Registration Dossier

Administrative data

Description of key information

First key event (molecular initiating event): Weight of evidence. Test method according to the OECD 442C Guideline with GLP. The test item showed a mean depletion of lysine and cysteine peptides of 7.07%, reflecting a low reactivity and thus a positive prediction of DPRA.

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 item may be classified as not skin sensitizer using the KeratinoSensTM test method.

Third key event (activation of dendritic cells): Weight of evidence. Test method according to the OECD 442E Guideline with GLP. Under the experimental conditions the test item may be classified as skin sensitizer using the h-CLAT test method.

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 1.36, 1.34 and 1.02 at concentrations of test item 25%, 50% and 90%, respectively.

Conclusion: Based on no concordant results obtained from the three in vitro assays and based also on the observational and practical experience, e.g. in manufacture or handling the substance at test

facility, it was decided to conduct an in vivo LLNA assay. 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:
10 April 2017 - 13 April 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))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Details on study design:

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 acetonitrile.

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, LLC; Ref. Ac RFAACAA-COOH; batch no 160801; purity 94.82%)
Lysine peptide (supplier RS Synthesis, LLC; Ref. Ac-RFAAKAA-COOH; batch no 160801; purity 94.19%)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: test item in phosphate buffer (100 mM; pH 7.5 ± 0.05) for cysteine peptide and ammonium acetate buffer (100 mM; pH 10.2 ± 0.05) for lysine peptide.
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 acetonitrile, the test item and the positive control solvent, in order to check its influence on the peptide stability.

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 47.2 mg was pre-weighted in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution with acetonitrile.
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: 11 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 22 ml of phosphate buffer (100 mM; pH 7.5 ± 0.05).
Lysine solution: 14.8 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 28,6 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, samples were checked. No precipitate was observed.

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

HPLC ANALYSIS
-Apparatus: Waters e2695 HPLC; Waters 2489 UV detector; Cortecs column C18 2.7 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: 0.21 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.
The column used has 2.7 µm particle size when the column described in the OECD 442C has 3.5 µm particle size. According to the guideline, the set-up parameters 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 73.86% for cysteine peptide and 55.56% for lysine peptide.
Key result
Parameter:
other: %depletion in lysine peptide
Run / experiment:
mean of 3 repetitions
Value:
2.13
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: %depletion in cysteine peptide
Run / experiment:
Mean of 3 repetitions
Value:
12.01
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: mean %depletion in peptides
Run / experiment:
Mean of lysine and cysteine peptides
Value:
7.07
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive 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 0.511 mM for lysine and 0.502 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 was 0.86% which is lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.516 mM for lysine and 0.496 mM for cysteine which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the controls C was 1.16% which is lower than 15%.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 0.243% for cysteine and 0.43% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 73.86% for cysteine peptide and 55.56% 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 0.89% for cysteine and 0.29% 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

56.06

73.71

Repetition 2

55.31

73.73

Repetition 3

55.32

74.14

SD

(Standard Deviation)

0.430

0.243

Mean

55.56

73.86

Depletion

Validity criteria

40.2 < Depletion < 69.4

60.8 < Depletion < 100

CV

0.77%

0.33%

Table 4: Test item

 

Depletion in

Lysine Peptide %

Depletion in

Cysteine Peptide %

 

Repetition 1

1.97

11.06

 

Repetition 2

1.95

12.82

Mean Depletion %

Repetition 3

2.46

12.14

Mean

2.13

12.01

7.07

SD

(Standard Deviation)

0.29

0.89

 

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 item shows mean depletion of 2.13% for lysine and 12.01% for cysteine, i.e. an overall average of 7.07%, reflecting a low reactivity and thus a positive prediction of DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for the test item 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 acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile 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 shows mean depletion of 2.13% for lysine and 12.01% for cysteine, i.e. an overall average of 7.07%, reflecting a low reactivity and thus a positive prediction of DPRA.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
18 April 2017 - 28 April 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)
Deviations:
yes
Remarks:
Dilution strategy (see justification on "details on study design")
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Details on study design:

REAGENTS AND MEDIA
-DMSO (Supplier ref. D5879-1L, Sigma Aldrich; Batch no SZBG2170; purity ≥99.5%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 1852416)
-Non-heat inactivated foetal calf serum (Supplier ref. 10270098, Fisher Bioblock; Batch no 42Q9761K)
-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 25 in repetition 1 and passage 16 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 solution: 2000 µM (1X) in treatment medium 1% DMSO
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) 100 X plate (positive and negative control): A 100-fold concentrated dilutions series was prepared in 96-well plate:
-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 (in cytotoxicity repetition 2, negative control was distributed in row H).

