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Description of key information

OECD guideline, GLP compliant in chemico/in vitro studies were conducted. Negative results were obtained in the in chemico study. Positive results were obtained in the 2 in vitro study. Based on the weight-of-evidence available, the substance is predicted to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Nov - 23 Dec 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)
Version / remarks:
adopted in Feb 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Government of India, National Good Laboratory Practice (GLP) Compliance Monitoring Authority, India
Type of study:
activation of keratinocytes
Details on study design:
TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.
The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™ (Lot No. JRF/HaCaT/2016/01)
- Source: Givaudan, Switzerland
- Passage number: 21

CELL CULTURE CONDITIONS
- Type and identity of media:
Culture medium: Dulbecco`s Modified Eagle Medium (GlutaMAX™) supplemented with 9.1% fetal bovine calf serum and 500 µg/mL geneticin
Exposure medium: Dulbecco`s Modified Eagle Medium (GlutaMAX™) supplemented with 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 1.0

TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
Positive control
- Substance: trans cinnamaldehyde
- Final concentration: 4 – 64 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 1.0

NUMBER OF REPLICATIONS: three plates each in two independent experiments, 12-fold determination (test substance), 6-fold determination (control) and 5-fold determination (positive control) for each plate

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 570 nm

DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Passive Lysis Buffer (Promega, Lot No. 0000174757)
- Luciferase Assay System: Luciferase Assay Kit (Promega, Lot No. 0000237533)
- Device: plate reader
Positive control results:
The gene induction for the positive control trans cinnamaldehyde was found to be >1.5 at concentrations of 32 µM and 64 µM in both repetitions. The EC1.5 value for the positive control was found to be 22.97 µM and 25.29 µM in Experiment I and II, respectively (within the acceptable range of 7.5 to 30 µM). The average gene induction for the positive control at 64 µM was found to be 3.31 and 2.72 (which lies within the acceptable range of 2 and 8) for Experiment I and II, respectively.
Key result
Parameter:
other: maximum luciferase activity induction (Imax)
Run / experiment:
Experiment I
Value:
2.82
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Test item concentration: 31.25 µM; IC50 = 25.56 µM
Key result
Parameter:
other: maximum luciferase activity induction (Imax)
Run / experiment:
Experiment II
Value:
2.17
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Test item concentration: 31.25 µM; IC50 = 34.80 µM
Key result
Parameter:
other: EC1.5 (µM)
Run / experiment:
Mean of Experiment I and II
Value:
20.79
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
The EC1.5 mean values for the test substance was 20.79 µM, which was less than 1000 µM. The mean value of maximum induction (Imax) for the test substance was 2.50 at the test concentration of 31.25 µM, which was higher than 1.5 fold. The cellular viability was 87.64% and 41.76% at 15.63 and 31.25 µM, respectively, with induction of luciferase activity above 1.5 fold. Therefore, the evaluation criteria are met for the test substance.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control met the acceptance criteria and was correctly identified as non-sensitiser.
- Acceptance criteria met for positive control: The positive control met the acceptance criteria and was correctly identified as sensitiser.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation observed for the negative control during Experiment I and II were 14.65% and 15.30%, respectively, which was below 20%. Since variation between the replicates was less than 20%, results of this run were considered as valid.

Table 1: Summary of Mean Induction (Experiment I and II)

Concentration

(µM)

Test item

Mean

SD

0.98

1.07

0.17

1.95

1.05

0.26

3.91

1.09

0.14

7.81

1.15

0.00

15.63

1.04

0.12

31.25

2.50

0.46

62.5

1.24

0.23

125

0.00

0.00

250

-0.01

0.00

500

0.00

0.00

1000

0.00

0.00

2000

0.00

0.00

Interpretation of results:
other: skin sensitising potential based on the key event "activation of keratinocytes"
Conclusions:
Under the conditions of the test, it can be concluded, that the test substance is a sensitiser in KeratinoSens Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 - 22 Mar 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: Human Cell Line Activation test (h-CLAT))
Version / remarks:
adopted in Oct 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
National GLP Compliance Monitoring Authority, India
Type of study:
activation of dendritic cells
Details on study design:
TEST CELL LINE
- Source: THP-1 cells, procured from American Type Culture Collection, Lot No.: 63176297
- Passage number: 19, 21, 22, 24 and 25

