Registration Dossier

Administrative data

Description of key information

skin sensitisation (OECD 442C and 442E): positive

skin sensitisation (OECD 442B): positive

WoE conclusion from in vitro and in vivo skin sensitisation tests: skin sensitising

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:
27 Jun - 15 Jul 2016
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)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM
Type of study:
direct peptide binding assay
Details on study design:
TEST SYSTEM
- Supplier: AnaSpec
- Cysteine-containing peptide:
Alternative name: Ac-RFAACAA-OH
Batch number: 1556171
Molecular weight: 751.5 g/mol
Purity: 95%
Expiry date: 5 years
- Lysine-containing peptide:
Alternative name: Ac-RFAAKAA-OH
Batch number: 1556172
Molecular weight: 776 g/mol
Purity: 90 - 95%
Expiry date: 5 years

TEST METHOD
The DPRA is an in chemico test system proposed to address the molecular initiating event of the skin sensitisation adverse outcome pathway, namely protein reactivity, by substance towards model synthetic peptides containing either lysine or cysteine. The DPRA quantifies the free concentration of cysteine- or lysine-containing peptide following incubation with the test substance. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in the prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Supplier: SAFC
- Batch number: MKBR2427V
- Purity: >95%
- Expiry date: Feb 2019

STABILITY AND PRECISION CONTROL
Stability and precision controls of both peptides were prepared at a concentration of 0.5 mM.

POSITIVE CONTROL PREPARATION
The positive control was prepared at a concentration of 100 mM.

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

PEPTIDE STOCK SOLUTION PREPARATION
Cysteine-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
Lysine-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); Lysine-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: at least 22 h

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and control

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm with guard column Phenomenex AJO4286
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid in deionised water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 20, 21, 23, 23.5, 30
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Injection volume: 2 µL
- Column temperature: 30 °C
Key result
Parameter:
other: % depletion of cystein-containing peptide
Remarks:
mean of 3 replicates
Run / experiment:
≥ 22 h incubation
Value:
21.2
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: % depletion of lysine-containing peptide
Remarks:
mean of 3 replicates
Run / experiment:
≥ 22 h incubation
Value:
8.11
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Key result
Parameter:
other: % overall mean depletion
Run / experiment:
≥ 22 h incubation
Value:
14.6
Vehicle controls validity:
not examined
Negative controls validity:
not examined
Positive controls validity:
valid
Other effects / acceptance of results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: No co-elution of the test substance occurred during the assay. The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was confirmed.

ACCEPTANCE OF RESULTS:
All analytical acceptance criteria for each peptide were met.

Table 5. Mean peptide depletion of cysteine-containing peptide.

 

 

Peak area (µV.sec)

Peptide concentration

(µg/mL)*

Peptide depletion (%)**

Mean depletion ± CV (%)

 

Positive control

252119

107.69

71.0

 

70.9 ± 0.33

253767

108.40

70.8

253194

108.15

70.9

Test substance

706138

304.09

18.8

 

21.2 ± 3.43

690228

297.21

20.6

729603

284.07

24.1

 CV: Coefficient of Variation

*: Samples prepared at a concentration of 376 μg/mL (0.5 mM)

**: Calculated against a mean reference control peak area of 869620 μV.sec (n = 6)

Table 6. Mean peptide depletion of lysine-containing peptide.

 

 

Peak area (µV.sec)

Peptide concentration

(µg/mL)*

Peptide depletion (%)**

Mean depletion ± CV (%)

 

Positive control

-      ***

-

-

 

57.4 ± 0.06

337876

168.77

57.5

338241

168.95

57.4

 

Test substance

736966

365.25

7.2

 

8.11 ± 0.87

727300

360.49

8.42

725044

359.38

8.70

CV: Coefficient of Variation

*: Samples prepared at a concentration of 388 μg/mL (0.5 mM)

**: Calculated against a mean reference control peak area of 794160 μV.sec (n = 6)                            

***: Result not reported due to atypical chromatography.

 

 

Interpretation of results:
other: DPRA prediction: positive (low reactivity) according to OECD 442C
Executive summary:

Under the conditions of the Direct Peptide Reactivity Assay the test substance showed low reactivity and therefore is considered positive for skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2 May - 21 Jun 2016
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)
Version / remarks:
December 2015
Deviations:
yes
Remarks:
cytotoxicity measurement and estimation of the CV75 value was performed by XTT test instead of flow cytometry
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of study:
activation of dendritic cells
Details on study design:
TEST CELL LINE
- Source: American Type Culture Collection
- Passage number: 24 (XTT test); 5 - 7 (h-CLAT)

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) pyruvate and 1% (v/v) L-glutamine
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5

The following information relate to details on XTT
The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4 - tetrazolium)-bis-(4–methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable concentration showing 75% cell viability (CV75).