2) 4X dilution plate (positive and negative control): The 100 X plate was diluted 25 fold in a new plate (4 X).

3) Preparation of the 1X dilution for the test item:
The test item was placed in the row D.
1100 µl of treatment medium, 1% DMSO were distributed columns 1 to 10 in a masterblock. 2200 µl of the 2000 µM solution were placed in column 12 then the series dilutions were prepared by transferring 1100 µl of the column 12 in the column 11 and so until the column 1. Dilutions were mixed by repeated pipetting at least 3 times, between each concentration.

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
-Negative and positive control: 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).
-Test item: In the 5 seeded plates, the medium was aspirated. The 1X masterblock was replicated 5 times: 200 µl from the 1X masterblock were placed in each of the three white plates and in the two transparent plates. The plates (1X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37ºC ± 1°C, 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, 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.

CITOTOXICITY 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, 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). After a 10 minutes homogenization, the absorbance was measured at 540 nm.

DEVIATIONS FROM OECD GUIDELINE:
The dilution strategy is different from the OECD 442D TG (paragraph 22). Given the slight solubility of the test item in water (i.e. 828 mg/l / 5.36 mM), the dilution was prepared directly 1X in treatment medium-1% DMSO at 2000 µM. This has no impact on the study result because the test item was tested in the expected final condition (i.e. 2000 µM as maximal concentration).
Positive control results:
Repetition 1: The maximal average induction of luciferase activity (Imax) was 7.90 at a concentration of 64 µM. The mean value EC1.5 was 8.24 µM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 2.98 at a concentration of 64 µM. The mean value EC1.5 was 19.12 µM.
Key result
Parameter:
other: Imax
Run / experiment:
Repetition 1
Value:
1.45
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
Repetition 2
Value:
1.3
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: 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 13.6% and for repetition 2 was 9.0% 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 7.90 and 2.98 in each repetition which are between 2 and 8. The EC1.5 values were 8.24 and 19.12 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: Positive control

Cinnamaldehyde

4 µM

8 µM

16 µM

32 µM

64 µM

EC1.5

Imax

Rep 1

1.37

1.48

2.22

3.66

7.90

8.24

7.90

Rep 2

1.15

1.19

1.22

2.67

2.98

19.12

2.98

Mean

1.26

1.33

1.72

3.17

5.44

12.55*

5.44

*geometric mean

Table 2: Test item

VIABILITY

INDUCTION

IC30(µM)

IC50(µM)

Imax

Linear EC1.5 (µM)

EC1.5 Lin/Log (µM)

Rep 1

>2000

>2000

1.45

-

-

Rep 2

>2000

>2000

1.30

-

-

Mean

 -

 -

1.37

-

-

Geometric mean

>2000

>2000

 -

-

-

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

83,24

87,90

89,96

88,81

84,00

78,35

79,19

81,10

85,07

64,07

99,05

98,36

Rep 2

78,54

97,11

93,36

87,63

85,32

84,20

85,95

80,54

83,72

76,87

79,66

80,22

Viability

80,89

92,50

91,66

88,22

84,66

81,28

82,57

80,82

84,40

70,47

89,35

89,29

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

1,21

1,06

1,11

1,15

1,13

1,09

1,13

1,13

1,30

1,16

1,20

1,45

Rep 2

0,98

1,10

1,13

1,12

1,22

1,21

1,26

1,20

1,22

1,30

1,19

1,19

Induction

1,09

1,08

1,12

1,14

1,18

1,15

1,20

1,17

1,26

1,23

1,19

1,32

SD

0,16

0,03

0,02

0,02

0,06

0,08

0,10

0,05

0,05

0,10

0,01

0,18

Table 5: Student t-test

Rep 1

0,436

0,485

0,503

0,069

0,379

0,520

0,183

0,399

0,183

0,073

0,097

0,003

Rep 2

0,784

0,082

0,174

0,050

0,048

0,053

0,031

0,006

0,057

0,001

0,020

0,038

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 item may be classified as not skin sensitizer using the KeratinoSensTM test method.
Executive summary:

