,98

1,95

3,91

7,81

15,63

31,25

62,50

125

250

500

1000

2000

Rep 1

1,05

1,12

1,16

1,34

1,25

1,19

1,28

0,64

0,01

0,00

0,00

0,00

Rep 2

0,93

1,10

1,07

1,08

1,17

1,09

0,99

0,00

0,00

0,00

0,00

0,00

Induction

0,99

1,11

1,12

1,21

1,21

1,14

1,14

0,32

0,00

0,00

0,00

0,00

SD

0,08

0,01

0,06

0,18

0,05

0,07

0,21

0,45

0,00

0,00

0,00

0,00

Table 5: Student t-test

Rep 1

0,579

0,141

0,060

0,004

0,076

0,062

0,023

0,161

0,000

0,000

0,000

0,000

Rep 2

0,232

0,414

0,182

0,441

0,044

0,334

0,897

0,000

0,000

0,000

0,000

0,000
Interpretation of results:
other: KeratinoSensTM test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442D.
Conclusions:
Under the experimental conditions the test substance may be classified as not skin sensitizer using the KeratinoSensTM test method.
Executive summary:

The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours ± 1 hour at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 2 plates (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 1 hour at 37ºC, 5% CO2. 2 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. For the test item, the calculated Imax values were lower than 1.5, thus a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
According to column 2 of REACH Annex VII, the available in vitro test method addressing the key event activation of dentritic cells (h-CLAT method, OECD TG 442E) is not applicable for this substance because it tends to produce false negative results with substances with a Log Kow greater than 3.5, as it is the case.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19 September 2018 – 02 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.51 (Skin sensitisation. local lymph node assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA:J
Remarks:
CBA/JRj
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941Le Genest Saint Isle).
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 10-11 weeks old.
- Weight at study initiation: average weight 21.6-24.1 g (treated groups and control group)
- Housing: the animals were housed individually (to avoid any test item absorption by oral route) in a suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Teklad Global 16% Protein Rodent Diet (ENVIGO 2016) ad libitum
- Water: tap water from public distribution system ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas - Eurofins (FRANCE)
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC-25ºC.
- Humidity (%): 30%-70%.
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light (7.00 to 19.00) / 12 h darkness
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
100%, 50% and 25%.
No. of animals per dose:
4
Details on study design:
PRE-SCREEN TESTS: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item undiluted (100%) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1 to day 6. The bodyweight of the mice were recorded on Day 1 (prior to dosing) and on Day 6.

- Compound solubility: Not specified.
- Irritation: No sign of excessive irritation was noted in the animal treated at the tested concentration of 100%.
- Systemic toxicity: No mortality and no signs of systemic toxicity were noted.
- Ear thickness measurements: values were within the acceptable range (Table 1)
- Erythema scores: no signs of erithema were observed (Table 1).

MAIN STUDY
Groups of four mice were treated with the test item undilluted (100%) and diluted at 50% and 25% in Acetone/olive oil (4:1, v/v) based on the results of the pre-screen test. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Clinical observations: all animals were observed daily on Days 1, 2, 3 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body weight: the bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighted in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighted.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin sensitisation. Local Lymph Node Assay:BrdU-ELISA
- Criteria used to consider a positive response: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 - Batch No. 29134900). 100 µL of the suspension of lymph node cells (LNC) was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 µL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.
The BrdU labelling index was defined as: BrdU labelling index = (ABSem - ABS blankem) - (ABSref - ABS blankref).
SI = Mean BrdU labelling index/mouse Treated Group / Mean BrdU labelling index/mouse Control Group.
The test item is regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (SI value between 1.6 and 1.9) is declared positive. Any test item failing to produce a SI>1.6 is classified as a "non-sensitiser".
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was detemined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6 -fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 - d) / (b - d)] x (a - c)
Legend: a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c

According to Regulation (EC) No. 286/2011, the positive test item will be classified in subcategory 1A or 1B in accordance with:
If the EC value ≤ 2, the test item will be classified in "sub-category 1A".
If the EC value > 2, the test item will be classified in "sub-category 1B".