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: The test substance was soluble in DMSO up to and including 500 µg/mL.
3.91, 7.81, 15.63, 31.25, 62.5, 125, 250 and 500 µg/mL

NUMBER OF REPLICATIONS: 4 samples per dose group were tested in two independent experiments

MEASUREMENT
- Device: microplate reader (Versamax Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm

EVALUATION CRITERIA
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance as compared to the solvent control is calculated using the formula:
Relative absorbance = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance vehicle control) - (absorbance blank)]

To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:
CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])
a) Conc. > 75: max. measured concentration with the % of vehicle control > 75%
b) Conc. < 75: min. measured concentration with the % of vehicle control < 75%
c) % > 75: relative absorbance at a) in %
d) % < 75: relative absorbance at b) in %

ACCEPTANCE CRITERIA
The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the negative control is ≥ 0.5
- mean viability of the vehicle control is ≥ 70%

The following information relate to details on h-CLAT
The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: DMSO
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene in DMSO
- Concentration: 2 and 3 µg/mL

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: Since a CV75 value could not be determined the highest soluble test substance concentration was multiplied with 1.2 (600 µg/mL)
167.4, 200.9, 241.1, 289.4, 347.2, 416.7, 500 and 600 µg/mL

NUMBER OF REPLICATIONS: at least in triplicates for the different stainings in two independent experiments

STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C
- Duration: 30 ± 5 min

MEASUREMENT
- Device: FACSCalibur (Becton Dickinson)

EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration:
RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance isotype control cells) / (MFI of vehicle control cells) - (MFI of vehicle isotype control cells)] x 100
MFI: geometric mean fluorescence intensity (GeoMean)

The cell viability is calculated as follows for each concentration:
Cell viability (%) = (mean cytotox of vehicle control cells) / (mean cytotox of test substance treated cells) x 100
Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.

The test substance is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of negative control is adjusted to 100% and the cell viability of the vehicle control should be more than 90% in comparison to the negative control.
- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For negative and vehicle controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test substance resulting in negative outcome, the cell viability at the 1.2 × CV75 value should be less than 90%. If the cell viability at the 1.2 × CV75 value is more than 90% for a positive tested test substance, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test substance are accepted independent by the cell viability.
- The cell viability of at least 4 doses in each experiment should be ≥50%.
Key result
Parameter:
other: relative fluorescence intensity of CD54 (%)
Run / experiment:
24 h incubation
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: relative fluorescence intensity of CD86 (%)
Run / experiment:
24 h incubation
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Phase separation: Due to phase separation observed in the two highest concentrations of the first experiment and the highest concentration of the second experiment, these results were excluded from the evaluation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
Cytotoxic effects were not observed following incubation with the test substance up to the highest soluble concentration (500 µg/mL). The viability of the cells spaced within a range of 102.67 to 130.87% in the first experiment and within a range of 89.08 to 111.77% in the second experiment (threshold of cytotoxicity: < 75%). Due to the lack of cytotoxicity, a CV75 value could not be calculated.
h-CLAT
The cell viability was not negatively affected (viability > 75%) in both independent experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was >50%.

Table 1: Results of the first XTT test

 

Concentration [µg/mL]

Mean absorbance*

Blank

Absorbance in % of vehicle control**

Medium control

-

0.973 ± 0.031

0.311

110.48

Vehicle control

-

0.922 ± 0.055

0.323

100.00

Test substance

3.91

1.000 ± 0.076

0.308

115.48

7.81

0.924 ± 0.067

0.312

102.12

15.63

0.924 ± 0.074

0.309

102.67

31.25

0.956 ± 0.030

0.303

108.91

62.5

0.968 ± 0.055

0.310

109.84

125

1.044 ± 0.096

0.316

121.58

250

1.068 ± 0.088

0.315

125.74

500

1.101 ± 0.062

0.317

130.87

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 2: Results of the second XTT test