The KeratinoSensTM test method was performed for the test item 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, 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
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
31 March 2018 – 04 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (Annex I: In vitro skin sensitisation: human cell line activation test (h-CLAT))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Test item formed suspension in saline at the concentration of 100 mg/mL. Later phase separation was also observed. When dissolved in DMSO, test item was found to be completely soluble at the concentration of 500 mg/mL. Therefore, DMSO was selected as the solvent for this study.

Details on study design:
Details on study design:

TEST SYSTEM
-Cell line: THP-1 cell line procured from American Type Culture Collection (ATCC) (Batch nº 63176297)
-Culture: cells were cultured at 37ºC, 5% CO2 in RPMI-1640 (Gibco, Lot #1897264) with 2 mM L-glutamine and 25 mM HEPES (according to h-CLAT DB-ALM protocol No 158) supplemented with 10% v/v FBS (Fetal Bovine Serum, Gibco, Lot#1841109), 0.05 mM 2-Mercaptoethanol (Gibco, Lot #1710209, 1852695) and appropriate antibiotics: 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Lot #1894156, 1924795).
-Passage number: cells were used at passage 15, 16, 18, 20, 22 and 27.
-Reactivity check: Yes, performed 2 weeks after thawing with cells of passage 15 on positive controls DNCB and nickel sulphate, and negative control lactic acid.

CONTROLS
-Positive control: 2, 4- Dinitrochlorobenzene (CAS 97-00-7; Sigma Aldrich batch no STBD7009V; purity 99.9%)
-Medium Control: complete medium
-Solvent Control: DMSO diluted to 0.4% in complete medium, which was tested at a final concentration of 0.2 % in the plate.

PREPARATION OF CELLS
-Pre-culture: Before assay, THP-1 cells were pre-cultured in culture flasks for 72 h when seeded at a density of 0.1 × 10^6 cells/mL and for 48 h when seeded at a density of 0.2 × 10^6 cells/mL. Cells were maintained in suspension at densities from 0.1 to 0.8 × 10^6 cells/mL.
-cells suspension: Pre-cultured cells were collected by centrifugation (130 x g, 4 oC, 5 min) and re-suspended in fresh culture medium at a density of 2 × 10^6 cells/mL.
-Nº of culture plates: For dose range finding (DRF) assay, cells were distributed into a 96-well flat-bottom plate with 80 μL. For CD86/CD54 expression measurement, cells were distributed into a 24 well flat-bottom plate with 500 μL.
-Cell density: 1.6 × 10^5 cells/well (DRF assay) and 1 × 10^6 cells/well (CD86/CD54 expression measurement).

DOSE FINDING ASSAY
-Preparation of test item: Stock solution of concentration 500 mg/mL of the test item was first prepared. Further ten stock solutions were prepared by 2-fold serial dilutions using DMSO (0.98–500 mg/mL). These stock solutions were further diluted 250-fold into culture medium (to obtain working solutions).
-Preparation of negative control: Along with the medium control, the solvent control used was DMSO, tested at a final concentration of 0.2% in the plate.
-Application of test item and control: The working solutions of test item and control were mixed in a 1:1 (v/v) ratio with the cell suspensions prepared in the 96-well flat-bottom plate (final range of concentrations of test item in the plate was 1.95–1000 μg/mL). The treated plates were incubated for 24 hours at 37 ± 1ºC under 5 ± 1% CO2.
-cell staining: After 24 hours of exposure, cells were transferred into 96-well V-bottom plate and collected by centrifugation (250 x g, 4ºC, 5 min). The supernatants were discarded and the remaining cells were resuspended with 200 µL of staining buffer (phosphate buffered saline (Sigma, Lot #SLBS1525V) containing 0.1% bovine serum albumin (HiMedia, Lot #0000268629)). Cells were washed twice with 200 µL of staining buffer. Finally, cells were resuspended in 200 µL of Propidium iodide (PI, Sigma, Lot #SLBM6232V)-staining buffer (final concentration of PI = 0.625 μg/mL).
-Cytotoxicity measurement and estimation of CV75 value:
Apparatus: flow cytometer (FACSVerseTM), Becton Dickinson (BD)
CV75 value: calculated according to equation stated in OECD TG 442E.
-Number of experiments: 3 runs. A third repetition was conducted for confirmation since results of first and second experiments were discordant. Based on the results of second and third experiments, CV75 value of test item was determined.