TREATMENT PREPARATION AND ADMINISTRATION:
Groups of four mice were treated with the test item undiluted (100%) and diluted at 50% and 25% in Acetone/olive oil (4:1 v/v) (AOO). The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
On day 5 (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
The Brdu solution was prepared by weighing 103.70 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. HMBF7815V)in a glass sample bottle and adding 10.37 mL of physiological saline. Then, the preparation was magnetically stirred during 20 minutes to obtain a colourless solution just before the treatment.
On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.
From each mouse, a single-cell suspension through of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of PBS (Ca2 +/Mg2+ - free) into a well of a multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1 -0.2.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
EC1.6= 24.66%. The substance has to be classified in category 1 "Skin sensitisation".
Key result
Parameter:
SI
Value:
1.31
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.33
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.13
Test group / Remarks:
100%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100% (Tables 5 and 6).

DETAILS ON STIMULATION INDEX CALCULATION
No stimulation index of more than 1.6 was recorded whatever the tested concentration. The Stimulation Index (SI) calculated by individual approach was 1.31, 1.33 and 1.13 for the treated groups at 25%, 50% and 100%, respectively (Table 4).

EC3 CALCULATION
The EC1.6 cannot be determined in this study.

CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity were noted in the test and control animals during the test (Table 2).
Slight dryness of the skin was noted in all animals treated at 100% on day 6 (Table 2).

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period (Table 3).

Table 1. Preliminary screening test: Clinical observation, bodyweight and mortality data

 

Concentration %

Animal

Bodyweight (g)

DAY

Day 1

Day 6

1

2

3

4

5

6

100%

Sf1112

20.5

20.7

0

0

0

0

0

0

0: No sign of systemic toxicity and no sign of erythema

 

 

Ear thickness (mm) on day 1

Ear thickness (mm) on day 3

Ear thickness (mm) on day 6

Ear weight (mg) on day 6

Weight Lymph nodes (mg)

Sf1112

0.20

0.21

0.21

29.7

9.0

 

The concentration of 100% was chosen as the highest concentration for the main study.

 Table 2. Main study: Individual clinical observation and mortality data

 

Groups

Test item

Amimals

Day 1

Day 2

 Day 3

Day 4

Day 5

Day 6

1

AOO

Nº Sf 1113

0

0

0

0

0

0

Nº Sf 1114

0

0

0

0

0

0

Nº Sf 1115

0

0

0

0

0

0

Nº Sf 1116

0

0

0

0

0

0

2

25%

Nº Sf 1118

0

0

0

0

0

0

Nº Sf 1119

0

0

0

0

0

0

Nº Sf 1120

0

0

0

0

0

0

Nº Sf 1121

0

0

0

0

0

0

3

50%

Nº Sf 1123

0

0

0

0

0

0

Nº Sf 1124

0

0

0

0

0

0

Nº Sf 1125

0

0

0

0

0

0

Nº Sf 1126

0

0

0

0

0

0

4

100%

Nº Sf 1128

0

0

0

0

0

0*

Nº Sf 1129

0

0

0

0

0

0*

Nº Sf 1130

0

0

0

0

0

0*

Nº Sf 1131

0

0

0

0

0

0*

0: No sign of systemic toxicity

(*): slight dryness

AOO: Acetone/olive oil (4:1 v/v)

 

 Table 3. Individual body weight and body weight gain

Groups

Test item

Animals No.

Body weight (g)

Body weight gain
(g)

Day 1

Day 6

1

AOO

Sf1113

20.7

21.2

0.5

Sf1114

22.7

24.4

1.7

Sf1115

19.8

20.3

0.5

Sf1116

24.0

23.4

-0.6

MEAN

21.8

22.3

0.5

Standard-deviation

1.9

1.9

0.9

2

25%

Sf1118

26.1

25.6

-0.5

Sf1119

25.6

25.6

0.0

Sf1120

24.5

24.8

0.3

Sf1121

20.2

20.5

0.3

MEAN

24.1

24.1

0.0

Standard-deviation

2.7

2.4

0.4

3

50%

Sf1123

22.7

24.0

1.3

Sf1124

19.4

19.8

0.4

Sf1125

22.5

21.9

-0.6

Sf1126

21.7

22.6

0.9

MEAN

21.6

22.1

0.5

Standard-deviation

1.5

1.8

0.8

4

100%

Sf1128

23.0

23.6

0.6

Sf1129

23.0

23.6

0.6

Sf1130

22.8

23.0

0.2

Sf1131

20.6

21.8

1.2

MEAN

22.4

23.0

0.7

Standard-deviation

1.2

0.8

0.4

AOO: Acetone/olive oil

 

Table 4. BrdU index & Stimulation index (individual data)

 

Groups

Test item

Animals No.