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES, 0.05 mM 2-mercaptoethanol and 100 U/mL penicillin/100 µg/mL streptomycin
- Temperature (°C): 37 ± 1
- CO2 (%): 5.0 ± 1

CONTROLS
Negative control
- Substance: culture medium
Vehicle control
- Substance: 0.2% dimethyl sulfoxide (DMSO)
Positive control
- Substance: 4 µg/mL 2,4-dinitrochlorobenzene (DNCB)

EXPOSURE CONDITIONS
- Exposure duration: 23.5 h

TEST CONCENTRATIONS: In the dose range finding assay the following concentrations 1.95, 3.91, 7.81, 15.62, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL were tested to determine the cytotoxic potential of the test substance. Based on the results of the dose range finding assay, the following concentrations 8.08, 9.70, 11.64, 13.97, 16.76, 20.12, 24.14 and 28.97 µg/mL were used for the measurement of the expression levels of CD86/CD54.

NUMBER OF REPLICATIONS: single measurement in two independent experiments

CYTOTOXICITY
- Method: Cytotoxicity was determined using propidium iodide staining by flow cytometry.
Cell viability = Number of living cells / Total number of acquired cells x 100
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
The CV75 = [((75 - c) x Log(b)) - ((75 - a) x Log(d))] / (a - c)
a: minimum value of cell viability over 75%
c: maximum value of cell viability below 75%
b and d: concentrations showing the value of cell viability a and c, respectively

MEASUREMENT
- Device: Becton Dickinson

STAINING
- Antibodies: FITC mouse anti-human CD86 Antibody (Clone: Fun-1) (BD Pharmingen, Lot #6348610), Monoclonal mouse anti-human CD54, ICAM-1 antibody/FITC (Clone 6.5 B5) (Dako, Lot #20044016) and FITC reagent mouse IgG1/FITC antibody (Dako (Lot #20046409)

EVALUATION CRITERIA
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction was considered POSITIVE if at least one of the following conditions was met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction was considered negative:
i. The RFI of CD86 is equal to or greater than 150% at any tested concentration (with cell viability ≥ 50%);
ii. The RFI of CD54 is equal to or greater than 200% at any tested concentration (with cell viability ≥ 50%).
Based on the results obtained, if the first two runs were both positive for CD86 and/or were both positive for CD54, the h-CLAT prediction was considered POSITIVE and a third run was not conducted. Similarly, if the first two runs were negative for both markers, the h-CLAT prediction was considered NEGATIVE, without the need for a third run. If however, the first two runs were not concordant for at least one of the markers (CD54 or CD86), a third run was needed and the final prediction was based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, if two independent runs were conducted and one was only positive for CD86 (referred to as P1) and the other was only positive for CD54 (referred to as P2), a third run was required. If this third run was negative for both markers (referred to as N), the h-CLAT prediction was considered negative. On the other hand, if the third run was positive for either marker (P1 or P2) or for both markers (referred to as P12), the h-CLAT prediction was considered positive.