 

Concentration [µg/mL]

Mean absorbance*

Blank

Absorbance in % of vehicle control**

Medium control

-

1.011 ± 0.092

0.309

120.38

Vehicle control

-

0.884 ± 0.046

0.301

100.00

Test substance

3.91

0.878 ± 0.037

0.309

97.45

7.81

0.834 ± 0.074

0.314

89.08

15.63

0.859 ± 0.053

0.307

94.58

31.25

0.979 ± 0.069

0.306

115.28

62.5

0.892 ± 0.070

0.310

99.85

125

0.964 ± 0.092

0.312

111.77

250

0.960 ± 0.059

0.316

110.33

500

0.879 ± 0.064

0.308

97.91

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 3. Results of the first h-CLAT run

 

Concentration [µg/mL]

Antibody / ISO

MFI GeoMean (FITC)

MFI - ISO

Csto(Geo) GeoMean (7-AAD)

Mean Cyto

RFI (%)

Viability (%)

Medium control

-

ISO

2.33

-

2.07

2.0

-

100.0

CD54

3.01

0.68

1.88

100.0

CD86

4.34

2.01

1.90

100.0

Vehicle control

-

ISO

2.35

-

1.88

1.9

-

100.0

CD54

3.24

0.89

1.81

100.0

CD86

4.00

1.65

1.91

100.0

Positive control

2

ISO

2.18

-

3.13

2.5

-

74.4

CD54

3.39

1.21

2.20

136.0

CD86

5.65

3.47

2.20

210.3

3

ISO

2.67

-

3.38

2.4

-

77.3

CD54

8.64

5.97

2.11

670.8

CD86

11.07

8.40

1.75

509.1

Test substance

167.4

ISO

2.23

-

2.59

2.2

-

85.2

CD54

3.93

1.70

1.98

191.0

CD86

4.60

2.37

2.00

143.6

200.9

ISO

2.32

-

2.09

1.9

-

99.3

CD54

3.92

1.60

1.69

179.8

CD86

4.26

1.94

1.86

117.6

241.1

ISO

2.21

-

2.13

1.9

-

99.5

CD54

4.12

1.91

1.85

214.6

CD86

4.90

2.69

1.65

163.0

289.4

ISO

2.20

-

3.06

2.5

-

75.7

CD54

3.92

1.72

2.51

193.3

CD86

5.37

3.17

1.83

192.1

347.2

ISO

2.35

-

2.60

2.4

-

78.1

CD54

3.64

1.29

2.13

144.9

CD86

4.05

1.70

2.44

103.0

416.7

ISO

2.27

-

2.35

2.3

-

80.3

CD54

3.93

1.66

2.11

186.5

CD86

4.03

1.76

2.51

106.7

500PS

ISO

2.27

-

2.34

2.1

-

90.2

CD54

3.92

1.65

1.91

185.4

CD86

4.53

2.26

1.96

137.0

600PS

ISO

2.27

-

2.11

2.1

-

90.5

CD54

4.00

1.73

2.27

194.4

CD86

4.47

2.20

1.81

133.3

*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).

PS: phase separation

 

Table 4. Results of the second h-CLAT run

 

Concentration [µg/mL]

Antibody / ISO

MFI GeoMean (FITC)

MFI - ISO

Csto(Geo) GeoMean (7-AAD)

Mean Cyto

RFI (%)

Viability (%)