BrdU-index
(DO Indiv)

BrdU-index
(DO mean)

BrdU-index
mean*

Stimulation Index S.I.
(indiv ± Standard deviation)

1

AOO

Sf1113

0.262
0.266
0.228

0.252

0.275

n.a

Sf1114

0.228
0.236
0.196

0.220

n.a

Sf1115

0.235
0.295
0.313

0.281

n.a

Sf1116

0.337
0.336
0.368

0.347

n.a

2

25%

Sf1118

0.456
0.351
0.405

0.404

0.361

1.47 ± 0.19

Sf1119

0.337
0.280
0.353

0.323

1.17 ± 0.14

Sf1120

0.312
0.351
0.359

0.340

1.24 ± 0.09

Sf1121

0.385
0.347
0.394

0.375

1.36 ± 0.09

3

50%

Sf1123

0.349
0.350
0.309

0.336

0.367

1.22 ± 0.09

Sf1124

0.256
0.358
0.451

0.355

1.29 ± 0.35

Sf1125

0.316
0.342
0.361

0.339

1.23 ± 0.08

Sf1126

0.412
0.435
0.461

0.436

1.59 ± 0.09

4

100%

Sf1128

0.288
0.259
0.283

0.276

0.310

1.00 ± 0.06

Sf1129

0.259
0.211
0.230

0.233

0.85 ± 0.09

Sf1130

0.338
0.349
0.375

0.354

1.29 ± 0.07

Sf1131

0.299
0.398
0.434

0.377

1.37 ± 0.25

*: mean:Σindividual value / 4

AOO: Acetone/olive oil

  

Table 5. Individual Ear thickness and irritation level.

Groups

Test item

Animals No.

Day 1
ear thickness
(mm)

Day 3
ear thickness
(mm)

Day 6
ear thickness
(mm)

Ear thickness 

increase D3/D1
%

Ear thickness 

increase D6/D1
%

1

AOO

Sf1113

0.20

0.20

0.21

0.0

5.0

Sf1114

0.21

0.21

0.20

0.0

-4.8

Sf1115

0.21

0.22

0.21

4.8

0.0

Sf1116

0.19

0.20

0.20

5.3

5.3

MEAN

0.20

0.21

0.21

2.51

1.38

Standard-deviation

0.01

0.01

0.01

2.90

4.75

2

25%

Sf1118

0.20

0.22

0.20

10.0

0.0

Sf1119

0.21

0.22

0.21

4.8

0.0

Sf1120

0.20

0.20

0.20

0.0

0.0

Sf1121

0.21

0.20

0.20

-4.8

-4.8

MEAN

0.21

0.21

0.20

2.5

-1.2

Standard-deviation

0.01

0.01

0.00

6.3

2.4

3

50%

Sf1123

0.20

0.20

0.21

0.0

5.0

Sf1124

0.21

0.21

0.21

0.0

0.0

Sf1125

0.20

0.20

0.21

0.0

5.0

Sf1126

0.20

0.21

0.21

5.0

5.0

MEAN

0.20

0.21

0.21

1.3

3.7

Standard-deviation

0.00

0.01

0.00

2.5

2.5

4

100%

Sf1128

0.21

0.21

0.21

0.0

0.0

Sf1129

0.21

0.22

0.21

4.8

0.0

Sf1130

0.21

0.22

0.22

4.8

4.8

Sf1131

0.21

0.21

0.22

0.0

4.8

MEAN

0.21

0.22

0.22

2.4

2.4

Standard-deviation

0.00

0.01

0.01

2.7

2.7

AOO: Acetone/olive oil

 

  

Table 6. Individual Ear biopsy weight and lymph node weight.

Groups

Test

item

Animals No.

ear weight
Day 6 (mg)

% of ear weight
increased/group1

Lymph

nodes (mg)

1

AOO

Sf1113

28.4

 

5.5

Sf1114

28.5

5.4

Sf1115

32.1

5.4

Sf1116

27.8

4.8

MEAN

29.2

5.3

Standard-

deviation

2.0

0.3

2

25%

Sf1118

29.8

-2.6

8.3

Sf1119

28.9

8.3

Sf1120

27.2

6.0

Sf1121

27.9

6.8

MEAN

28.5

7.4

Standard-

deviation

1.1

1.1

3

50%

Sf1123

30.9

1.9

9.8

Sf1124

30.0

8.2

Sf1125

29.7

8.1

Sf1126

28.4

8.5

MEAN

29.8

8.7

Standard-

deviation

1.0

0.8

4

100%

Sf1128

31.0

6.8

10.4

Sf1129

28.6

7.9

Sf1130

31.0

9.1

Sf1131

34.2

10.7

MEAN

31.2

9.5

Standard-

deviation

2.3

1.3

AOO: Acetone/olive oil

 

 

Table 7. Summary of result – skin irritation

 

Groups

Test item

Ear thickness increase D6/D1 (%)

Biopsy ear weight Increase (%)

Excessive irritation#

1

AOO

1.4

n.a

No

2

25%

-1.2

-2.6

No

3

50%

3 .7

1.9

No

4

100%

2.4

6.8

No

#: O.E.C.D. criteria: % increase in ear thickness higher than 25%, score of erythema higher than 3.