CD86/CD54 EXPRESSION MEASUREMENT
-Preparation of test item: The test item was first diluted to the concentration corresponding to 500-fold of the 1.2 × CV75, as determined in the dose range finding assay. Then, 1.2-fold serial dilutions were performed in DMSO to obtain the stock solutions (eight concentrations ranging from 500 × 1.2 × CV75 to 500 × 0.335 × CV75) to be tested. These stock solutions were further diluted 250-fold in culture medium (working solutions).
-Preparation of positive control: A 10 mg/mL stock solution of DNCB was prepared in DMSO and diluted to 2 mg/mL. This was further diluted 250-fold in culture medium to obtain working solution of concentration 8 μg/mL. The working solution was used for treatment with the cell suspension in 1:1 ratio (final range of concentration in the plate was 4.0 μg/mL).
-Preparation of negative control: Along with the medium control, the solvent control used was DMSO, tested at a final concentration of 0.2% in the plate.
-Application of test item and controls: The working solutions of test item and controls prepared (500 µL) were mixed with 500 µL of suspended cells (1 x 10^6 cells) in a 1:1 ratio in a 24-well plate (final range of concentrations of test item in the plate was 101–362 μg/mL) and incubated for 23 hours and 30 minutes at 37± 1ºC under 5± 1% CO2.
-cell staining: After 23 hours and 30 minutes of exposure, cells were transferred from 24-well plates into sample tubes, collected by centrifugation (250 x g, 4ºC, 5 min) and then washed twice with 1 mL of staining buffer. After washing, cells were blocked with 600 µL of blocking solution (staining buffer containing 0.01% (w/v) globulin (Cohn Fraction II, III Human, lyophilized barrier, Sigma, Lot# 017K7650V)) and incubated for 15 minutes on ice. After blocking, cells were split in three aliquots of 180 µL into a 96-well V-bottom plate.
The three groups of cells were centrifuged and the cell pellets were stained with 50 µL of FITC-labelled anti-CD86 (BD Pharmingen, Lot #6348610), anti-CD54 (Dako, Lot #20044016) or mouse IgG1 (isotype) antibodies (Dako, Lot #20046409) for 30 minutes on ice. The antibodies were used by diluting 6:50 (v/v, for CD86) or 3:50 (v/v, for CD54) and IgG1 with staining buffer. After incubation, the cells were centrifuged (250 x g, 4ºC, 5 min) and were washed with 200 µL of staining buffer three times. Finally, cells were resuspended in 200 µL of PI-staining buffer (final concentration of PI = 0.625 µg/mL).
- CD86/CD54 measurement and cell viability:
Apparatus: flow cytometer (FACSVerseTM), Becton Dickinson (BD)
CD86/CD54 expression: Relative Fluorescence Intensity (RFI) was used as an indicator of CD86 and CD54 expression calculated according to equation stated in OECD TG 442E.
EC150/EC200 values: calculated according to equation stated in OECD TG 442E.
-Number of experiments: 3 runs. For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs for CD86/CD54 expression measurement were performed.