Medium control

-

ISO

2.18

-

1.73

1.8

-

100.0

CD54

3.01

0.83

1.73

100.0

CD86

3.07

0.89

1.80

100.0

Vehicle control

-

ISO

2.13

-

1.76

1.6

-

100.0

CD54

3.14

1.01

1.61

100.0

CD86

3.42

1.29

1.49

100.0

Positive control

2

ISO

2.13

-

2.38

1.8

-

91.0

CD54

6.33

4.20

1.65

415.8

CD86

9.61

7.48

1.31

579.8

3

ISO

2.13

-

2.49

1.7

-

97.2

CD54

10.94

8.81

1.10

872.3

CD86

10.45

8.32

1.41

645.0

Test substance

167.4

ISO

1.95

-

2.36

2.1

-

78.9

CD54

4.46

2.51

2.05

248.5

CD86

4.45

2.50

1.75

193.8

200.9

ISO

1.99

-

2.02

1.8

-

87.9

CD54

4.48

2.49

1.89

246.5

CD86

4.25

2.26

1.62

175.2

241.1

ISO

1.92

-

2.28

1.9

-

83.2

CD54

4.44

2.52

1.88

249.5

CD86

4.52

2.60

1.68

201.6

289.4

ISO

1.93

-

2.47

2.1

-

77.9

CD54

4.41

2.48

1.88

245.5

CD86

3.79

1.86

1.89

144.2

347.2

ISO

1.98

-

2.49

2.1

-

76.4

CD54

4.03

2.05

1.90

203.0

CD86

3.91

1.93

1.97

149.6

416.7

ISO

1.98

-

2.18

1.9

-

86.2

CD54

4.37

2.39

1.64

236.6

CD86

3.89

1.91

1.82

148.1

500

ISO

1.99

-

1.97

1.8

-

88.2

CD54

4.37

2.38

1.77

235.6

CD86

3.94

1.95

1.77

151.2

600PS

ISO

1.90

-

2.29

2.0

-

80.2

CD54

4.33

2.43

1.87

240.6

CD86

4.02

2.12

1.90

164.3

*: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥150% and CD54 ≥200%).

PS: phase separation

Interpretation of results:
other: positive according to OECD 442E
Conclusions:
Under the conditions of the Human Cell Line Activation Test the test substance increased the expression of both cell surface markers, CD86 and CD54, associated with the process of activation of monocytes and dendritic cells. Based on this result, the h-CLAT prediction is considered positive for skin sensitisation.
Executive summary:

Dendritic cell response of the test substance was determinded by a Human Cell Line Activation Test according to OECD 442E in compliance with GLP. The test substance increased the expression of both cell surface markers, CD86 and CD54, associated with the process of activation of monocytes and dendritic cells. Based on this result, the h-CLAT prediction is considered positive for skin sensitisation.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
03 Jan - 13 Jun 2017
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)
Version / remarks:
adopted: 22 Jul 2010
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
other:
Remarks:
CBA/N
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 9 weeks
- Weight at study initiation: 17.1 - 21.7 g (dose range finding study); 17.2 - 20.1 g (main study - set 1); 16.8 - 21.7 g (main study - set 2)
- Housing: 2 to 3 animals per cage in polysulfone cages (200W x 320D x 140H mm)
- Diet: pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water: tap water (filtered and irradiated by UV-light), ad libitum
- Acclimation period: 4 - 5 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 - 24.2 (dose range finding study); 20.7–23.6 (main study - set 1); 21.2–23.0 (main study - set 2)
- Humidity (%): 41.3–59.1 (dose range finding study); 41.8–54.8 (main study - set 1); 45.1–53.9% (main study - set 2)
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Dose range finding study: 5, 10, 25, 50% (w/v) and 100%
Main experiment: Set 1: 25, 50% (w/v) and 100%; Set 2: 1, 5, 10 (w/v)
No. of animals per dose:
2 (dose range finding study), 5 (main study)
Details on study design:
RANGE-FINDING STUDY: Dose selection was based on the consecutive doses and dose levels are selected from a series of appropriate concentrations such as 5, 10, 25, 50 and 100%.
- Compound solubility: The test substance was dissolved in aceton/olive oil in a preliminary solubility test. Therefore, aceton/olive oil was utilized as vehicle for this study.
Two animals were observed per dose group. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing (Day 1) and on the day of necropsy, Day 6. Both ears of each mouse were observed for erythema and scored for 6 days. Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 and Day 6. Additionally, on Day 6, ear weight was determined by balance.
- Irritation: not specified
- Systemic toxicity: not specified
- Ear thickness measurements: not specified
- Erythema scores: not specified

MAIN STUDY: Based on the result of the dose range finding study, the high dose level for the main study was selected at 100%. Two additional low dose levels (25 and 50%) and a positive and negative control were tested in the main study. An additional main experiment with 3 lower doses (1, 5 and 10%) was also conducted.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ELISA BrdU
- Criteria used to consider a positive response: A stimulation index (SI) was calculated for each group using the BrdU labelling index of each test group divided by the BrdU labelling index of the vehicle control group. SI < 1.6 is considered as negative result. SI ≥ 1.6 is considered as positive result.