AOO: Acetone/olive oil

 

Table 8. BrdU index & Stimulation index per group and calculation of EC1.6of the positive control (study performed 04 July 2018 – 11 July 2018)

 

Groups

Test item

BrdU-index (mean*)

Stimulation Index SI (mean + standard deviation)

Result

EC1.6 value

1

AOO

0.637

n.a.

n.a.

n.a.

2

5%

0.614

0.96±0.10

negative

24.66%

3

10%

0.744

1.17±0.17

negative

4

25%

1.023

1.61±0.20

positive

AOO: Acetone/olive oil

 

 

 

 

 

 

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
The test item does not have to be classified as a skin sensitizer, under the tested conditions in the LLNA assay (OECD 442B). The Stimulation Index (SI) calculated by individual approach were 1.31, 1.33 and 1.13 at concentrations of test item 25%, 50% and 100%, respectively.

Executive summary:

The skin sensitisation potential of the test item was tested in the LLNA assay according to OECD 442B and E.U. B.51 method, following GLP. Firstly, a preliminary screening test was performed using one mouse treated with 25 μL of the test item undiluted for three consecutive days. No mortality or signs of systemic toxicity and no signs of excessive irritation were noted in the preliminary study. In the main test, three groups of four mouse CBA/J were treated for three consecutive days with 50 µL (25 µL per ear) of the test item undiluted (100%) and diluted at concentrations of 50% and 25% in Acetone/olive oil (4:1, v/v). A control group was treated with Acetone/olive oil (4:1, v/v). On day 5, 0.5 mL of BrdU solution (10mg/mL) was injected by intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was deremined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. Only slight dryness of the skin was noted in all animals treated at 100% on day 6. No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 100%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 1.31, 1.33 and 1.13 for the treated groups at 25%, 50% and 100% respectively. Therefore, the EC1.6 could not be determined due to the absence of SI value higher than 1.6. Under these experimental conditions, the test item does not have to be classified as a skin sensitizer in accordance with the CLP Regulation (EC) no. 1272/2008.

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

Skin sensitisation (in chemico): First key event (molecular initiating event): Weight of evidence. A DPRA skin sensitization test was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. Cinnamaldehyde dissolved in acetonitrile was used as positive control. The test item showed mean depletion of 0.39% for lysine and 2.42% for cysteine, i.e. an overall average of 1.40%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Skin sensitisation (in vitro): Second key event (Activation of keratinocytes): Weight of evidence. The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control 1% DMSO were incubated for 48 hours at 37ºC, 5% CO2. The calculated Imax values were lower than 1.5, thus a negative result can be predicted for this key event.

Skin sensitisation (in vitro): Third key event (activation of dendritic cells): Data waiving (study scientifically not necessary). According to column 2 of REACH Annex VII, the available in vitro test method addressing the key event activation of dentritic cells (h-CLAT method, OECD TG 442E) is not applicable for this substance because it tends to produce false negatice results with substances with a Log Kow greater than 3.5, as it is the case.

Skin sensitisation (in vivo): Weight of evidence. The skin sensitisation potential of the test item was tested in the LLNA assay according to OECD 442B and E.U. B.51 method, following GLP. Three groups of four mouse CBA/J were treated for three consecutive days with 50 µL (25 µL per ear) of the test item undiluted (100%) and diluted at concentrations of 50% and 25% in Acetone/olive oil (4:1, v/v). A control group was treated with Acetone/olive oil (4:1, v/v). The Stimulation Index (SI) calculated by individual approach was 1.31, 1.33 and 1.13 for the treated groups at 25%, 50% and 100% respectively. Therefore, the EC1.6 could not be determined due to the absence of SI value higher than 1.6. Under these experimental conditions, the test item does not have to be classified as a skin sensitizer in accordance with the CLP Regulation(EC) no. 1272/2008.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for skin sensitization according to CLP Regulation no. 1272/2008.