ACCEPTANCE CRITERIA
i. The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
ii. In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥ 200%).
iii. For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
iv. In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
v. For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.
Positive control results:
The RFI values of positive control 2, 4-Dinitrochlorobenzene in both experiments were found to be 741.67% and 610.66% for CD86 marker and 595.53% and 688.08% for CD54 marker, which met the assay acceptance criteria (RFI CD86 ≥ 150%, RFI CD54 ≥ 200%). The cell viability of positive control was >80% in both the experiments.
Key result
Parameter:
other: EC150 (µg/mL)
Run / experiment:
Mean of Experiment I and II
Value:
5.32
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC200 (µg/mL)
Run / experiment:
Mean of Experiment I and II
Value:
13.04
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: CV75 (µg/mL)
Run / experiment:
Mean of Experiment I and II
Value:
24.14
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The RFI values of the solvent control for CD86 were 88.50 and 116.05 and for CD54 53.59 and 124.79 in Experiment I and II, respectively, and thus the values did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 95.3 and 96.1% and thus > 90%.
- Acceptance criteria met for positive control:
The RFI values of the solvent control for CD86 were 741.67 and 610.66 and for CD54 595.53 and 688.08 in Experiment I and II, respectively, and thus the values exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 83.4 and 81.8% and thus > 50%.

Table 1: Cell viability, MFI and RFI values of the test substance in CD86/CD54 expression measurements

Expt.1

Expt.2

Conc (µg/mL)

% Cell Viability

MFI

% RFI

% Cell Viability

MFI

% RFI

IgG1

CD86

CD54

CD86

CD54

IgG1

CD86

CD54

CD86

CD54

28.97

61.1

97.2

124

116

89.33

76.42

42.1 %

116

137

136

60.52

132.45

24.14

90.8

96.4

135

156

128.67

242.28

84.8 %

116

145

185

83.57

456.95

20.12

93.6

94.5

142

158

158.33

258.13

91.1 %

112

148

179

103.75

443.71

16.76

94.3

93.7

147

151

177.67

232.93

92.7 %

110

150

182

115.27

476.82

13.97

94.6

92.8

136

145

144.00

212.20

92.8 %

109

153

164

126.80

364.24

11.64

92.7

90.3

145

135

182.33

181.71

91.7 %

108

154

175

132.56

443.71

9.70

93.6

87.3

154

124

222.33

149.19

91.1 %

104

160

137

161.38

218.54

8.08

93.9

81.5

147

109

218.33

111.79

91.8 %

97.2

152

123

157.93

170.86

Table 2: Individual CV75, EC150 and EC200 of different experiments

Parameters Expt.1 Expt.2 Average values (in µg/mL)
CV75 (in µg/mL) 28.62 19.67 24.14
EC150 (in µg/mL) 0.36 5.32 5.32*
EC200 (in µg/mL) 13.04 9.07 13.04*

* = Higher calculated EC values out of the two experiments were used as average values

Interpretation of results:
other: skin sensitising potential based on the key event "activation of dentritic cells"
Conclusions:
Under the conditions of the test, it can be concluded, that the test substance is a sensitiser in the human Cell Line Activation Test (h-CLAT). The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Since 2 out of 3 key events resulted in a positive result, the test substance was considered to be a skin sensitiser. The available data meet the criteria for classification in Skin Sens. 1, H317 according to Regulation (EC) No 1272/2008.
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 - 18 Nov 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))
Version / remarks:
adopted in Feb 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Government of India, National Good Laboratory Practive Compliance Monitoring Authority, India
Type of study:
direct peptide binding assay
Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

TEST METHOD
The DPRA is an in chemico method, which quantifies the remaining concentration of cysteine or lysine containing peptide, following 24 ± 2 hours incubation with the test item at 25 ± 2.5 °C. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are calculated and used in a prediction model which allow assigning the test item to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers.

TEST SYSTEM
- Supplier of synthetic peptides: RS Synthesis, Louisville, USA
- Peptide stock solution preparation: Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the approprate peptide in appropriate buffer solution.
Cysteine-containing peptide: 9.270 mg cysteine was dissolved in 18.503 mL phosphate buffer (pH 7.5).
- Concentration: 0.667 mM
Lysine-containing peptide: 9.784 mg lysine was dissolved in 18.888 mL ammonium acetate buffer (pH 10.2).
- Concentration: 0.667 mM

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as a 100 mM solution in acetonitrile.