DEVIATIONS FROM OECD GUIDELINE: No.
Positive control results:
% cell viability: ≥ 50% (all experiments)
% RFI CD86: ≥ 150% (all experiments)
% RFI CD54: ≥ 200% (all experiments)
See results on “Any other information on results incl. tables”

Key result
Parameter:
other: CV75 (µg/mL)
Run / experiment:
Average of experiment 2 and 3
Value:
302
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI of CD86 (%)
Run / experiment:
Experiment 1
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54 (%)
Run / experiment:
Experiment 1
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD86 (%)
Run / experiment:
Experiment 2
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54 (%)
Run / experiment:
Experiment 2
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD86
Run / experiment:
Experiment 3
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54 (%)
Run / experiment:
Experiment 3
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC150 (µg/mL)
Run / experiment:
Experiment 2
Value:
465
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC200 (µg/mL)
Run / experiment:
Experiment 1
Value:
141
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC200 (µg/mL)
Run / experiment:
Experiment 2
Value:
94
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC200 (µg/mL)
Run / experiment:
Experiment 3
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: EC200 Median value
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 442E 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 controls: yes, the cell viabilities of medium and solvent controls were found to be >90 % in all three experiments. In the solvent control, % RFI CD86 and % RFI CD54 were found to be ≤ 150% and ≤ 200% respectively in all three experiments. The MFI ratio of medium and solvent controls for both CD86 and CD54 to isotype control were found to be >105% in all three experiments.
- Acceptance criteria met for positive control: Yes, the RFI values of DNCB were found to be 244.66%, 575.47% and 270.27% for CD86 marker and 785.19%, 549.02% and 206.35% for CD54 marker, which met the assay acceptance criteria (RFI CD86 ≥ 150%, RFI CD54 ≥ 200%). The cell viability of positive control was found to be >70 % in all three experiments.
- Acceptance criteria met for test item: yes, the cell viability of test item was found to be >50% in 8/8 concentrations in all three experiments.




Table 1: Results of CV75, EC150 and EC200

Sr. No.

Observed CV75 (in µg/mL)

Observed EC150 for CD86

(in µg/mL)

Observed EC200 for CD54

(in µg/mL)*

Expt. 1 Prediction

Expt. 2 Prediction

Expt. 3 Prediction

Final Prediction

1

302

Negative

100

Positive (P2)

Positive (P12)

Positive (P2)

Positive

Table 2: Cell Viability, MFI and RFI Values of Positive Control in Expression Study

Expt. No.

% Cell Viability

MFI

% RFI CD86

% RFI CD54

Observed values

Expected values

IgG1

CD86

CD54

Observed values

Expected values

Observed values

Expected values

1

85.6

≥ 50%

87.8

262

215

244.66

≥ 150%

785.19

≥ 200%

2

73.4

111

172

167

575.47

549.02

3

77.4

105

245

183

270.27

206.35

Table 3: Cell Viability and MFI Values of Medium Control in Different Experiments

Expt. No.

% Cell Viability

MFI

Observed values (in %)

Expected values (in %)

IgG1

CD86

CD54

1

93.7

>90

82.1

142

90.7

2

94.4

87.3

112

98.4

3

91.8

92.8

149

131

Table 4: Cell Viability, MFI and RFI Values of DMSO Control in Different Experiments

Expt. No.

% Cell Viability

MFI

RFI CD86

RFI CD54

Observed values (in %)

Expected values (in %)

Observed values (in %)

Expected values (in %)

Observed values (in %)

Expected values (in %)

IgG1

CD86

CD54

1

92.1

>90

81.8

153

98

118.86

<150

188.37

<200

2

94.9

87.3

97.9

97.5

42.91

91.89

3

92.5

92.2

144

130

92.17

98.95

Table 5: Cell Viability, MFI and RFI Values of Test Item in CD86/CD54 Expression Measurement

Expt.1

Expt.2

Expt.3

Conc (µg/mL)