The EC1.6 value was used to classify the test substance according to ECETOC Potency classification as follows:
EC1.6 Value (%) ≥ 10 to ≤ 100 -> Weak
EC1.6 Value (%) ≥ 1 to ≤ 10 -> Moderate
EC1.6 Value (%) ≥ 0.1 to ≤ 1 -> Strong
EC1.6 Value (%) < 0.1 -> Extreme
Ryan et al. Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutaneous and Ocular Toxicology, 2007, 26: 135-145.

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. Two days after the third application on Day 5, an intraperitoneal injection of 0.5 mL (5 mg/mouse) of BrdU solution (10 mg/mL) was made. Approximately 24 h after BrdU injection, the mice were sacrificed and draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal. A single cell suspension was prepared by separation through a nylon mesh. In each case, the target volume of the cell suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1 - 0.2 in the negative control group. BrdU was measured by ELISA using a commercial kit. Briefly, 100 µL of the lymph node cell suspension was added to the wells of a microplate in triplicate. After fixation and denaturation of the cell suspension, anti-BrdU antibody was added to each well. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was added. Absorbance was measured at 370 nm with a reference wavelength of 492 nm.

Negative control animals were dosed with the vehicle, acetone/olive oil solution.

OBSERVATIONS:
Animals were observed daily for mortality, general condition, clinical signs of toxicity and signs of local irritation (erythema) at the test site.
 
Body weight:
Individual body weights were recorded on Day 1 prior to dosing and on Day 6 prior to necropsy.

Ear thickness:
Ear thickness was measured using a thickness gauge on Day 1 prior to dosing, Day 3 (approximately 48 h after the first dose) and Day 6 (the day of necropsy).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index.
Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group. Since it was not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.
Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.
Positive control results:
Body weights: The mean body weight was 18.6 - 18.9 g (set 1) and 19.8 - 20.4 g (set 2). There were no significant differences when compared to the negative control group.
Erythema scores: The mean erythema score was 0 - 3 (set 1) and 0 - 1 (set 2). There were significant increases when compared to the negative control group (set 1 and 2: p<0.01for Days 3, 4, 5 and 6)
Ear thickness: The mean ear thickness was 0.20 - 0.21 mm (set 1) and 0.19 - 0.21 mm (set 2) . There were significant increases when compared to the negative control group (set 1: p<0.01for days 1, 3 and 6 and set 2: p < 0.05 for Days 3 and 6).
Ear weights: The mean ear weight was 16.5 mg (set 1) and 14.1 mg (set 2). For set 1 there were no significant differences when compared to the negative control group. For set 2 there was asignificant increase when compared to the negative control group (p<0.01).
Stimulation Index: The mean stimulation index was 2.84 (set 1) and 1.81 (set 2) . There was a significant increase when compared to the negative control group (p<0.01).
Key result
Parameter:
SI
Value:
1.88
Test group / Remarks:
100%
Key result
Parameter:
SI
Value:
2.18
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
1.83
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
1.23
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
5% (w/v)
Key result
Parameter:
SI
Value:
1.21
Test group / Remarks:
1% (w/v)
Cellular proliferation data / Observations:
IRRITATION, EAR THICKNESS AND EAR WEIGHTS:
Erythema Score: Set 1: In the negative control group, the mean erythema score was 0 from Day 1 to Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean erythema scores were in the ranges of 0–1, 0–1 and 0–1, respectively. There were significant increases when compared to the negative control group (p<0.01: Days 4, 5 and 6 (25, 50 and 100%)).
Set 2: In the negative control group, the mean erythema score was 0 from Day 1 to Day 6 after dosing. In the test substance groups at 1, 5 and 10%, the mean erythema scores were in the ranges of 0, 0 and 0, respectively. There were no significant differences when compared to the negative control group.
Ear thickness: Set 1: In the negative control group, the mean ear thickness was in the range of 0.19–0.20 mm from Day 1 to Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean ear thickness was in the ranges of 0.19–0.20 mm, 0.19–0.20 mm and 0.20–0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 3 (50%), p<0.01: Day 1 (50 and 100%), Day 3 (100%), Day 6 (100%)).
Set 2: In the negative control group, the mean ear thickness was in the range of 0.19 mm from Day 1 to Day 6 after dosing. In the test substance groups at 1, 5 and 10%, the mean ear thickness was in the ranges of 0.19, 0.19 and 0.19 mm, respectively. There were no significant differences when compared to the negative control group.
Ear weights: Set 1: In the negative control group, the mean ear weight was 14.8 mg. In the test substance groups at 25, 50 and 100%, the mean ear weights were 14.7, 14.8 and 15.0 mg, respectively. There were no significant differences when compared to the negative control group.
Set 2: In the negative control group, the mean ear weight was 12.2 mg. In the test substance groups at 1, 5 and 10%, the mean ear weights were 12.1, 12.6 and 12.6 mg, respectively. There were no significant differences when compared to the negative control group.