CO-ELUTION CONTROL
- Co-elution control was prepared without peptide, to verify if the test item absorbs at 220 nm and has a similar retention time as a peptide and/or interfere with the data analysis.

REFERENCE CONTROLS
Two types of "Reference Controls" were included on each day of analysis. Reference control is a peptide solution where the test item is replaced by the solvent used to dissolve test item.
- Reference control A was prepared with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
Preparation of reference control A (0.5 mM): 750 µL cysteine/lysine peptide (0.667 mM) + 250 µL of acetonitrile
- Reference control B was prepared with acetonitrile and its replicates were injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
Preparation of reference control B: Same as preparation for reference control A (but run before and after 24 ± 2 hours incubation at 25 ± 2.5 °C in the dark)

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: minimum of 24 ± 2 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for test substance and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Shimadzu with UV Detector
- Analytical Column: ZorbaX SB-C18 (2.1 mm x 100 mm x 3.5 micron) with Guard Column Phenomenex Security Guard C18 (4 mm x 2 mm)
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in Milli Q water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Column temperature: 30 ± 2 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Injection volume: 5 μL
- peptide standards: calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, and a buffer blank were measured.
Positive control results:
Cinnamic aldehyde was used as positive control in each assay with cysteine and lysine peptides. Values of the mean percent peptide depletion value of positive control cinnamic aldehyde was 79% for cysteine peptide and 54% for lysine peptide, respectively.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
2
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Peak Area and Retention Time of Positive Control and Test Item

Cysteine Peptide

Sample ID

Rep-1

Area (AU)

RT

(min)

Rep-2

Area (AU)

RT

(min)

Rep-3

Area (AU)

RT

(min)

Cinnamic aldehyde

437677

9.46

417586

9.448

420800

9.454

 Test Item

1969407

9.419

1926517

9.426

1932687

9.408

Co-elution control- Test Item

ND

ND

-

-

-

-

Lysine Peptide

Cinnamic aldehyde

681685

6.52

687649

6.506

695988

6.536

Test Item

1526868

6.496

1498531

6.487

1522569

6.492

Co-elution control- Test Item

ND

ND

-

-

-

-

Rep = Replicate, AU = Arbitrary Units, RT = Retention Time, min = Minute,ND = Not Detected,   - = Not applicable

Table 2: Positive Control and Test Item Percent Peptide Depletion

Percent Peptide Depletion (Cysteine)

Positive Control Name/

Test Item Code

Rep-1

Area

(AU)

Rep-2

Area

(AU)

Rep-3

Area

(AU)

Average

%

Depletion

SD

RCV

Expected

Depletion

Cinnamic aldehyde

437677

417586

420800

425354

79

10792.06

2.54

60.8-100

Test Item

1969407

1926517

1932687

1942870

2

23187.57

1.19

 

Co-elution control- Test Item

ND

-

-

-

-

-

-

-

Percent Peptide Depletion (Lysine)

Cinnamic aldehyde

681685

687649

695988

688441

54

7184.29

1.04

40.2-69

Test Item

1526868

1498531

1522569

1515989

0

15271.39

1.01

 

Co-elution control- Test Item

ND

-

-

-

-

-

-

-

Rep = Replicate, AU = Arbitrary Units, SD = Standard Deviation, RCV = Relative Coefficient of Variability, ND = Not Detected, - = Not applicable

Interpretation of results:
other: no skin sensitising potential based on the key event "protein reactivity"
Conclusions:
Under the conditions of the test, the test substance did not show reactivity towards selected proteins. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance meets the criteria for classification as Skin Sensitisation 1 (H317) under CLP regulation (EC 1272/2008, as amended) as 2 out of 3 in vitro/chemico studies concluded a positive a result.


It is not possible subcategorise Category 1 to 1A or 1B as there are no data available to determine potency.