% Cell Viability

MFI

% RFI

% Cell Viability

MFI

% RFI

% Cell Viability

MFI

% RFI

IgG1

CD86

CD54

CD86

CD54

IgG1

CD86

CD54

CD86

CD54

IgG1

CD86

CD54

CD86

CD54

362

84.3

85.4

143

413

80.90

2022.22

82.9

86.8

137

300

473.58

2090.20

79.7

93.5

133

368

76.25

726.19

302

92.9

79.5

117

363

52.67

1750.00

84.0

89

138

260

462.26

1676.47

79.4

90.5

126

326

68.53

623.02

252

92.2

78.3

115

333

51.54

1572.22

85.5

89.8

142

199

492.45

1070.59

80.8

92.9

126

266

63.90

457.94

210

94.0

78.8

122

349

60.67

1667.90

87.8

92.7

146

167

502.83

728.43

83.2

92.9

131

241

73.55

391.80

175

94.5

80.2

119

240

54.49

986.42

87.8

90.4

154

144

600.00

525.49

84.8

92.7

133

197

77.80

275.93

146

94.4

80.4

121

220

57.02

861.73

88.7

92.1

136

127

414.15

342.16

87.0

90.3

132

166

80.50

200.26

121

95.0

79.4

125

136

64.04

349.38

87.6

89.1

142

215

499.06

1234.31

84.7

91.4

126

566

66.80

1255.56

101

94.6

79.3

107

164

38.90

522.84

87.9

91.2

144

143

498.11

507.84

85.1

89.3

124

193

66.99

274.34

Interpretation of results:
other: h-CLAT 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 442E.
Conclusions:
Under the experimental conditions the test item dextro alpha fenchol may be classified as skin sensitizer using the in vitro h-CLAT test method.


Executive summary:

The h-CLAT test method was performed for dextro alpha fenchol as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442E, under GLP conditions. After solubility test, DMSO was selected as solvent. Concurrent negative controls, one consisting of solvent and other consisting of complete medium alone without test item were included in each assay. 2, 4- Dinitrochlorobenzene (DNCB) was used as a positive control at a single concentration of 4.0 µg/mL in DMSO. Three independent Dose Range Finding (DRF) experiments were performed at a final concentration range in the plate of 1.95–1000 μg/mL. The CV75 value of test item was found to be 302 µg/mL. Based on this value, three independent experiments of CD86/CD54 expression measurement (expression study) were performed at eight concentrations ranging from 101 to 362 μg/mL. Negative and positive controls met the specified acceptance criteria for the controls. Furthermore, the cell viability of test item was found to be >50% in 8/8 concentrations in all three experiments. In the first experiment, positive response was obtained for only CD54 marker, hence the prediction was “P2”. In second experiment, prediction obtained was “P12”, as positive response was observed for both CD86 and CD54 markers. While, in third experiment a positive response was obtained again for only CD54 marker, hence the prediction obtained was “P2”. Based on the results of three experiments, the test item was concluded as sensitizer in hCLAT assay. The median EC200 value of test item was found to be 100 µg/mL.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27 June 2018 – 24 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Qualifier:
according to
Guideline:
other: EU Method B.51 (Skin sensitisation. local lymph node assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
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: 9 weeks old.
- Weight at study initiation: average weight 21.0-22.7 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:
90%, 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 diluted at 90% in Acetone/olive oil (4:1 v/v) (AOO) 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: A preliminary solubility test was performed and Acetone/olive oil (4:1 v/v) was chosen as vehicle since it produced the most suitable formulation at the required concentration.
- Irritation: No sign of excessive irritation was noted at the tested concentration of 90%.
- 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 diluted at 90%, 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 weighed.

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)
The test item will be 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 will be 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:
The test item was freshly prepared in Acetone/olive oil (4:1, v/v). Groups of four mice were treated with the test item diluted at 90%, 50% and 25% in Acetone/olive oil (4:1, v/v). The mice were treated by daily application of 25 µL of the appropiate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1,2,3). 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 150.70 mg and 155.80 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. HMBF5970V) added to 2 glass bottles of respectively 15.07 mL and 15.58 mL of physiological saline. The preparations were magnetically stirred then pooled 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:
1.34
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.36
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.02
Test group / Remarks:
90%
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 90% (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 1.34, 1.36 and 1.02 for the treated groups at 25%, 50% and 90%, 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).
No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 90%.
Therefore, the test item has to be considered as not excessively irritant at these concentrations.