DETAILS ON STIMULATION INDEX CALCULATION:
SI values were calculated to be above 1.6 at all concentrations in the main study-SET1, but SI values were calculated to be <1.6 in the main study-SET2. Thus, EC1.6 value was calculated according to the following formula:
EC1.6 =c+[(1.6-d)/(b-d)] x (a-c)
a = The dose concentration with higher SI
b = The higher SI value
c = The dose concentration with lower SI
d = The lower SI value

EC1.6 = 23.4%.

CLINICAL OBSERVATIONS: There were no abnormal clinical signs or deaths in any dosing group during the observation period.

BODY WEIGHTS: Set 1: In the negative control group, the mean body weight was in the range of 19.0–18.9 g from Day 1 to Day 6 after dosing. In the test substance groups at 25, 50 and 100%, the mean body weights were in the ranges of 18.1– 18.7, 18.7–18.7 and 18.7–19.6 g, respectively. There were no significant differences when compared to the negative control group.
Set 2: In the negative control group, the mean body weight was in the range of 19.1–19.8 g from Day 1 to Day 6 after dosing. In the test substance groups at 1, 5 and 10%, the mean body weights were in the ranges of 19.4– 19.2, 19.4–19.8 and 19.3–19.6 g, respectively. There were no significant differences when compared to the negative control group.




Interpretation of results:
other: Skin Sens Cat 1B according to Regulation (EC) No 1272/2008
Conclusions:
Under the conditions of the local lymph node assay, the test substance revealed a SI ≥ 1.6 at concentrations of 25, 50 and 100%. The calculated EC1.6 value was 23.4%. Therefore, the test substance is considered to be weak sensitiser.
Executive summary:

The potency of the skin sensitisation was investigated by a LLNA test using BrdU ELISA method according to OECD 442B in compliance with GLP. Treatment of CBA/N mice with the stest substance revealed stimulation indices of 1.21, 1.20, 1.23, 183, 2.18 and 1.88 at concentrations of 1, 5, 10, 25, 50 and 100%.

Under the conditions of this test, the test substance revealed a SI ≥ 1.6 at concentrations of 25, 50 and 100%.

The calculated EC1.6 value was 23.4%. Therefore, the test substance is considered to be weak sensitiser.
Endpoint conclusion
Additional information:

In vitro:

Protein reactivity of the test substance was determined by a Direct Peptide Reactivity Assay according to OECD 442C and in compliance with GLP (2016). The overall mean percent cysteine and lysine depletion is 14.6 and therefore the test substance is allocated to the “low reactivity” class (mean % depletion > 6.38 ≤ 22.62). Thus, the DPRA prediction for protein reactivity is considered positive.

Dendritic cell response of the test substance was determined by a Human Cell Line Activation Test (h-CLAT) according to OECD 442E and in compliance with GLP (2016). The test substance increased the expression of the cell surface marker CD86 and CD54 to > 150% and > 200%, respectively in at least one dose of 2 independent run data. Thus, the h-CLAT prediction for activation of dendritic cells is considered positive.

In vivo:

The skin sensitisation potential of the test substance was investigated in vivo by a LLNA test using the BrdU ELISA method according to OECD Guideline 442B in compliance with GLP (2017). Treatment of CBA/N mice with the test substance revealed stimulation indices of 1.21, 1.20, 1.23, 1.83, 2.18 and 1.88 at concentrations of 1, 5, 10, 25, 50 and 100%, respectively. EC1.6 value was calculated to be 23.4% and therefore the test substance is considered to be a weak skin sensitiser.

Conclusion

Based on a weight of evidence approach considering two positive results in the in vitro test battery and a positve result in the LLNA-BrdU, the test substance is considered to be a weak skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation meets the criteria for classification as Skin Sens 1B (H317) according to Regulation (EC) 1272/2008.