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

90%

Sf0803

22.3

23.7

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)

Sf0803

0.22

0.22

0.21

27.8

6.0

 

The concentration of 90% 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 0843

0

0

0

0

0

0

Nº Sf 0844

0

0

0

0

0

0

Nº Sf 0846

0

0

0

0

0

0

Nº Sf 0847

0

0

0

0

0

0

2

10%

Nº Sf 0848

0

0

0

0

0

0

Nº Sf 0849

0

0

0

0

0

0

Nº Sf 0850

0

0

0

0

0

0

Nº Sf 0851

0

0

0

0

0

0

3

25%

Nº Sf 0853

0

0

0

0

0

0

Nº Sf 0854

0

0

0

0

0

0

Nº Sf 0855

0

0

0

0

0

0

Nº Sf 0856

0

0

0

0

0

0

4

50%

Nº Sf 0858

0

0

0

0

0

0

Nº Sf 0859

0

0

0

0

0

0

Nº Sf 0860

0

0

0

0

0

0

Nº Sf 0861

0

0

0

0

0

0

0: No sign of systemic toxicity

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

Sf0843

21.0

20.5

-0.5

Sf0844

20.7

23.2

2.5

Sf0846

23.7

23.7

0.0

Sf0847

21.1

21.2

0.1

MEAN

21.6

22.2

0.5

Standard-deviation

1.4

1.5

1.3

2

25%

Sf0848

20.0

20.3

0.3

Sf0849

21.9

21.9

0.0

Sf0850

21.0

21.0

0.0

Sf0851

21.1

22.0

0.9

MEAN

21.0

21.3

0.3

Standard-deviation

0.8

0.8

0.4

3

50%

Sf0853

21.7

22.2

0.5

Sf0854

20.2

20.5

0.3

Sf0855

21.7

21.7

0.0

Sf0856

22.2

22.8

0.6

MEAN

21.5

21.8

0.4

Standard-deviation

0.9

1.0

0.3

4

90%

Sf0858

24.9

24.6

-0.3

Sf0859

20.8

22.4

1.6

Sf0860

23.2

23.8

0.6

Sf0861

21.7

21.6

-0.1

MEAN

22.7

23.1

0.5

Standard-deviation

1.8

1.4

0.9

AOO: Acetone/olive oil

 

Table 4. BrdU index & Stimulation index per group and calculation of EC1.6

 

Groups

Test item

BrdU-index (mean*)

Stimulation Index SI (mean + standard deviation)

Result

EC1.6 value

1

AOO

0.470

n.a.

n.a.

n.a.

2

25%

0.630

1.34±0.41

negative

n.a

3

50%

0.641

1.36±0.29

negative

4

90%

0.481

1.02±0.07

negative

*: 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

Sf0843

0.20

0.21

0.19

5.0

-5.0

Sf0844

0.21

0.21

0.20

0.0

-4.8

Sf0846

0.21

0.20

0.20

-4.8

-4.8

Sf0847

0.20

0.19

0.20

-5.0

0.0

MEAN

0.21

0.20

0.20

-1.19

-3.63

Standard-deviation

0.01

0.01

0.01

4.73

2.42

2

25%

Sf0848

0.19

0.19

0.20

0.0

5.3

Sf0849

0.21

0.20

0.19

-4.8

-9.5

Sf0850

0.20

0.21

0.20

5.0

0.0

Sf0851

0.20

0.21

0.20

5.0

0.0

MEAN

0.20

0.20

0.20

1.3

-1.1

Standard-deviation

0.01

0.01

0.01

4.7

6.2

3

50%

Sf0853

0.21

0.21

0.20

0.0

-4.8

Sf0854

0.20

0.19

0.20

-5.0

0.0

Sf0855

0.19

0.19

0.21

0.0

10.5

Sf0856

0.20

0.21

0.20

5.0

0.0

MEAN

0.20

0.20

0.20

0.0

1.4

Standard-deviation

0.01

0.01

0.00

4.1

6.5

4

90%

Sf0858

0.19

0.19

0.21

0.0

10.5

Sf0859

0.20

0.19

0.22

-5.0

10.0

Sf0860

0.21

0.20

0.20

-4.8

-4.8

Sf0861

0.19

0.21

0.21

10.5

10.5

MEAN

0.20

0.20

0.21

0.2

6.6

Standard-deviation

0.01

0.01

0.01

7.3

7.6

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

Sf0843

26.7

 

4.6

Sf0844

29.2

4.8

Sf0846

28.4

5.6

Sf0847

30.9

6.3

MEAN

28.8

5.3

Standard-deviation

1.7

0.8

2

25%

Sf0848

25.3

0.1

4.8

Sf0849

26.9

6.6

Sf0850

28.2

5.5

Sf0851

34.9

8.8

MEAN

28.8

6.4

Standard-deviation

4.2

1.7

3

50%

Sf0853

26.5

1.3

6.6

Sf0854

29.8

6.6

Sf0855

27.4

6.1

Sf0856

33.0

16.9

MEAN

29.2

9.1

Standard-deviation

2.9

5.2

4

90%

Sf0858

31.3

2.9

6.3

Sf0859

27.0

5.0

Sf0860

28.6

6.5

Sf0861

31.6

5.1

MEAN

29.6

5.7

Standard-deviation

2.2

0.8

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

-3.6

n.a

No

2

25%

-1.1

0.1

No

3

50%

1.4

1.3

No

4

90%

6.6

2.9

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 1.34, 1.36 and 1.02 at concentrations of test item 25%, 50% and 90%, 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 diluted at 90% in Acetone/olive oil (4:1 v/v) 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 diluted at concentrations of 25%, 50% and 90% 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. No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 90%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 1.34, 1.36 and 1.02 for the treated groups at 25%, 50% and 90% respectively. Therefore, the EC1.6 cannot 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:

First key event (molecular initiating event): Weight of evidence. A DPRA skin sensitization test was performed for the test item 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 acetonitrile as solvent. Reference controls A, B and C were prepared with acetonitrile 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 shows mean depletion of 2.13% for lysine and 12.01% for cysteine, i.e. an overall average of 7.07%, reflecting a low reactivity and thus a positive prediction of DPRA.

Second key event (Activation of keratinocytes): Weight of evidence. The KeratinoSensTM test method was performed for the test item 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, calculated Imax values were lower than 1.5, thus a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Third key event (activation of dendritic cells): Weight of evidence. The h-CLAT test method was performed for dextro alpha fenchol as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442E, under GLP conditions. After solubility test, DMSO was selected as solvent. Concurrent negative controls, one consisting of solvent and other consisting of complete medium alone without test item were included in each assay. 2, 4- Dinitrochlorobenzene (DNCB) was used as a positive control at a single concentration of 4.0 µg/mL in DMSO. Three independent Dose Range Finding (DRF) experiments were performed at a final concentration range in the plate of 1.95–1000 μg/mL. The CV75 value of test item was found to be 302 µg/mL. Based on this value, three independent experiments of CD86/CD54 expression measurement (expression study) were performed at eight concentrations ranging from 101 to 362 μg/mL. Negative and positive controls met the specified acceptance criteria for the controls. Furthermore, the cell viability of test item was found to be >50% in 8/8 concentrations in all three experiments. In the first experiment, positive response was obtained for only CD54 marker, hence the prediction was “P2”. In second experiment, prediction obtained was “P12”, as positive response was observed for both CD86 and CD54 markers. While, in third experiment a positive response was obtained again for only CD54 marker, hence the prediction obtained was “P2”. Based on the results of three experiments, the test item was concluded as sensitizer in hCLAT assay. The median EC200 value of test item was found to be 100 µg/mL.

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. Firstly, a preliminary screening test was performed using one mouse treated with 25 μL of the test item diluted at 90% in Acetone/olive oil (4:1 v/v) 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 diluted at concentrations of 25%, 50% and 90% 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. No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 90%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 1.34, 1.36 and 1.02 for the treated groups at 25%, 50% and 90% respectively. Therefore, the EC1.6 cannot 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.

Conclusion: Based on no concordant results obtained from the three in vitro assays and based also on the observational and practical experience, e.g. in manufacture or handling the substance at test

facility, it was decided to conduct an in vivo LLNA assay. Based on weight of evidence approach, the test substance does not need to be classified as skin sensitizer.

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.